WO2025184574A1

WO2025184574A1 - Treatment of eye diseases and diagnostics

Info

Publication number: WO2025184574A1
Application number: PCT/US2025/017947
Authority: WO
Inventors: Steven A. GROSS
Original assignee: Stoke Therapeutics Inc
Current assignee: Stoke Therapeutics Inc
Priority date: 2024-03-01
Filing date: 2025-02-28
Publication date: 2025-09-04
Anticipated expiration: 2026-09-01

Abstract

Provided herein, in some aspects, are methods of treatment that involve using a vision test (e.g., low-contrast visual acuity test) score as a biomarker of disease progression. In some aspects, provided herein are compositions and methods relating to antisense oligomers that modulate splicing of OPA1 pre-mRNA. In some aspects, provided herein are methods of treatment relating to antisense oligomers or vector encoding the antisense oligomer as a therapeutic agent and use of a vision test (e.g., low-contrast visual acuity test) score as a biomarker for assessing patient eligibility for the treatment, prognostics, and/or adjustment in treatment regimen.

Description

TREATMENT OF EYE DISEASES AND DIAGNOSTICS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/560,497, filed March 1, 2024, and U.S. Provisional Application No. 63/560,506, filed March 1, 2024, each of which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Autosomal dominant optic atrophy (ADOA) is one of the most commonly diagnosed optic neuropathies. This optic nerve disease is associated with structural and functional mitochondrial deficits that lead to degeneration of the retinal ganglion cells and progressive, irreversible loss of vision. A majority of ADOA patients carry mutations in OPA1 and most mutations lead to haploinsufficiency (Uenaers G. et al. Orphanet J Rare Dis 2012). OPA1 encodes a mitochondrial GTPase with a critical role in mitochondrial fusion, ATP synthesis and apoptosis. Currently, there are no noninvasive assessments for mitochondrial dysfunction in ADOA patients and there is a need for such diagnostics.

[0003] ADOA is seen as essentially a mitochondrial disorder originating from a nuclear-encoded gene OPA1 , and most ADOA patients carry mutations in this gene, resulting in haploinsufficiency with approximately 50% reduction in cellular OPA1 protein level. OPA1 can localize to the mitochondrial inner membrane, and reduced levels of OPA1 can impair mitochondrial function, leading to retinal ganglion cell loss and progressive, irreversible visual loss. In some cases, oxidative phosphorylation, a cellular process that can take place in mitochondria, is found to be impaired in the fibroblasts from ADOA patients with a heterozygous mutation in OPA1. Considering the centrality of mitochondrial function in ADOA, having a biomarker to determine mitochondrial function in vivo would be helpful to examine the efficacy of treatment or target engagement and evaluate disease severity or disease stage, but the development of such a biomarker and assessment of ADOA patient eye condition both pose unique challenges. Vitreous sampling after administration is difficult because this procedure requires vitrectomy, which is invasive and is associated with nontrivial risks such as retinal detachment and vision loss. Systemic assessments are also not representative of the condition of the ocular targets after intravitreal delivery of treatment.

[0004] Mitochondria contain flavoproteins, which can carry out essential functions in electron transport during cellular respiration. Flavoproteins in mitochondria have been found to emit green light when stimulated with cobalt blue light. Specifically, in the presence of retinal oxidative stress, mitochondrial flavoproteins can exhibit increased fluorescence measured as emitted green light (peak emission at 520- 540 nm) when stimulated by blue light (peak excitation at 430-470 nm). For eye diseases and conditions that present with mitochondrial dysfunction, mitochondrial flavoproteins are expected to exhibit increased fluorescence intensity upon blue light stimulation.

SUMMARY

[0005] Described herein, in some aspects, is a method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutic agent, wherein the subject has a vision test score within a reference value range and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer.

[0006] Described herein, in some aspects, is a method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising: (1) determining a vision test score of the subject; (2) identifying the subject as an eligible subject for treatment when the vision test score determined in (1) is within a reference value range; and (3) administering to the eligible subject a pharmaceutical composition comprising a therapeutic agent, wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer.

[0007] Described herein, in some aspects, is a method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising administering to the subject a pharmaceutical composition according to a dosing regimen selected based at least in part on a vision test score that the subject has, wherein the pharmaceutical composition comprises a therapeutic agent, and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer.

[0008] In some embodiments, the vision test score is measured before the subject receives administration of the pharmaceutical composition. In some embodiments, the vision test score is measured after the subject receives administration of one or more prior doses of the pharmaceutical composition.

[0009] Described herein, in some aspects, is a method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising: (1) determining a vision test score of the subject; (2) selecting a dosing regimen for a pharmaceutical composition for the subject based at least in part on the vision test score determined in (1), wherein the pharmaceutical composition comprises a therapeutic agent, and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer; and (3) administering the pharmaceutical composition to the subject according to the selected dosing regimen.

[0010] Described herein, in some aspects, is a method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising: (1) administering to the subject a pharmaceutical composition according to a dosing regimen, wherein the pharmaceutical composition comprises a therapeutic agent, and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer; (2) after (1), determining a vision test score of the subject; (3) adjusting the dosing regimen for the pharmaceutical composition based at least in part on the vision test score determined in (2); and (4) administering the pharmaceutical composition to the subject according to the dosing regimen adjusted in (3). In some embodiments, the dosing regimen for the pharmaceutical composition is selected based at least in part on a vision test score measured prior to the administering in (1). In some embodiments, the dosing regimen comprises frequency of administration of the pharmaceutical composition, dose of the pharmaceutical composition per a single administration, time interval between administrations of the pharmaceutical composition, duration of treatment with the pharmaceutical composition, or administration route for the pharmaceutical composition.

[0011] In some embodiments, the vision test score is within a reference value range. In some embodiments, the vision test score is determined based at least in part on result from a Best Corrected Visual Acuity (BCVA) test of one or both eyes of the subject. In some embodiments, the vision test score is determined based at least in part on a parameter selected from the group consisting of Best Corrected Visual Acuity (BCVA) letter score; Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score; high-contrast Best Corrected Visual Acuity (HC BCVA) letter score; low-contrast Best Corrected Visual Acuity (LC BCVA) letter score; and any combinations thereof. In some embodiments, the vision test score is determined based at least in part on a low-contrast Best Corrected Visual Acuity (LC BCVA) letter score. In some embodiments, the vision test score is determined based at least further in part on a parameter selected from the group consisting of flavoprotein fluorescence intensity; Humphrey 10-2 Visual Field test Mean Deviation (MD); Humphrey 10-2 Visual Field test Pattern Standard Deviation (PSD); Visual Acuity Score (VAS); Quality of life questionnaires (NEI-VFQ-25, IVI-C, EQ-5D, EQ-5D-Y); Refraction test result; Minnesota Reading (MNRead) acuity chart score; Pelli-Robson chart score; slit lamp examination result; intraocular pressure using a Tonopen; perimetry; dilated fiindoscopy result; retinal nerve fiber layer (RNFL) measurement; optical coherence tomography (OCT) result; and macular ganglion cell layer/inner plexiform layer (GCL/IPL) thickness measurements; Curve Width (CW) measurement; Electroretinogram (ERG) result; fundus photography result; and any combinations thereof.

[0012] In some embodiments, the BCVA comprises testing in a defined sequence for (1) ocular refraction from a fixed distance, and (2) visual acuity from a fixed distance. In some embodiments, the testing comprises using at least one Original Sloan Early Treatment Diabetic Retinopathy Study (ETDRS) chart. In some embodiments, the ETDRS chart comprises a chart selected from the group consisting of Chart R (Precision Vision 2110) to measure refraction, Chart 1 (Pression Vision 2111) to test the right eye (OD), and Chart 2 (Precision Vision 2112) to test the left eye (OS). In some embodiments, the defined sequence comprises testing for ocular refraction with Chart R before testing for visual acuity with either Chart 1 or Chart 2. In some embodiments, the fixed distance comprises a distance of about 4 meters or about 1 meter from the eyes of the subject to the front of the chart. In some embodiments, the testing for ocular refraction comprises a distance of about 4 meters from the eyes of the subject to the front of the chart. In some embodiments, the testing for visual acuity comprises at least a first distance of about 4 meters from the eyes of the subject to the front of the chart. In some embodiments, the testing for visual acuity comprises a second distance of about 1 meter from the eyes of the subject to the front of the chart. In some embodiments, the BCVA further comprises calculating a letter score comprising a sum of a total number of letters correctly identified by the subject at 4 meters, plus 30; or calculating a letter score comprising a sum of a total number of letters correctly identified by the subject at 1 meter. In some embodiments, the ETDRS chart is a High-Contrast (HC) ETDRS chart. In other embodiments, the ETDRS chart is a Low-Contrast (LC) (2.5%) ETDRS chart. In some instances, the subject has decrease in the Low-Contrast (LC) (2.5%) ETDRS letter score of at least 5 letters after about 12 months as compared to the subject’s baseline Low-Contrast (LC) (2.5%) ETDRS letter score prior to the administering.

[0013] In some embodiments, the vision test score is determined based at least in part on result from a Humphrey 10-2 Visual Field test of one or both eyes of the subject.

[0014] In some embodiments, the vision test score is determined based at least in part on a parameter selected from the group consisting of flavoprotein fluorescence intensity; Humphrey 10-2 Visual Field test Mean Deviation (MD); Humphrey 10-2 Visual Field test Pattern Standard Deviation (PSD); Visual Acuity Score (VAS); Quality of life questionnaires (NEI-VFQ-25, IVI-C, EQ-5D, EQ-5D-Y); Refraction test result; Minnesota Reading (MNRead) acuity chart score; Pelli-Robson chart score; slit lamp examination result; intraocular pressure using a Tonopen; perimetry; dilated fundoscopy result; retinal nerve fiber layer (RNFL) measurement; optical coherence tomography (OCT) result; and macular ganglion cell layer/inner plexiform layer (GCL/IPL) thickness measurements; Curve Width (CW) measurement; Electroretinogram (ERG) result; fundus photography result; and any combinations thereof. In some embodiments, the vision test score is determined based at least further in part on a parameter selected from the group consisting of Best Corrected Visual Acuity (BCVA) letter score; Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score; high-contrast Best Corrected Visual Acuity (HC BCVA) letter score; low-contrast Best Corrected Visual Acuity (LC BCVA) letter score; and any combinations thereof.

[0015] In some embodiments, the vision test score is indicative of a level of visual acuity in the eye of the subject. In some embodiments, the reference value range is a range lower than a vision test score of a healthy control subject. In some embodiments, the reference value range is a range lower than an average vision test score measured from a population of healthy control subjects.

[0016] In some embodiments, when the pharmaceutical composition is tested on a population of test subjects suffering the disease or condition, a vision test score measured from the test subjects in the population is determined to have a correlation with therapeutic efficacy of the pharmaceutical composition in the test subjects, and wherein the reference value range is a range associated with the therapeutic efficacy of the pharmaceutical composition at a reference level according to the correlation. In some embodiments, the genotype of the subject is unknown prior to the administration, n some embodiments, the genotype of the subject is unknown prior to the determining. In some embodiments, the dosing regimen is not selected based on the genotype of the subject.

[0017] In some embodiments, about 0.005 to about 20 mg of the antisense oligomer is administered to one eye of the subject. In some embodiments, about 0.005 mg to about 15 mg, about 0.005 mg to about 10 mg, about 0.005 mg to about 5 mg, about 0.005 mg to about 1 mg, about 0.01 mg to about 15 mg, about 0.01 mg to about 10 mg, about 0.01 mg to about 5 mg, about 0.01 mg to about 2.5 mg, about 0.01 mg to about 1.0 mg, about 0.01 mg to about 0.5 mg, about 0.01 mg to about 0.25 mg, about 0.01 mg to about 0. 1 mg, about 0.01 mg to about 0.05 mg, about 0.05 mg to about 10 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 2.5 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.25 mg, about 0.05 mg to about 0.1 mg, about 0. 1 mg to about 5 mg, about 0.1 mg to about 2.5 mg, about 0.1 mg to about 1.0 mg, about 0.1 mg to about 0.5 mg, or about 0.1 mg to about 0.25 mg of the antisense oligomer is administered to one eye of the subject. In some embodiments, about 0.005 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.2 mg, about 0.5 mg, about 0.75 mg, about 1.0 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2.0 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, about 5.0 mg, about 5.5 mg, about 6.0 mg, about 7.0 mg, about 8.0 mg, about 9.0 mg, about 10 mg, about 12.5 mg, about 15 mg, about 17.5 mg, or about 20 mg of the antisense oligomer is administered to one eye of the subject. In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0.1 mg to about 1.5 mg, about 0. 1 mg to about 1.4 mg, about 0.1 mg to about 1.2 mg, about 0. 1 mg to about 1.0 mg, about 0.1 mg to about 0.8 mg, about 0. 1 mg to about 0.7 mg, about 0.1 mg to about 0.5 mg, about 0.1 mg to about 0.3 mg, about 0.2 mg to about 1.5 mg, about 0.2 mg to about 1.4 mg, about 0.2 mg to about 1.2 mg, about 0.2 mg to about 1.0 mg, about 0.2 mg to about 0.8 mg, about 0.2 mg to about 0.7 mg, about 0.2 mg to about 0.5 mg, about 0.3 mg to about 1.5 mg, about 0.3 mg to about 1.4 mg, about 0.3 mg to about 1.2 mg, about 0.3 mg to about 1.0 mg, about 0.3 mg to about 0.8 mg, about 0.3 mg to about 0.7 mg, about 0.3 mg to about 0.5 mg, about 0.5 mg to about 1.5 mg, about 0.5 mg to about 1.4 mg, about 0.5 mg to about 1.2 mg, about 0.5 mg to about 1.0 mg, about 0.5 mg to about 0.8 mg, about 0.5 mg to about 0.7 mg, about 0.7 mg to about 1.5 mg, about 0.7 mg to about 1.4 mg, about 0.7 mg to about 1.2 mg, about 0.7 mg to about 1.0 mg, about 0.8 mg to about 1.5 mg, about 0.8 mg to about 1.4 mg, about 0.8 mg to about 1.2 mg, about 0.8 mg to about 1.0 mg, about 1.0 mg to about 1.5 mg, about 1.0 mg to about 1.4 mg, about 1.0 mg to about 1.2 mg, about 1.2 mg to about 1.5 mg, or about 1.2 mg to about 1.4 mg of the antisense oligomer. In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0.1 mg, about 0.2 mg, about 0.3 mg, 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, or about 1.5 mg of the antisense oligomer.

[0018] In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition in a volume of about 5 pl to about 250 pl, about 10 pl to about 250 pl, about 20 pl to about 250 pl, about 30 pl to about 250 pl, about 40 pl to about 250 pl, about 50 pl to about 250 pl, about 60 pl to about 250 pl, about 70 pl to about 250 pl, about 80 pl to about 250 pl, about 100 pl to about 250 pl, about 120 pl to about 250 pl, about 150 pl to about 250 pl, about 160 pl to about 250 pl, about 180 pl to about 500 pl, about 200 pl to about 250 pl, about 220 pl to about 250 pl, about 5 pl to about 220 pl, about 10 pl to about 220 pl, about 20 pl to about 220 pl, about 30 pl to about 220 pl, about 40 pl to about 220 pl, about 50 pl to about 220 pl, about 60 pl to about 220 pl, about 70 pl to about 220 pl, about 80 pl to about 220 pl, about 100 pl to about 220 pl, about 120 pl to about 220 pl, about 150 pl to about 220 pl, about 160 pl to about 220 pl, about 180 pl to about 220 pl, about 5 pl to about 200 pl, about 10 pl to about 200 pl, about 20 pl to about 200 pl, about 30 pl to about 200 pl, about 40 pl to about 200 pl, about 50 pl to about 200 pl, about 60 pl to about 200 pl, about 70 pl to about 200 pl, about 80 pl to about 200 pl, about 100 pl to about 200 pl, about 120 pl to about 200 pl, about 150 pl to about 200 pl, about 160 pl to about 200 pl, about 180 pl to about 200 pl, about 5 pl to about 180 pl, about 10 pl to about 180 pl, about 20 pl to about 180 pl, about 30 pl to about 180 pl, about 40 pl to about 180 pl, about 50 pl to about 180 pl, about 60 pl to about 180 pl, about 70 pl to about 180 pl, about 80 pl to about 180 pl, about 100 pl to about 180 pl, about 120 pl to about 180 pl, about 150 pl to about 180 pl, about 5 pl to about 150 pl, about 10 pl to about 150 pl, about 20 pl to about 150 pl, about 30 pl to about 150 pl, about 40 pl to about 150 pl, about 50 pl to about 150 pl, about 60 pl to about 150 pl, about 70 pl to about 150 pl, about 80 pl to about 150 pl, about 100 pl to about 150 pl, about 120 pl to about 150 pl, about 5 pl to about 150 pl, about 10 pl to about 120 pl, about 20 pl to about 120 pl, about 30 pl to about 120 pl, about 40 pl to about 120 pl, about 50 pl to about 120 pl, about 60 pl to about 120 pl, about 70 pl to about 120 pl, about 80 pl to about 120 pl, about 100 pl to about 120 pl, about 5 pl to about 100 pl, about 10 pl to about 100 pl, about 20 pl to about 100 pl, about 30 pl to about 100 pl, about 40 pl to about 100 pl, about 50 pl to about 100 pl, about 60 pl to about 100 pl, about 70 pl to about 100 pl, about 80 pl to about 100 pl, about 5 pl to about 80 pl, about 10 pl to about 80 pl, about 20 pl to about 80 pl, about 30 pl to about 80 pl, about 40 pl to about 80 pl, about 50 pl to about 80 pl, about 60 pl to about 80 pl, about 5 pl to about 60 pl, about 10 pl to about 60 pl, about 20 pl to about 60 pl, about 30 pl to about 60 pl, about 40 pl to about 60 pl, or about 50 pl to about 60 pl. In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition in a volume of about 5 pl, about 8 pl, about 10 pl, about 12 pl, about 15 pl, about 18 pl, about 20 pl, about 25 pl, about 28 pl, about 30 pl, about 35 pl, about 40 pl, about 45 pl, about 48 pl, about 50 pl, about 55 pl, about 60 pl, about 65 pl, about 70 pl, about 75 pl, about 80 pl, about 90 pl, about 100 pl, about 120 pl, about 150 pl, about 160 pl, about 180 pl, about 200 pl, about 220 pl, or about 250 pl.

[0019] In some embodiments, the method comprises administering the pharmaceutical composition to both left eye and right eye of the subject. In some embodiments, the method comprises administering the pharmaceutical composition at the same dose to both the left eye and the right eye of the subject. In some embodiments, the method comprises administering the pharmaceutical composition at different doses to the left eye and the right eye of the subject.

[0020] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NO: 6-275 or 280-299. In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 280-283, 288, and 290-292. In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 92-96, and 166-168. In some embodiments, the antisense oligomer modulates splicing of a nonsense-mediated RNA decay-inducing exon (NMD exon) from a pre-mRNA in a cell of the subject, wherein the pre-mRNA encodes the OPA1 protein and comprises the NMD exon, thereby modulating a level of processed mRNA that is processed from the pre-mRNA, and modulating expression of the OPA1 protein in the cell. In some embodiments, the antisense oligomer: (a) binds to a targeted portion of the pre-mRNA; (b) modulates binding of a factor involved in splicing of the NMD exon; or (c) a combination of (a) and (b).

[0021] In some embodiments, the targeted portion of the pre-mRNA is proximal to the NMD exon. In some embodiments, the targeted portion of the pre-mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of 5’ end of the NMD exon. In some embodiments, the targeted portion of the pre-mRNA is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotides upstream of 5’ end of the NMD exon. In some embodiments, the targeted portion of the pre-mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of 3’ end of the NMD exon. In some embodiments, the targeted portion of the pre-mRNA is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, or about 1 nucleotide(s) downstream of 3’ end of the NMD exon.

[0022] In some embodiments, the targeted portion of the pre-mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of genomic site GRCh38/ hg38: chr3 193628509.

[0023] In some embodiments, the targeted portion of the pre-mRNA is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of genomic site GRCh38/ hg38: chr3 193628509.

[0024] In some embodiments, the targeted portion of the pre-mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of genomic site GRCh38/ hg38: chr3 193628616.

[0025] In some embodiments, the targeted portion of the pre-mRNA is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of genomic site GRCh38/ hg38: chr3 193628616.

[0026] In some embodiments, the targeted portion of the pre-mRNA is located in an intronic region between two canonical exonic regions of the pre-mRNA, and wherein the intronic region contains the NMD exon. In some embodiments, the targeted portion of the pre-mRNA at least partially overlaps with the NMD exon. In some embodiments, the targeted portion of the pre-mRNA at least partially overlaps with an intron upstream or downstream of the NMD exon. In some embodiments, the targeted portion of the pre-mRNA comprises 5’ NMD exon-intron junction or 3’ NMD exon-intron junction. In some embodiments, the targeted portion of the pre-mRNA is within the NMD exon. In some embodiments, the targeted portion of the pre-mRNA comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides of the NMD exon. In some embodiments, the NMD exon comprises a sequence with at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO: 279. In some embodiments, the NMD exon comprises a sequence of SEQ ID NO: 279. In some embodiments, the targeted portion of the pre-mRNA is within the nonsense -mediated RNA decay-inducing exon GRCh38/ hg38: chr3 193628509 to 193628616. In some embodiments, the targeted portion of the pre-mRNA is upstream or downstream of the nonsense-mediated RNA decay-inducing exon GRCh38/ hg38: chr3 193628509 to 193628616. In some embodiments, the targeted portion of the pre-mRNA comprises an exon-intron junction of exon GRCh38/ hg38: chr3 193628509 to 193628616. [0027] In some embodiments, the OPA1 protein expressed from the processed mRNA is a full-length OPA1 protein or a wild-type OPA1 protein. In some embodiments, the OPA1 protein expressed from the processed mRNA is a functional OPA1 protein. In some embodiments, the OPA1 protein expressed from the processed mRNA is at least partially functional as compared to a wild-type OPA1 protein. In some embodiments, the OPA1 protein expressed from the processed mRNA is at least partially functional as compared to a full-length wild-type OPA1 protein.

[0028] In some embodiments, the method promotes exclusion of the NMD exon from the pre-mRNA. In some embodiments, the exclusion of the NMD exon from the pre-mRNA in the cell contacted with the antisense oligomer is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6- fold, about 1. 1 to about 7-fold, about 1. 1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5- fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5- fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5 -fold, or at least about 10-fold, compared to in the absence of the antisense oligomer. In some embodiments, the method results in an increase in the level of the processed mRNA in the cell. In some embodiments, the level of the processed mRNA in the cell contacted with the antisense oligomer is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5 -fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5 -fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4- fold, at least about 5 -fold, or at least about 10-fold, compared to in the absence of the antisense oligomer. [0029] In some embodiments, the method results in an increase in the expression of the OPA1 protein in the cell. In some embodiments, a level of the OPA1 protein expressed from the processed mRNA in the cell contacted with the antisense oligomer is increased by about 1. 1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5- fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9- fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5 -fold, at least about 4-fold, at least about 5 -fold, or at least about 10-fold, compared to in the absence of the antisense oligomer.

[0030] In some embodiments, the therapeutic agent further comprises a gene editing molecule. In some embodiments, the gene editing molecule comprises CRISPR-Cas9.

[0031] In some embodiments, the therapeutic agent comprises the antisense oligomer, and wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the therapeutic agent comprises the antisense oligomer, and wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2’-O-methyl moiety, a 2 ’-Fluoro moiety, or a 2’-O-methoxyethyl moiety. In some embodiments, the therapeutic agent comprises the antisense oligomer, and wherein the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomer comprises a 5’- methylcytosine (5’-MeC). In some embodiments, each cytosine of the antisense oligomer is a 5’- methylcytosine (5’-MeC). In some embodiments, the antisense oligomer comprises a 5 ’-methyluracil (5’- MeU). In some embodiments, each cytosine or thymidine of the antisense oligomer is a 5 ’-methyluracil (5’-MeU). In some embodiments, the antisense oligomer comprises a phosphorothioate linkage. In some embodiments, each intemucleoside linkage of the ASO is a phosphorothioate linkage. In some embodiments, the antisense oligomer comprises a locked nucleic acid (LNA).

[0032] In some embodiments, the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

[0033] In some embodiments, the therapeutic agent comprises the antisense oligomer, and the antisense oligomer has any one of the following chemical structures:

or a pharmaceutically acceptable salt thereof.

[0034] In some embodiments, the antisense oligomer has any of the following structures: [0035] In some embodiments, the therapeutic agent comprises the vector, and wherein the vector comprises a viral vector encoding the antisense oligomer. In some embodiments, the viral vector comprises an adenoviral vector, adeno-associated viral (AAV) vector, lentiviral vector, Herpes Simplex Virus (HSV) viral vector, or retroviral vector.

[0036] In some embodiments, the pharmaceutical composition is a liquid composition. In some embodiments, the method comprises administering the pharmaceutical composition as a bolus injection over 1 to 60 minutes, 1 to 50 minutes, 1 to 40 minutes, 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes,

1 to 5 minutes, or 1 to 3 minutes. In some embodiments, the method comprises administering the pharmaceutical composition as a bolus injection.

[0037] In some embodiments, the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, or water for injection. In some embodiments, the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, and water for injection. In some embodiments, the antisense oligomer is solubilized or diluted in an isotonic solution. In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate-buffered solution with at least pH 5.8. In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate-buffered (pH 6.6 - 7.6) solution. In some embodiments, the pharmaceutical formulation does not comprise a preservative.

[0038] In some embodiments, the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml to about 200 mg/ml. In some embodiments, the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml to about 200 mg/ml, about 5 mg/ml to about 200 mg/ml, about 10 mg/ml to about 200 mg/ml, about 15 mg/ml to about 200 mg/ml, about 20 mg/ml to about 200 mg/ml, about 25 mg/ml to about 200 mg/ml, about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml,

2 mg/ml to about 150 mg/ml, about 5 mg/ml to about 150 mg/ml, about 10 mg/ml to about 150 mg/ml, about 15 mg/ml to about 150 mg/ml, about 20 mg/ml to about 150 mg/ml, about 25 mg/ml to about 150 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, 2 mg/ml to about 100 mg/ml, about 5 mg/ml to about 100 mg/ml, about 10 mg/ml to about 100 mg/ml, about 15 mg/ml to about 100 mg/ml, about 20 mg/ml to about 100 mg/ml, about 25 mg/ml to about 100 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, 2 mg/ml to about 80 mg/ml, about 5 mg/ml to about 80 mg/ml, about 10 mg/ml to about 80 mg/ml, about 15 mg/ml to about 80 mg/ml, about 20 mg/ml to about 80 mg/ml, about 25 mg/ml to about 80 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, 2 mg/ml to about 60 mg/ml, about 5 mg/ml to about 60 mg/ml, about 10 mg/ml to about 60 mg/ml, about 15 mg/ml to about 60 mg/ml, about 20 mg/ml to about 60 mg/ml, about 25 mg/ml to about 60 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, about 40 mg/ml to about 60 mg/ml, 2 mg/ml to about 40 mg/ml, about 5 mg/ml to about 40 mg/ml, about 10 mg/ml to about 40 mg/ml, about 15 mg/ml to about 40 mg/ml, about 20 mg/ml to about 40 mg/ml, or about 25 mg/ml to about 40 mg/ml. In some embodiments, the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 12 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 18 mg/ml, about 20 mg/ml, about 22 mg/ml, about 24 mg/ml, about 26 mg/ml, about 28 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 80 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

[0039] In some embodiments, the pharmaceutical composition is prepared by diluting a concentrate comprising the antisense oligomer. In some embodiments, the antisense oligomer is present in the concentrate at a concentration of about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, or about 40 mg/ml to about 60 mg/ml. In some embodiments, the antisense oligomer is present in the concentrate at a concentration of about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml. In some embodiments, the concentrate is aphosphate- buffered solution.

[0040] In some embodiments, the disease or condition is associated with a deficient amount or activity of the OPA protein. In some embodiments, the disease or condition comprises an eye disease or condition. In some embodiments, the disease or condition comprises a cardiovascular disease or condition. In some embodiments, the disease or condition comprises a neurological disease or condition. In some embodiments, the disease or condition comprises ADOA-plus; a mitochondrial disorder; glaucoma; normal tension glaucoma; Charcot-Marie-Tooth disease; mitochondria dysfunction; diabetic retinopathy; age-related macular degeneration; retinal ganglion cell death; mitochondrial fission-mediated mitochondrial dysfunction; progressive external ophthalmoplegia; deafness; ataxia; motor neuropathy; sensory neuropathy; myopathy; Behr syndrome; brain dysfunction; encephalopathy; peripheral neuropathy; fatal infantile mitochondrial encephalomyopathy; hypertrophic cardiomyopathy; spastic ataxic syndrome; sensory motor peripheral neuropathy; hypotonia; gastrointestinal dysmotility and dysphagia; optic atrophy; optic atrophy plus syndrome; Mitochondrial DNA depletion syndrome 14; late- onset cardiomyopathy; diabetic cardiomyopathy; Alzheimer’s Disease; focal segmental glomerulosclerosis; kidney disease; Huntington’s Disease; cognitive function decline in healthy aging; Prion diseases; late onset dementia and parkinsonism; mitochondrial myopathy; Leigh syndrome;

Friedreich’s ataxia; Parkinson’s disease; MELAS (Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes); pyruvate dehydrogenase complex deficiency; chronic kidney disease; Leber’s hereditary optic neuropathy; obesity; age-related systemic neurodegeneration; skeletal muscle atrophy; heart and brain ischemic damage; or massive liver apoptosis. In some embodiments, the disease or condition comprises Optic atrophy type 1. In some embodiments, the disease or condition comprises autosomal dominant optic atrophy (ADOA).

[0041] In some embodiments, the pharmaceutical composition is administered via intracerebroventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, implantation, or intravenous injection. In some embodiments, the pharmaceutical composition is administered via intravitreal injection.

[0042] In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a small molecule. In some embodiments, the additional therapeutic agent comprises an antisense oligomer. In some embodiments, the additional therapeutic agent comprises an ophthalmologic drug. In some embodiments, the subject is a human subject.

[0043] Described herein, in some aspects, is a method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutic agent, wherein the subject has a flavoprotein fluorescence (FPF) intensity score within a reference value range and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer.

[0044] Described herein, in some aspects, is a method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising: (1) determining a flavoprotein fluorescence (FPF) intensity score of the subject; (2) identifying the subject as an eligible subject for treatment when the FPF intensity score determined in (1) is within a reference value range; and (3) administering to the eligible subject a pharmaceutical composition comprising a therapeutic agent, wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer.

[0045] Described herein, in some aspects, is a method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising administering to the subject a pharmaceutical composition according to a dosing regimen selected based at least in part on a flavoprotein fluorescence (FPF) intensity score that the subject has, wherein the pharmaceutical composition comprises a therapeutic agent, and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer.

[0046] In some embodiments, the FPF intensity score is measured before the subject receives administration of the pharmaceutical composition. In some embodiments, the FPF intensity score is measured after the subject receives administration of one or more prior doses of the pharmaceutical composition.

[0047] Described herein, in some aspects, is a method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising: (1) determining a flavoprotein fluorescence (FPF) intensity score of the subject; (2) selecting a dosing regimen for a pharmaceutical composition for the subject based at least in part on the FPF intensity score determined in (1), wherein the pharmaceutical composition comprises a therapeutic agent, and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer; and (3) administering the pharmaceutical composition to the subject according to the selected dosing regimen.

[0048] Described herein, in some aspects, is a method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising: (1) administering to the subject a pharmaceutical composition according to a dosing regimen, wherein the pharmaceutical composition comprises a therapeutic agent, and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer; (2) after (1), determining a flavoprotein fluorescence (FPF) intensity score of the subject; (3) adjusting the dosing regimen for the pharmaceutical composition based at least in part on the FPF intensity score determined in (2); and (4) administering the pharmaceutical composition to the subject according to the dosing regimen adjusted in (3).

[0049] In some embodiments, the dosing regimen for the pharmaceutical composition is selected based at least in part on an FPF intensity score measured prior to the administering in (1).

[0050] In some embodiments, the dosing regimen comprises frequency of administration of the pharmaceutical composition, dose of the pharmaceutical composition per a single administration, time interval between administrations of the pharmaceutical composition, duration of treatment with the pharmaceutical composition, or administration route for the pharmaceutical composition.

[0051] In some embodiments, the FPF intensity score is within a reference value range. In some embodiments, the FPF intensity score is determined based at least in part on detection of FPF from one or both eyes of the subject. In some embodiments, the FPF intensity score is determined based at least further in part on a parameter selected from the group consisting of Best Corrected Visual Acuity (BCVA) letter score; Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score; high-contrast Best Corrected Visual Acuity (HC BCVA) letter score; low-contrast Best Corrected Visual Acuity (LC BCVA) letter score; Humphrey 10-2 Visual Field test Mean Deviation (MD); Humphrey 10-2 Visual Field test Pattern Standard Deviation (PSD); Visual Acuity Score (VAS); Quality of life questionnaires (NEI-VFQ-25, IVI-C, EQ-5D, EQ-5D-Y); Refraction test result; Minnesota Reading (MNRead) acuity chart score; Pelli-Robson chart score; slit lamp examination result; intraocular pressure using a Tonopen; perimetry; dilated fundoscopy result; retinal nerve fiber layer (RNFL) measurement; optical coherence tomography (OCT) result; and macular ganglion cell layer/inner plexiform layer (GCL/IPL) thickness measurements; Curve Width (CW) measurement; Electroretinogram (ERG) result; fundus photography result; and any combinations thereof.

[0052] In some embodiments, the detection of FPF is performed when exposing an eye of the subject for an amount of exposure time to an excitation flash. In some embodiments, the detection of FPF yields an average pixel intensity over a region of interest (RO I) of a detectable signal emitted in response to exposure to at least one excitation flash for an amount of exposure time. In some embodiments, the detectable signal is a fluorescent signal. In some embodiments, the fluorescent signal comprises a green light having a wavelength, or a combination of wavelengths, between about 520 nm and about 540 nm. In some embodiments, the excitation flash comprises a blue light having a wavelength, or combination of wavelengths, between about 430 nm and about 470 nm. In some embodiments, the wavelength of blue light is about 465 nm. In some embodiments, the amount of the exposure time is from about 1 to about 100 ms. In some embodiments, the amount of exposure time is about 60 ms. In some embodiments, the region of interest comprises at least one region selected from the group consisting of a macular-papillary (Mac) retinal nerve fiber layer (RNFL); an ocular global macula; an ocular Superior Nasal sector; an ocular Inferior Nasal sector; an ocular Nasal sector; an ocular Superior Temporal sector; an ocular Inferior Temporal sector; an ocular Temporal sector; and an ocular peripapillary retinal nerve fiber layer (pRNFL). In some embodiments, the region of interest comprises the global macula, the ocular Temporal Inferior sector, or the ocular Temporal sector, or any combination thereof. In some embodiments, the FPF intensity score is indicative of a level of mitochondrial dysfunction in the eye of the subject.

[0053] In some embodiments, the reference value range is a range lower than an FPF intensity score of a healthy control subject. In some embodiments, the reference value range is a range lower than an average FPF intensity score measured from a population of healthy control subjects. In some embodiments, when the pharmaceutical composition is tested on a population of test subjects suffering the disease or condition, an FPF intensity score measured from the test subjects in the population is determined to have a correlation with therapeutic efficacy of the pharmaceutical composition in the test subjects, and wherein the reference value range is a range associated with the therapeutic efficacy of the pharmaceutical composition at a reference level according to the correlation. In some embodiments, the genotype of the subject is unknown prior to the administration. In some embodiments, the genotype of the subject is unknown prior to the determining. In some embodiments, the dosing regimen is not selected based on the genotype of the subject.

[0054] In some embodiments, about 0.005 to about 20 mg of the antisense oligomer is administered to one eye of the subject. In some embodiments, about 0.005 mg to about 15 mg, about 0.005 mg to about 10 mg, about 0.005 mg to about 5 mg, about 0.005 mg to about 1 mg, about 0.01 mg to about 15 mg, about 0.01 mg to about 10 mg, about 0.01 mg to about 5 mg, about 0.01 mg to about 2.5 mg, about 0.01 mg to about 1.0 mg, about 0.01 mg to about 0.5 mg, about 0.01 mg to about 0.25 mg, about 0.01 mg to about 0. 1 mg, about 0.01 mg to about 0.05 mg, about 0.05 mg to about 10 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 2.5 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.25 mg, about 0.05 mg to about 0.1 mg, about 0. 1 mg to about 5 mg, about 0.1 mg to about 2.5 mg, about 0.1 mg to about 1.0 mg, about 0.1 mg to about 0.5 mg, or about 0.1 mg to about 0.25 mg of the antisense oligomer is administered to one eye of the subject. In some embodiments, about 0.005 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.2 mg, about 0.5 mg, about 0.75 mg, about 1.0 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2.0 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, about 5.0 mg, about 5.5 mg, about 6.0 mg, about 7.0 mg, about 8.0 mg, about 9.0 mg, about 10 mg, about 12.5 mg, about 15 mg, about 17.5 mg, or about 20 mg of the antisense oligomer is administered to one eye of the subject.

[0055] In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0. 1 mg to about 1.5 mg, about 0.1 mg to about 1.4 mg, about 0. 1 mg to about 1.2 mg, about 0.1 mg to about 1.0 mg, about 0.1 mg to about 0.8 mg, about 0. 1 mg to about 0.7 mg, about 0.1 mg to about 0.5 mg, about 0.1 mg to about 0.3 mg, about 0.2 mg to about 1.5 mg, about 0.2 mg to about 1.4 mg, about 0.2 mg to about 1.2 mg, about 0.2 mg to about 1.0 mg, about 0.2 mg to about 0.8 mg, about 0.2 mg to about 0.7 mg, about 0.2 mg to about 0.5 mg, about 0.3 mg to about 1.5 mg, about 0.3 mg to about 1.4 mg, about 0.3 mg to about 1.2 mg, about 0.3 mg to about 1.0 mg, about 0.3 mg to about 0.8 mg, about 0.3 mg to about 0.7 mg, about 0.3 mg to about 0.5 mg, about 0.5 mg to about 1.5 mg, about 0.5 mg to about 1.4 mg, about 0.5 mg to about 1.2 mg, about 0.5 mg to about 1.0 mg, about 0.5 mg to about 0.8 mg, about 0.5 mg to about 0.7 mg, about 0.7 mg to about 1.5 mg, about 0.7 mg to about 1.4 mg, about 0.7 mg to about 1.2 mg, about 0.7 mg to about 1.0 mg, about 0.8 mg to about 1.5 mg, about 0.8 mg to about 1.4 mg, about 0.8 mg to about 1.2 mg, about 0.8 mg to about 1.0 mg, about 1.0 mg to about 1.5 mg, about 1.0 mg to about 1.4 mg, about 1.0 mg to about 1.2 mg, about 1.2 mg to about 1.5 mg, or about 1.2 mg to about 1.4 mg of the antisense oligomer. In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0. 1 mg, about 0.2 mg, about 0.3 mg, 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, or about 1.5 mg of the antisense oligomer.

[0056] In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition in a volume of about 5 pl to about 250 pl, about 10 pl to about 250 pl, about 20 pl to about 250 pl, about 30 pl to about 250 pl, about 40 pl to about 250 pl, about 50 pl to about 250 pl, about 60 pl to about 250 pl, about 70 pl to about 250 pl, about 80 pl to about 250 pl, about 100 pl to about 250 pl, about 120 pl to about 250 pl, about 150 pl to about 250 pl, about 160 pl to about 250 pl, about 180 pl to about 500 pl, about 200 pl to about 250 pl, about 220 pl to about 250 pl, about 5 pl to about 220 pl, about 10 pl to about 220 pl, about 20 pl to about 220 pl, about 30 pl to about 220 pl, about 40 pl to about 220 pl, about 50 pl to about 220 pl, about 60 pl to about 220 pl, about 70 pl to about 220 pl, about 80 pl to about 220 pl, about 100 pl to about 220 pl, about 120 pl to about 220 pl, about 150 pl to about 220 pl, about 160 pl to about 220 pl, about 180 pl to about 220 pl, about 5 pl to about 200 pl, about 10 pl to about 200 pl, about 20 pl to about 200 pl, about 30 pl to about 200 pl, about 40 pl to about 200 pl, about 50 pl to about 200 pl, about 60 pl to about 200 pl, about 70 pl to about 200 pl, about 80 pl to about 200 pl, about 100 pl to about 200 pl, about 120 pl to about 200 pl, about 150 pl to about 200 pl, about 160 pl to about 200 pl, about 180 pl to about 200 pl, about 5 pl to about 180 pl, about 10 pl to about 180 pl, about 20 pl to about 180 pl, about 30 pl to about 180 pl, about 40 pl to about 180 pl, about 50 pl to about 180 pl, about 60 pl to about 180 pl, about 70 pl to about 180 pl, about 80 pl to about 180 pl, about 100 pl to about 180 pl, about 120 pl to about 180 pl, about 150 pl to about 180 pl, about 5 pl to about 150 pl, about 10 pl to about 150 pl, about 20 pl to about 150 pl, about 30 pl to about 150 pl, about 40 pl to about 150 pl, about 50 pl to about 150 pl, about 60 pl to about 150 pl, about 70 pl to about 150 pl, about 80 pl to about 150 pl, about 100 pl to about 150 pl, about 120 pl to about 150 pl, about 5 pl to about 150 pl, about 10 pl to about 120 pl, about 20 pl to about 120 pl, about 30 pl to about 120 pl, about 40 pl to about 120 pl, about 50 pl to about 120 pl, about 60 pl to about 120 pl, about 70 pl to about 120 pl, about 80 pl to about 120 pl, about 100 pl to about 120 pl, about 5 pl to about 100 pl, about 10 pl to about 100 pl, about 20 pl to about 100 pl, about 30 pl to about 100 pl, about 40 pl to about 100 pl, about 50 pl to about 100 pl, about 60 pl to about 100 pl, about 70 pl to about 100 pl, about 80 pl to about 100 pl, about 5 pl to about 80 pl, about 10 pl to about 80 pl, about 20 pl to about 80 pl, about 30 pl to about 80 pl, about 40 pl to about 80 pl, about 50 pl to about 80 pl, about 60 pl to about 80 pl, about 5 pl to about 60 pl, about 10 pl to about 60 pl, about 20 pl to about 60 pl, about 30 pl to about 60 pl, about 40 pl to about 60 pl, or about 50 pl to about 60 pl. In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition in a volume of about 5 pl, about 8 pl, about 10 pl, about 12 pl, about 15 pl, about 18 pl, about 20 pl, about 25 pl, about 28 pl, about 30 pl, about 35 pl, about 40 pl, about 45 pl, about 48 pl, about 50 pl, about 55 pl, about 60 pl, about 65 pl, about 70 pl, about 75 pl, about 80 pl, about 90 pl, about 100 pl, about 120 pl, about 150 pl, about 160 pl, about 180 pl, about 200 pl, about 220 pl, or about 250 pl.

[0057] In some embodiments, the method comprises administering the pharmaceutical composition to both left eye and right eye of the subject. In some embodiments, the method comprises administering the pharmaceutical composition at the same dose to both the left eye and the right eye of the subject. In some embodiments, the method comprises administering the pharmaceutical composition at different doses to the left eye and the right eye of the subject.

[0058] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NOS: 6-275 or 280-299. In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 280-283, 288, and 290-292. In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 92-96, and 166-168.

[0059] In some embodiments, the antisense oligomer modulates splicing of a nonsense-mediated RNA decay-inducing exon (NMD exon) from a pre-mRNA in a cell of the subject, wherein the pre-mRNA encodes the OPA1 protein and comprises the NMD exon, thereby modulating a level of processed mRNA that is processed from the pre-mRNA, and modulating expression of the OPA1 protein in the cell. In some embodiments, the antisense oligomer: (a) binds to a targeted portion of the pre-mRNA; (b) modulates binding of a factor involved in splicing of the NMD exon; or (c) a combination of (a) and (b). [0060] In some embodiments, the targeted portion of the pre-mRNA is proximal to the NMD exon. In some embodiments, the targeted portion of the pre-mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of 5’ end of the NMD exon. In some embodiments, the targeted portion of the pre-mRNA is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotides upstream of 5’ end of the NMD exon. In some embodiments, the targeted portion of the pre-mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of 3’ end of the NMD exon. In some embodiments, the targeted portion of the pre-mRNA is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, or about 1 nucleotide(s) downstream of 3’ end of the NMD exon.

[0061] In some embodiments, the targeted portion of the pre-mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of genomic site GRCh38/ hg38: chr3 193628509.

[0062] In some embodiments, the targeted portion of the pre-mRNA is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of genomic site GRCh38/ hg38: chr3 193628509.

[0063] In some embodiments, the targeted portion of the pre-mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of genomic site GRCh38/ hg38: chr3 193628616.

[0064] In some embodiments, the targeted portion of the pre-mRNA is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of genomic site GRCh38/ hg38: chr3 193628616.

[0065] In some embodiments, the targeted portion of the pre-mRNA is located in an intronic region between two canonical exonic regions of the pre-mRNA, and wherein the intronic region contains the NMD exon. In some embodiments, the targeted portion of the pre-mRNA at least partially overlaps with the NMD exon. In some embodiments, the targeted portion of the pre-mRNA at least partially overlaps with an intron upstream or downstream of the NMD exon. In some embodiments, the targeted portion of the pre-mRNA comprises 5’ NMD exon-intron junction or 3’ NMD exon-intron junction. In some embodiments, the targeted portion of the pre-mRNA is within the NMD exon. In some embodiments, the targeted portion of the pre-mRNA comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides of the NMD exon. In some embodiments, the NMD exon comprises a sequence with at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO: 279. In some embodiments, the NMD exon comprises a sequence of SEQ ID NO: 279.

[0066] In some embodiments, the targeted portion of the pre-mRNA is within the nonsense -mediated RNA decay-inducing exon GRCh38/ hg38: chr3 193628509 to 193628616. In some embodiments, the targeted portion of the pre-mRNA is upstream or downstream of the nonsense-mediated RNA decayinducing exon GRCh38/ hg38: chr3 193628509 to 193628616. In some embodiments, the targeted portion of the pre-mRNA comprises an exon-intron junction of exon GRCh38/ hg38: chr3 193628509 to 193628616.

[0067] In some embodiments, the OPA1 protein expressed from the processed mRNA is a full-length OPA1 protein or a wild-type OPA1 protein. In some embodiments, the OPA1 protein expressed from the processed mRNA is a functional OPA1 protein. In some embodiments, the OPA1 protein expressed from the processed mRNA is at least partially functional as compared to a wild-type OPA1 protein. In some embodiments, the OPA1 protein expressed from the processed mRNA is at least partially functional as compared to a full-length wild-type OPA1 protein.

[0068] In some embodiments, the method promotes exclusion of the NMD exon from the pre-mRNA. In some embodiments, the exclusion of the NMD exon from the pre-mRNA in the cell contacted with the antisense oligomer is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6- fold, about 1. 1 to about 7-fold, about 1. 1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5- fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5- fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5 -fold, or at least about 10-fold, compared to in the absence of the antisense oligomer. In some embodiments, the method results in an increase in the level of the processed mRNA in the cell. In some embodiments, the level of the processed mRNA in the cell contacted with the antisense oligomer is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5 -fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5 -fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4- fold, at least about 5 -fold, or at least about 10-fold, compared to in the absence of the antisense oligomer. [0069] In some embodiments, the method results in an increase in the expression of the OPA1 protein in the cell. In some embodiments, a level of the OPA1 protein expressed from the processed mRNA in the cell contacted with the antisense oligomer is increased by about 1. 1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5- fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9- fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5 -fold, at least about 4-fold, at least about 5 -fold, or at least about 10-fold, compared to in the absence of the antisense oligomer.

[0070] In some embodiments, the therapeutic agent further comprises a gene editing molecule. In some embodiments, the gene editing molecule comprises CRISPR-Cas9.

[0071] In some embodiments, the therapeutic agent comprises the antisense oligomer, and wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the therapeutic agent comprises the antisense oligomer, and wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2’-O-methyl moiety, a 2 ’-Fluoro moiety, or a 2’-O-methoxyethyl moiety. In some embodiments, the therapeutic agent comprises the antisense oligomer, and wherein the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomer comprises a 5’- methylcytosine (5’-MeC). In some embodiments, each cytosine of the antisense oligomer is a 5’- methylcytosine (5’-MeC). In some embodiments, the antisense oligomer comprises a 5 ’-methyluracil (5’- MeU). In some embodiments, each cytosine or thymidine of the antisense oligomer is a 5 ’-methyluracil (5’-MeU). In some embodiments, the antisense oligomer comprises a phosphorothioate linkage. In some embodiments, each intemucleoside linkage of the ASO is a phosphorothioate linkage. In some embodiments, the antisense oligomer comprises a locked nucleic acid (LNA).

[0072] In some embodiments, the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

[0073] In some embodiments, the therapeutic agent comprises the antisense oligomer, and the antisense oligomer has any one of the following chemical structures:

or a pharmaceutically acceptable salt thereof.

[0074] In some embodiments, the therapeutic agent comprises the vector, and wherein the vector comprises a viral vector encoding the antisense oligomer. In some embodiments, the viral vector comprises an adenoviral vector, adeno-associated viral (AAV) vector, lentiviral vector, Herpes Simplex Virus (HSV) viral vector, or retroviral vector.

[0075] In some embodiments, the pharmaceutical composition is a liquid composition. In some embodiments, the method comprises administering the pharmaceutical composition as a bolus injection over 1 to 60 minutes, 1 to 50 minutes, 1 to 40 minutes, 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes,

1 to 5 minutes, or 1 to 3 minutes. In some embodiments, the method comprises administering the pharmaceutical composition as a bolus injection. In some embodiments, the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, or water for injection. In some embodiments, the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, and water for injection. In some embodiments, the antisense oligomer is solubilized or diluted in an isotonic solution. In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate-buffered solution with at least pH 5.8. In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate-buffered (pH 6.6 - 7.6) solution. In some embodiments, the pharmaceutical formulation does not comprise a preservative.

[0076] In some embodiments, the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml to about 200 mg/ml. In some embodiments, the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml to about 200 mg/ml, about 5 mg/ml to about 200 mg/ml, about 10 mg/ml to about 200 mg/ml, about 15 mg/ml to about 200 mg/ml, about 20 mg/ml to about 200 mg/ml, about 25 mg/ml to about 200 mg/ml, about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml,

[0077] In some embodiments, the pharmaceutical composition is prepared by diluting a concentrate comprising the antisense oligomer. In some embodiments, the antisense oligomer is present in the concentrate at a concentration of about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, or about 40 mg/ml to about 60 mg/ml. In some embodiments, the antisense oligomer is present in the concentrate at a concentration of about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml. In some embodiments, the concentrate is a phosphate- buffered solution.

[0078] In some embodiments, the disease or condition is associated with a deficient amount or activity of the OPA protein. In some embodiments, the disease or condition comprises an eye disease or condition. In some embodiments, the disease or condition comprises a cardiovascular disease or condition. In some embodiments, the disease or condition comprises a neurological disease or condition. In some embodiments, the disease or condition comprises ADOA-plus; a mitochondrial disorder; glaucoma; normal tension glaucoma; Charcot-Marie-Tooth disease; mitochondria dysfunction; diabetic retinopathy; age-related macular degeneration; retinal ganglion cell death; mitochondrial fission-mediated mitochondrial dysfunction; progressive external ophthalmoplegia; deafness; ataxia; motor neuropathy; sensory neuropathy; myopathy; Behr syndrome; brain dysfunction; encephalopathy; peripheral neuropathy; fatal infantile mitochondrial encephalomyopathy; hypertrophic cardiomyopathy; spastic ataxic syndrome; sensory motor peripheral neuropathy; hypotonia; gastrointestinal dysmotility and dysphagia; optic atrophy; optic atrophy plus syndrome; Mitochondrial DNA depletion syndrome 14; late- onset cardiomyopathy; diabetic cardiomyopathy; Alzheimer’s Disease; focal segmental glomerulosclerosis; kidney disease; Huntington’s Disease; cognitive function decline in healthy aging; Prion diseases; late onset dementia and parkinsonism; mitochondrial myopathy; Leigh syndrome;

[0079] In some embodiments, the pharmaceutical composition is administered via intracerebroventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, implantation, or intravenous injection. In some embodiments, the pharmaceutical composition is administered via intravitreal injection.

[0080] In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a small molecule. In some embodiments, the additional therapeutic agent comprises an antisense oligomer. In some embodiments, the additional therapeutic agent comprises an ophthalmologic drug. In some embodiments, the subject is a human subject.

INCORPORATION BY REFERENCE

[0081] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0082] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0083] FIG. 1A depicts a schematic representation of a target mRNA that contains a nonsense-mediated mRNA decay-inducing exon (NMD exon mRNA) and therapeutic agent-mediated exclusion of the nonsense-mediated mRNA decay-inducing exon to increase expression of the full-length target protein or functional RNA. In particular, FIG. 1A shows an example schematic of a Novel NMD exon inclusion event (designated as Exon X) identified in the OP Al gene which leads to the introduction of a premature termination codon (PTC) resulting in a non-productive mRNA transcript degraded by nonsense-mediated decay (NMD). The schematic also shows the location of the two primers used to detect both the productive and non-productive mRNA transcripts by RT-PCR.

[0084] FIG. IB illustrates expression of OPA1 transcripts containing the NMD exon in HEK293 cells treated with increasing amounts of cycloheximide. Treatment of HEK293 cells with cycloheximide for 3 hours increases non-productive mRNA transcripts.

[0085] FIG. 1C illustrates RT-PCR data from non-productive transcript in RNA isolated from retinal tissue punches from macular and peripheral regions of both eyes of a human donor. OD: oculus dexter (right eye), OS: oculus sinister (left eye).

[0086] FIG. 2A illustrates RT-PCR data for non-productive OPA1 mRNAs in HEK293 cells after treatment with ASO-1 and cycloheximide.

[0087] FIG. 2B illustrates quantification of productive OPA1 mRNAs in HEK293 cells after treatment with ASO-1 in the absence of cycloheximide.

[0088] FIG. 2C illustrates protein expression of OPA1 in HEK293 cells after treatment with ASO-1 in the absence of cycloheximide.

[0089] FIG. 2D is an EC50 evaluation of ASO-1 in HEK293 cells and illustrates a representative doseresponse curve fit based on levels of non-productive transcript measured when various doses of ASO-1 were applied to HEK293 cells.

[0090] FIGS. 3A-3B represent data from western blot analyses showing reduced OPA1 protein levels in 6VN / ~ HEK293 cells compared to isogenic OPA1^+/+ HEK293 cells. FIG. 3A shows exemplary western blots. FIG. 3B is a histogram showing quantified data corresponding to the western blot of FIG. 3A.

[0091] FIGS. 4A-4E show that ASO-1 increases OPA1 mRNA and OPA1 protein expression in OPAl⁺ ~ HEK293 cells. FIGS. 4A-4B are histograms of data from OPA1^+/+ HEK293 cells (FIG. 4A) and OPAl^+/~ HEK293 cells (FIG. 4B) treated with Vehicle or 10 pM ASO-1 by gymnotic delivery for 72 hours and assessed for effect on OPA1 mRNA expression. FIGS. 4C-4D are histograms of corresponding data from OPA1^+/+ HEK293 cells (FIG. 4C) or O7 7^+/ HEK293 cells (FIG. 4D) for OPA1 protein expression 72 hours post treatment. FIG. 4E is a representative western blot image for data in FIGS. 4C-4D.

[0092] FIGS. 5A-5D show that ASO-1 reduces non-productive exon inclusion and increases OPA1 expression in ADOA patient fibroblast cells. FIG. 5A contains OPA1 variant information for the three ADOA patients designated as F34, F35 and F36. FIGS. 5B-5C are histograms representing data for WT and all three patient fibroblast cells were treated with ASO-1 and harvested for analysis of non-productive splicing (FIG. 5B) and OPA1 mRNA levels (FIG. 5C). FIG. 5D shows a histogram of OPA1 protein levels determined by western blot after transfection and normalized to -actin.

[0093] FIGS. 6A-6D are exemplary histograms of oxygen consumption rate that demonstrate ADOA patient-derived fibroblast cells have reduced mitochondrial function. Basal respiration (FIG. 6A), ATP- linked respiration (FIG. 6B), maximal respiration (FIG. 6C) and spare respiratory capacity (FIG. 6D) are shown.

[0094] FIGS. 7A-7D are histograms that show ASO-1 increases mitochondrial function in ADOA patient fibroblast cells. Oxygen consumption rate measurements were taken from patient fibroblast cells treated with 20, 40 or 60 nM ASO-1 or Vehicle. Basal respiration (FIG. 7A), ATP-linked respiration (FIG. 7B), maximal respiration (FIG. 7C) and spare respiratory capacity (FIG. 7D) are shown.

[0095] FIGS. 8A-8D show that surrogate antisense oligonucleotide (ASO) ST- 1102 increases OPA1 protein in Wild-Type (WT) rabbit retinal tissue following single intravitreal (IVT) injection. FIG. 8A illustrates the study design for the in vivo rabbit experiment of Example 9. FIG. 8B are histograms representing RT-PCR data measuring non-productive splicing using rabbit retinal RNA. FIG. 8C shows histograms of quantitated western blot data for OPA1 protein normalized to actin. FIG. 8D shows a histogram of ST-1102 detected in retinal tissue on Day 29 by HELISA at all three dose levels.

[0096] FIG. 9 shows RT-PCR gel images from ST-1102 injected rabbit retinas.

[0097] FIG. 10 shows western blot images from ST-1102 injected rabbit retinas.

[0098] FIGS. 11A-11C show histograms demonstrating that intravitreal administration of ASO-1 induces dose-related reduction in nonsense-mediated decay exon inclusion and increased OPA1 protein with sustained tissue exposure in the retina of cynomolgus monkeys. FIG. HA is a histogram of qPCR data measuring the presence of non-productive exon-containing RNA transcripts in retinal tissue relative to total OPA1 transcripts. FIG. 11B is a histogram representing OPA1 protein quantitated in retinal lysates by ELISA and normalized to total protein in the sample. FIG. 11C is a histogram representing ASO-1 levels in retinal tissue quantitated by HELISA.

[0099] FIG. 12A-12B are images representative of ASO-1 and OPA1 protein in retinal ganglion cells (RGCs) after intravitreal administration to cynomolgus monkeys. FIG. 12A shows RNAscope™ staining with a specific probe to ASO-1 was performed on MDF-fixed, paraffin-embedded cynomolgus monkey eyes to demonstrate cellular location of ASO-1 (red) in RGCs. Increased ASO-1 signal is correlated with increased dose. Nuclei were counterstained with hematoxylin (blue). FIG. 12B shows immunofluorescence staining with OPA1 antibody indicating apparent dose-related protein increase (red) in the RGCs in/near the fovea. Nuclei are stained with DAPI (blue) and magnification was 40X.

[0100] FIG. 13A-13D show that there is dose-related increase in ASO-1 in retinal ganglion cells (RGCs) after intravitreal (IVT) administration to cynomolgus monkeys. FIGS. 13A-13B are histograms representing quantitated copies of mRNA per cell (FIG. 13A) and percent positive RGCs (FIG. 13B) in or near foveal RGCs. FIGS. 13C-13D are histograms representing quantitated copies of mRNA per cell (FIG. 13C) and percent positive RGCs (FIG. 13D) in the peripheral retina.

[0101] FIG. 14A-14B show quantitation of OPA1 protein immunofluorescence in cynomolgus monkey retinal ganglion cells (RGCs) after intravitreal ASO-1 administration. Fluorescence (FL) intensity was quantitated in the RGC layer in foveal/near foveal (FIG. 14A) and peripheral (FIG. 14B) retina.

[0102] FIG. 15 is a scatterplot showing the correlation between baseline visual acuity (logMAR, or log of the minimum angle of resolution) and age (in years). Each point represents the mean logMAR from both eyes of each patient. N=48.

[0103] FIG. 16 is a heatmap showing the relationship between BCVA tests of either high contrast or low contrast (2.5%, 5%), plotted against various ophthalmological readouts: global ganglion cell layer (GCL.g), Nasal ganglion cell layer (GCL.n), Temporal ganglion cell layer (GCL.t), Humphrey Visual Field (H), Reading speed (Rd Spd), global retinal nerve fiber layer (RNFL.g), Nasal retinal nerve fiber layer (RNFL.n), and Temporal retinal nerve fiber layer (RNFL.t). Numerical values within the figure are correlation coefficients. logMAR at low contrast (2.5%) appears highly correlated with Global, Nasal, and Temporal GCL, and Global RNFL. LogMAR and low contrast logMAR also appear to both be highly correlated with Humphrey Visual Field Mean Deviation.

[0104] FIG. 17 is a scatterplot showing the correlation between baseline visual acuity (logMAR) and thickness of the Nasal Ganglion Cell Layer-Inner Plexiform Layer (GCL/IPL) (pm). A Nasal GCL/IPL threshold value of about 53 pm appears to be present. Each point represents the mean logMAR from both eyes of each patient. N=47. Slopes for Nasal GCL > 53 and < 53 pm are different. Nasal GCL > 53 pm = no change. Nasal GCL < 53 pm logMAR increases when Nasal GCL decreases.

[0105] FIG. 18 is a scatterplot showing the correlation between Delta Letter Scores (derived by subtracting 2.5% LC BCVA from HC BCVA) and thickness of the Global Ganglion Cell Layer-Inner Plexiform Layer (GCL/IPL) (pm). Each point represents the mean logMAR from both eyes of each patient. N=48. Delta HC/LC BCVA to Global GCL/IPL thickness shows a relatively flat slope. There appears to be poor correlation between HC/LC BCVA Delta and GCL/IPL thickness, suggesting excess Delta may be due to factors(s) other than Global GCL/IPL.

[0106] FIG. 19 is a scatterplot showing the correlation between Humphrey 10-2 Visual Field Mean Deviation and age (in years). Each point represents the mean logMAR from both eyes of each patient. N=40. For patients < 27y, MD changes minimally (slight decrease in absolute value with age). For patients > 27y, MD increases in absolute value linearly.

[0107] FIGS. 20A-20C are line charts illustrating that there were minimal changes in visual acuity and RNFL thickness in FALCON participants over the course of 12 months. FIG. 20A is a line chart showing High-Contrast Early Treatment of Diabetic Retinopathy Study (ETDRS) Letter Acuity, and FIG. 20B is a line chart showing Low-Contrast (2.5%) ETDRS Letter Acuity of three age cohorts (8-17 years old, 18-40 years old, and 41-60 years old) and all cohorts. The x-axis shows the visit month and y-axis shows the ETDRS letter change in FIG. 20A and FIG. 20B. FIG. 20C is a line chart showing the change in global RNFL thickness of FALCON participants over 12 months for the three age cohorts. The x-axis shows the visit month and y-axis shows the change in RNFL thickness with a Standard Error (SE) of ±2.

[0108] FIGS. 21A-21B are line charts illustrating that FALCON participants with higher baseline high- contrast visual acuity had a greater decline in low-contrast visual acuity at 12 months. FIG. 21A is a line chart showing ETDRS letter change in Low-Contrast (LC) (2.5%) Visual Acuity (VA) (y-axis) at various visit months (x-axis) for patients with initially better visual acuity (higher baseline High-Contrast (HC) VA). FIG. 21B is a line chart showing the ETDRS letter change in LC (2.5%) VA (y-axis) over the course of 12 months (x-axis) for all patients completing the 12-month study visit.

[0109] FIGS. 22A-22C are line charts showing the percentage change in Photopic Negative Response (PhNR) base ratio (peak to trough) at 12 months for FALCON participants with a baseline BCVA of <0.3 logMAR (FIG. 22A), a baseline BCVA >0.3 to <0.6 logMAR (FIG. 22B), and a baseline BCVA >0.6 to <0.9 logMAR (FIG. 22C).

[0110] FIGS. 23A-23H are line charts showing the High-Contrast (HC) ETDRS Letter Score Changes (FIGS. 23A-23D) and Low-Contrast (LC) (2.5%) ETDRS Letter Score Changes (FIGS. 23E-23H) of FALCON participants over the course of 12 months. HC Letter Score Changes for participants with Initial Visual Acuity (“Va”) <0.3 logMAR (FIG. 23A), initial visual acuity of >0.3 to <0.6 logMAR (FIG. 23B), initial visual acuity of >0.6 to <0.9 logMAR (FIG. 23C), and initial visual acuity of >1 logMAR (FIG. 23D). LC Letter Score Changes for participants with Initial Visual Acuity (“Va”) <0.3 logMAR (FIG. 23E), initial visual acuity of >0.3 to <0.6 logMAR (FIG. 23F), initial visual acuity of >0.6 to <0.9 logMAR (FIG. 23G), and initial visual acuity of >1 logMAR (FIG. 23H). Letter Score Changes are shown on the y-axes and Visit Months shown on the x-axes. “N” indicates sample size, and the change in Letter Scores (“Ltrs”) for each condition are provided to the right side of each chart.

[oni] FIG. 24 is a histogram showing the percentage of FALCON participants with a greater than 5- Letter loss at 12 months. The left column shows data for the percentage of participants who experienced a greater than 5 -Letter loss with High-Contrast (HC) ETDRS. The right column shows data for the percentage of participants who experienced a greater than 5 -Letter loss with Low-Contrast (LC) ETDRS. N indicates the group size out of 46 participants.

[0112] FIGS. 25A-25B show FALCON participant ETDRS BCVA Letter Loss data. FIG. 25A shows the number of ETDRS BCVA letters lost at 12 months compared to baseline for participants divided according to various levels of initial visual acuity: <0.3 LogMAR (FIG. 25A, first column), >0.3 to <0.6 logMAR (FIG. 25A, second column), >0.6 to <0.9 logMAR (FIG. 25A, third column), and >1 logMAR (FIG. 25 A, fourth column). FIG. 25B shows the distribution of patients (percentage of participants) who lost more than 5 letters (“> 5 letter loss”) at 12 months compared to baseline, divided according to various levels of initial visual acuity: <0.3 LogMAR (FIG. 25B, first column), >0.3 to <0.6 logMAR (FIG. 25B, second column), >0.6 to <0.9 logMAR (FIG. 25B, third column), and >1 logMAR (FIG. 25B, fourth column).

[0113] FIG. 26 shows exemplary Low-Contrast (2.5%) BCVA letter acuity of individual patients who had more than five (“>5”) letters lost and were evaluated for their OPA1 mutation type (e.g., nonsense mutation, missense mutation).

[0114] FIG. 27 shows ETDRS Visual Acuity Score (VAS) letter change with High-Contrast (HC) and Low-Contrast (LC) (2.5%) at 12 months correlated with participants who had missense mutations in OPAL From the 47 participants, 37 participants (79%) had a nonsense mutation, 8 participants (17%) had a missense mutation, and 2 participants (4%) had a splicing error.

[0115] FIGS. 28A-28B show Letter Change data from High-Contrast (HC) (FIG. 28A) and Low- Contrast (2.5%) (LC) (FIG. 28B) at 12 months for participants who had a missense mutation in the GTPase (diamonds), C-terminal coil-coil (squares), and dynamin (triangles) OPA1 domains.

[0116] FIGS. 29A-29D show maximum reading speed (words per minute) data from Minnesota Maximum Reading Speed (MnRead) assessments over 12 months for 8- to 17-year-olds (FIG. 29A), 18- to 40-year-olds (FIG. 29B), 41- to 60-year-olds (FIG. 29C), and all patients (FIG. 29D).

[0117] FIG. 30 is an image of a Flavoprotein Fluorescence (FPF) Report generated by OcuMet Beacon comprising patient information (e.g., patient identifier, birthdate, eye image date, eye image time, and the eye examined); an infrared image of the fundus in a wide-angle view; a Region of Interest (ROI) box (box with dashed lines) highlighting the area of the retina captured on a metabolic FPF image; the (metabolic) FPF image, which is a colorized image of the ROI that shows the measured FPF signal, wherein the highest signal value is represented with red and the lowest signal value is represented with black; an FPF score representing the average level of flavoprotein fluorescence in the Region of Interest (ROI) (box with dashed lines); a histogram of pixel values binned to form a frequency distribution from within the ROI; and a Curve Width (CW) value, which is the degree to which the FPF signal varies across the ROI. [0118] FIG. 31A-31B are algorithm data maps derived from Optical Coherence Tomography (OCT) maps. FIG. 31A shows the region of the macular-papillary (Mac) retinal nerve fiber layer (RNFL) that is analyzed. FIG. 31B shows that the analysis region of the peripapillary (Ppy) optic nerve is divided into five sectors: 1: Temporal (T); 2: Superior (S); 3: Nasal (N); 4: Inferior (I); and 5: Center (C).

[0119] FIG. 32 is a detailed flavoprotein fluorescence (FPF) report comprising retinal images of each measured eye (e.g., right eye, or oculus dexter (OD); and left eye, or oculus sinister (OS)) and false- colored maps of these images (green represents normal FPF, yellow represents a moderate increase in FPF, and red represents significant increase in FPF). From the retinal images and false-colored maps, (1) an optic nerve rim flavoprotein fluorescence profile is generated for the assessed sectors of each measured eye: Temporal (TMP); Superior (SUP); Nasal (NAS); and Inferior (INF), and (2) false-colored maps corresponding to the Optic Nerve Hypoplasia (ONH) stress index of each measured sector of each eye. [0120] FIG. 33 is a schematic showing the progression of dose administration in cohort A. Safety Monitoring Committee (SMC) evaluation is indicated by “SMC.” Four-pointed stars indicate that 6 or fewer patients may be added to a dose level that is well-tolerated and has fewer than two patients experiencing dose-limiting toxicities (DLT). Five-pointed stars indicate that three additional patients may be added for safety assessment and the SMC may recommend de-escalation to a lower dosage.

[0121] FIG. 34 is a schematic showing the progression of dose administration in cohort B. Five-pointed stars indicate that three additional patients may be added for safety assessment and the SMC may recommend de-escalation to a lower dosage in 3+3 fashion.

[0122] FIG. 35A-35C are scatterplots of flavoprotein fluorescence (FPF) measured in decibel grayscale units (dB GSU) plotted against peripapillary retinal nerve fiber layer (RNFL) thickness (pm) for the global optic disc (FIG. 35A), the Temporal Inferior sector (FIG. 35B), and the Temporal sector (FIG. 35C).

[0123] FIG. 36A-36D are scatterplots of participant age (in years) plotted against flavoprotein fluorescence (FPF) measured in decibel grayscale units (dB GSU). Data that has not been normalized to RNFL is plotted for the global optic disc (FIG. 36A), the Temporal Inferior sector (FIG. 36B), and the Temporal sector (FIG. 36C). Data that has been normalized to RNFL is plotted for the global optic disc (FIG. 36D), the Temporal Inferior sector (FIG. 36E), and the Temporal sector (FIG. 36F). Data for three age groups (younger than 18, between 18 and 40 years of age, and older than 40 years of age) is shown. [0124] FIG. 37A-37D are scatterplots of flavoprotein fluorescence (FPF) plotted against participant age (in years) for the global optic disc (FIG. 37A), the Temporal sector (FIG. 37B), the Temporal Inferior sector (FIG. 37C), and the macula (FIG. 37D). FPF is denoted in decibel grayscale units (dB GSU) for FIGS. 37A-37C, and in Linear GSU for FIG. 37D.

[0125] FIGS. 38A-38D are scatterplots of high-contrast (HC) and low-contrast (LC) Best Corrected Visual Acuity (BCVA) letter scores plotted against FPF. HC BCVA letter scores are plotted against FPF (in dB GSU), not normalized to RNFL in FIG. 38A. LC (2.5%) BCVA letter scores are plotted against FPF (in dB GSU), not normalized to RNFL in FIG. 38B. HC BCVA letter scores are plotted against FPF (in dB GSU), normalized to RNFL in FIG. 38C. LC (2.5%) BCVA letter scores are plotted against FPF (in dB GSU), normalized to RNFL in FIG. 38D. Data for three age groups (younger than 18, between 18 and 40 years of age, and older than 40 years of age) is shown.

[0126] FIGS. 39A-39B are scatterplots of Flavoprotein fluorescence (FPF) readings for the global optic disc (in units of decibel (dB) gray scale units (GSU), (FIG. 39A) and global macula (in units of linear gray scale units (GSU), (FIG. 39B) plotted against Humphrey 10-2 Visual Field (HVF) threshold test mean deviation (MD).

[0127] FIG. 40 shows a hierarchical cluster analysis performed using data compiled from all the assessments run during the BEACON study. Data for N=10/19 is shown, where each row represents data from a single patient and only data for patients completing all assessments were included. Analyses for three age groups (younger than 18, between 18 and 40 years of age, and older than 40 years of age) are presented.

[0128] FIG. 41 is a plot showing the relationship between Garway-Heath visual field sectors (Global (G); Temporal (T); Temporal Superior (TS); Nasal Superior (NS); Nasal (N); Nasal Inferior (NI); and Temporal Inferior (TI)) and optic disc flavoprotein fluorescence (FPF) for three age groups (study participants ages 8-17, ages 18-40, and ages 41-60).

[0129] FIG. 42 is a heat map showing correlations of various assessments with FPF regions. The assessments evaluated included LogMAR from Best Corrected Visual Acuity (BCVA) assessments, Visual Acuity Scores (VAS), LogMAR from Low-Contrast (2.5%, 5%, and 25%) BCVA assessments, VAS from Low-Contrast (2.5%, 5%, and 25%) assessments, and Humphrey Automated Perimetry Assessments with Mean Deviation (MD) and Pattern Standard Deviation (PSD) shown on the y-axis. The x-axis shows the flavoprotein fluorescence (FPF) in various visual fields including the global disc (“Disc Global”), global macular region (“Macular Global”), Nasal (N), Nasal Inferior (NI), Nasal Superior (NS), Temporal (T), Temporal Inferior (TI), and Temporal Superior (TS) sectors.

[0130] FIGS. 43A-43B show FPF scores (y-axis) for FALCON participants in the Global region (FIG. 43 A) and Temporal visual field sector (FIG. 43B) at baseline, 6 months, and 12 months (x-axis) for the various age groups: 8- to 17-year-olds (circles), 18- to 40-year-olds (squares), 41- to 60-year-olds (triangles), and all patients (diamonds). N indicates group size.

[0131] FIG. 44 shows the percentage change from baseline (y-axis) in participant FPF scores in the Temporal visual field sector at baseline, 6 months, and 12 months (x-axis) for the various age groups: 8- to 17-year-olds (circles), 18- to 40-year-olds (squares), 41- to 60-year-olds (triangles), and all patients (diamonds). Scores were normalized to RNFL thickness. N indicates group size. [0132] FIGS. 45A-45B show FPF scores (y-axis) for FALCON participants in the Temporal Inferior visual field sector with non-normalized (FIG. 45A) and normalized (FIG. 45B) data at baseline, 6 months, and 12 months (x-axis) for the various age groups: 8- to 17-year-olds (circles), 18- to 40-year- olds (squares), 41- to 60-year-olds (triangles), and all patients (diamonds). N indicates group size.

[0133] FIGS. 46A-46B show the percentage of change from baseline in FPF scores (y-axis) for FALCON participants in the Nasal visual field sector sorted by age groups (FIG. 46A) and initial BCVA (FIG. 46B) data at baseline, 6 months, and 12 months (x-axis) for the various age groups: 8- to 17-year- olds (circles), 18- to 40-year-olds (squares), 41- to 60-year-olds (triangles), and all patients (diamonds). N indicates group size.

[0134] FIG. 47 shows the percentage change from baseline in FPF scores (y-axis) for FALCON participants in the Macular region at baseline, 6 months, and 12 months (x-axis) for the various age groups: 8- to 17-year-olds (circles), 18- to 40-year-olds (squares), 41- to 60-year-olds (triangles), and all patients (diamonds). N indicates group size.

[0135] FIG. 48 shows the percentage change from baseline in FPF scores (y-axis) for FALCON participants in the Global Disc region at baseline, 6 months, and 12 months (x-axis) for the groups divided according to initial BCVA: <0.3 LogMAR (circles), >0.3 to <0.6 logMAR (squares), >0.6 to <1.0 logMAR (triangles), and >1 logMAR (diamonds).

DETAILED DESCRIPTION

[0136] In some aspects, described herein is a noninvasive method of assessing ADOA eye condition in vivo. In some aspects, described herein is a method of correlating a vision test score with eye condition in ADOA. Described herein is the use of vision test score as an in vivo biomarker for eye condition in ADOA. In some aspects, described herein is the use of vision test score as an in vivo biomarker for mitochondrial dysfunction in ADOA.

[0137] In some aspects, described herein is a method of correlating mitochondrial flavoprotein fluorescence, which is measured as average flavoprotein fluorescence (FPF) intensity in one eye and output as an FPF score, with eye condition in ADOA. Described herein is the use of mitochondrial flavoprotein fluorescence (FPF) as an in vivo biomarker for eye condition in ADOA. In some aspects, described herein is the use of mitochondrial flavoprotein fluorescence (FPF) as an in vivo biomarker for mitochondrial dysfunction in ADOA.

[0138] Alternative splicing events in the OP Al gene can lead to non-productive mRNA transcripts which in turn can lead to aberrant protein expression, and therapeutic agents which can target the alternative splicing events in the OP Al gene can modulate the expression level of functional proteins in ADOA patients and/or inhibit aberrant protein expression. Such therapeutic agents can be used to treat a condition caused by OPA1 protein deficiency.

[0139] One of the alternative splicing events that can lead to non-productive mRNA transcripts is the inclusion of an extra exon in the mRNA transcript that can induce nonsense-mediated mRNA decay. The present disclosure also provides compositions and methods for modulating alternative splicing of OPA1 to increase the production of protein-coding mature mRNA, and thus, translated functional 0PA1 protein. These compositions and methods include antisense oligomers (ASOs) that can cause exon skipping, e.g., pseudoexon skipping, and promote constitutive splicing of OPA1 pre-mRNA. In various embodiments, functional OPA1 protein can be increased using the methods of the disclosure to treat a condition caused by OPA1 protein deficiency. mRNA Splicing

[0140] Intervening sequences in RNA sequences or introns are removed by a large and highly dynamic RNA-protein complex termed the spliceosome, which orchestrates complex interactions between primary transcripts, small nuclear RNAs (snRNAs) and a large number of proteins. Spliceosomes assemble ad hoc on each intron in an ordered manner, starting with recognition of the 5’ splice site (5’ss) by U1 snRNA or the 3’splice site (3’ss) by the U2 pathway, which involves binding of the U2 auxiliary factor (U2AF) to the 3’ss region to facilitate U2 binding to the branch point sequence (BPS). U2AF is a stable heterodimer composed of a U2AF2-encoded 65 -kD subunit (U2AF65), which binds the polypyrimidine tract (PPT), and a U2AFl-encoded 35-kD subunit (U2AF35), which interacts with highly conserved AG dinucleotides at 3‘ss and stabilizes U2AF65 binding. In addition to the BPS/PPT unit and 3’ss/5’ss, accurate splicing requires auxiliary sequences or structures that activate or repress splice site recognition, known as intronic or exonic splicing enhancers or silencers. These elements allow genuine splice sites to be recognized among a vast excess of cryptic or pseudo-sites in the genome of higher eukaryotes, which have the same sequences but outnumber authentic sites by an order of magnitude. Although they often have a regulatory function, the exact mechanisms of their activation or repression are poorly understood.

[0141] The decision of whether to splice or not to splice can be typically modeled as a stochastic rather than deterministic process, such that even the most defined splicing signals can sometimes splice incorrectly. However, under normal conditions, pre-mRNA splicing proceeds at surprisingly high fidelity. This is attributed in part to the activity of adjacent cis-acting auxiliary exonic and intronic splicing regulatory elements (ESRs or ISRs). Typically, these functional elements are classified as either exonic or intronic splicing enhancers (ESEs or ISEs) or silencers (ESSs or ISSs) based on their ability to stimulate or inhibit splicing, respectively. Although there is now evidence that some auxiliary cis-acting elements may act by influencing the kinetics of spliceosome assembly, such as the arrangement of the complex between U1 snRNP and the 5’ss, it seems very likely that many elements function in concert with transacting RNA-binding proteins (RBPs). For example, the serine- and arginine-rich family of RBPs (SR proteins) is a conserved family of proteins that have a key role in defining exons. SR proteins promote exon recognition by recruiting components of the pre-spliceosome to adjacent splice sites or by antagonizing the effects of ESSs in the vicinity. The repressive effects of ESSs can be mediated by members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and can alter recruitment of core splicing factors to adjacent splice sites. In addition to their roles in splicing regulation, silencer elements are suggested to have a role in repression of pseudo-exons, sets of decoy intronic splice sites with the typical spacing of an exon but without a functional open reading frame. ESEs and ESSs, in cooperation with their cognate trans-acting RBPs, represent important components in a set of splicing controls that specify how, where and when mRNAs are assembled from their precursors.

[0142] Alternative splicing is a regulated process during gene expression that can result in multiple isoforms of mature mRNA transcripts that are processed from a single primary mRNA transcript that is transcribed from a single gene, and the resultant multiple proteins that are translated from at least some of the multiple mature mRNA isoforms. In this process, particular exons of a gene may be included within or excluded from the final, processed mRNA produced from that gene. Consequently, the proteins translated from alternatively splices mRNAs will contain differences in their amino acid sequence and, in some cases, in their biological functions.

[0143] As described herein, an “alternatively spliced exon” can refer to an exon of a gene that can be either included or excluded naturally from a mature mRNA transcript, thus resulting in different protein products that are translated from the different mature mRNA transcripts. The inclusion or skipping of an alternatively spliced exon can take place naturally in a cell, either randomly, or in a regulated manner, e.g., subject to regulation by external physiological or pathological stimuli, or intracellular signaling. In some cases, the production of alternatively spliced mRNAs, e.g., the splicing of the alternatively spliced exon, is regulated by a system of trans-acting proteins that bind to cis-acting sites on the primary transcript itself. In some cases, an alternatively spliced exon is a coding exon, e.g., an exon that, when included in the mature mRNA transcript, is translated into an amino acid sequence as part of the protein product translated from the mature mRNA transcript. In some cases, the inclusion of an alternatively spliced exon in the mature mRNA transcript would maintain the canonical open reading frame as compared to a mature mRNA transcript without the alternatively spliced exon, e.g., the number of nucleotides in the alternatively spliced exon is divisible by 3.

[0144] The sequences marking the exon-intron boundaries are degenerate signals of varying strengths that can occur at high frequency within human genes. In multi -exon genes, different pairs of splice sites can be linked together in many different combinations, creating a diverse array of transcripts from a single gene. This is commonly referred to as alternative pre-mRNA splicing. Although most mRNA isoforms produced by alternative splicing can be exported from the nucleus and translated into functional polypeptides, different mRNA isoforms from a single gene can vary greatly in their translation efficiency. Those mRNA isoforms with premature termination codons (PTCs) at least 50 bp upstream of an exon junction complex are likely to be targeted for degradation by the nonsense-mediated mRNA decay (NMD) pathway. Mutations in traditional (BPS/PPT/3’ss/5’ss) and auxiliary splicing motifs can cause aberrant splicing, such as exon skipping or cryptic (or pseudo-) exon inclusion or splice-site activation, and contribute significantly to human morbidity and mortality. Both aberrant and alternative splicing patterns can be influenced by natural DNA variants in exons and introns.

[0145] Given that exon-intron boundaries can occur at any of the three positions of a codon, it is clear that only a subset of alternative splicing events can maintain the canonical open reading frame. For example, only exons that are evenly divisible by 3 can be skipped or included in the mRNA without any alteration of reading frame. Splicing events that do not have compatible phases will induce a frameshift. Unless reversed by downstream events, frameshifts can certainly lead to one or more PTCs, probably resulting in subsequent degradation by NMD. NMD is a translation-coupled mechanism that eliminates mRNAs containing PTCs. NMD can function as a surveillance pathway that exists in all eukaryotes. NMD can reduce errors in gene expression by eliminating mRNA transcripts that contain premature stop codons. Translation of these aberrant mRNAs could, in some cases, lead to deleterious gain-of-function or dominant-negative activity of the resulting proteins. NMD targets not only transcripts with PTCs but also a broad array of mRNA isoforms expressed from many endogenous genes, suggesting that NMD is a master regulator that drives both fine and coarse adjustments in steady-state RNA levels in the cell.

[0146] An NMD-inducing exon (“NIE” or “NMD exon”) is an exon or a pseudo-exon that is a region within an intron and can activate the NMD pathway if included in a mature RNA transcript. In constitutive splicing events, the intron containing an NMD exon is usually spliced out, but the intron or a portion thereof (e.g., NMD exon) may be retained during alternative or aberrant splicing events. Mature mRNA transcripts containing such an NMD exon may be non-productive due to frame shifts which induce the NMD pathway. Inclusion of an NMD exon in mature RNA transcripts may downregulate gene expression. mRNA transcripts containing an NMD exon may be referred to as “NIE-containing mRNA” or “NMD exon mRNA” in the current disclosure.

[0147] Cryptic (or pseudo- splice sites) have the same splicing recognition sequences as genuine splice sites but are not used in splicing reactions. They outnumber genuine splice sites in the human genome by an order of a magnitude and are normally repressed by thus far poorly understood molecular mechanisms. Cryptic 5 ’ splice sites have the consensus NNN/GUNNNN or NNN/GCNNNN where N is any nucleotide and / is the exon-intron boundary. Cryptic 3’ splice sites have the consensus NAG/N. Their activation is positively influenced by surrounding nucleotides that make them more similar to the optimal consensus of authentic splice sites, namely MAG/GURAGU and YAG/G, respectively, where M is C or A, R is G or A, and Y is C or U.

[0148] Splice sites and their regulatory sequences can be readily identified by a skilled person using suitable algorithms publicly available, listed for example, in Kralovicova, J. and Vorechovsky, I. (2007) Global control of aberrant splice site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition. Nucleic Acids Res., 35, 6399-6413 (www.ncbi.nlm.nih.gov/pmc/articles/PMC2095810/pdf/gkm680. pdf).

[0149] The cryptic splice sites or splicing regulatory sequences may compete for RNA -binding proteins, such as U2AF, with a splice site of the NMD exon. In some embodiments, an agent may bind to a cryptic splice site or splicing regulatory sequence to prevent binding of RNA-binding proteins and thereby favor binding of RNA-binding proteins to the NMD exon splice sites.

[0150] In some embodiments, the cryptic splice site may not comprise the 5 ’ or 3 ’ splice site of the NMD exon. In some embodiments, the cryptic splice site may be at least 10 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides or at least 200 nucleotides upstream of the NMD exon 5’ splice site. In some embodiments, the cryptic splice site may be at least 10 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, or at least 200 nucleotides downstream of the NMD exon 3’ splice site. Target Transcripts

[0151] In some embodiments, the methods and compositions of the present disclosure exploit the presence of NMD exon in the pre-mRNA transcribed from the OP Al gene. Splicing of the identified OPA1 NMD exon pre-mRNA species to produce functional mature OPA1 mRNA may be induced using an agent such as an ASO that stimulates exon skipping of an NMD exon. Induction of exon skipping may result in inhibition of an NMD pathway. The resulting mature OPA1 mRNA can be translated normally without activating NMD pathway, thereby increasing the amount of OPA1 protein in the patient’s cells and alleviating symptoms of a condition or disease associated with OPA1 deficiency, such as an eye disease or condition, Optic atrophy type 1, autosomal dominant optic atrophy (ADOA), ADOA-plus syndrome; a mitochondrial disorder; glaucoma; normal tension glaucoma; Charcot-Marie-Tooth disease; mitochondria dysfunction; diabetic retinopathy; age-related macular degeneration; retinal ganglion cell death; mitochondrial fission-mediated mitochondrial dysfunction; progressive external ophthalmoplegia; deafness; ataxia; motor neuropathy; sensory neuropathy; myopathy; Behr syndrome; brain dysfunction; encephalopathy; peripheral neuropathy; fatal infantile mitochondrial encephalomyopathy; hypertrophic cardiomyopathy; spastic ataxic syndrome; sensory motor peripheral neuropathy; hypotonia; gastrointestinal dysmotility and dysphagia; optic atrophy; optic atrophy plus syndrome; Mitochondrial DNA depletion syndrome 14; late-onset cardiomyopathy; diabetic cardiomyopathy; Alzheimer’s Disease; focal segmental glomerulosclerosis; kidney disease; Huntington’s Disease; cognitive function decline in healthy aging; Prion diseases; late onset dementia and parkinsonism; mitochondrial myopathy; Leigh syndrome; Friedreich’s ataxia; Parkinson’s disease; MELAS (Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes); pyruvate dehydrogenase complex deficiency; chronic kidney disease; Leber’s hereditary optic neuropathy; obesity; age-related systemic neurodegeneration; skeletal muscle atrophy; heart and brain ischemic damage; or massive liver apoptosis.

[0152] In some embodiments, the methods and compositions of the present disclosure exploit the alternative splicing of the pre-mRNA transcribed from the OP Al gene. In some cases, splicing of a coding exon, e.g., an alternatively spliced exon, e.g., OPA1 exon 7 (or an exon encoded by genomic region spanning from GRCh38/ hg38: chr3 193626092 to 193626202), can modulate the level of OPA1 protein expressed from the OP Al gene. As described herein, the term “OP Al exon 7” or grammatically equivalents thereof, is used interchangeably with the term “exon (GRCh38/ hg38: chr3 193626092 to 193626202)” or “an exon encoded by genomic region spanning from GRCh38/ hg38: chr3 193626092 to 193626202.” Without wishing to be bound by a certain theory, the presence or absence of an amino acid sequence encoded by exon 7 or exon (GRCh38/ hg38: chr3 193626092 to 193626202) can modulate the stability of the OPA1 protein. For instance, in some cases, the OPA1 protein encoded by a mature mRNA transcript that lacks exon 7 can have fewer proteolytic cleavage sites as compared to an OPA1 protein encoded by a corresponding mature mRNA transcript that has contains exon 7. In some cases, the OPA1 protein an OPA1 protein encoded by a corresponding mature mRNA transcript that has contains encoded by a mature mRNA transcript that lacks exon 7 is a functional protein. The OPA1 protein encoded by a mature mRNA transcript that lacks exon 7 can be at least partially functional as compared to an OPA1 protein encoded by a corresponding mature mRNA transcript that has contains exon 7. In some cases, the OPA1 protein encoded by a mature mRNA transcript that lacks exon 7 is at least partially functional as compared to a full-length wild-type OPA1 protein. In some cases, increase of OPA1 protein encoded by a mature mRNA transcript that lacks exon 7 in a cell can result in more functional OPA1 protein in the cell, due to the higher stability of the OPA1 protein lacking exon 7 and its at least partial functional equivalence.

[0153] In other embodiments, a coding exon of OP Al pre-mRNA other than exon 7 is targeted by an agent disclosed herein, which promotes exclusion of the coding exon other than exon 7. In these other embodiments, the agent that promotes exclusion of the coding exon other than exon 7 increases expression of OPA1 protein encoded by a mature mRNA transcript that lacks the excluded exon.

[0154] Alternative splicing of the OPA1 pre-mRNA species, e.g., skipping of a coding exon, e.g., an alternatively spliced exon, e.g., exon 7, to produce functional mature OPA1 protein may be induced using an agent such as an ASO that stimulates the exon skipping. Induction of exon skipping may result in modulation of levels of different alternatively spliced mRNA transcripts. The resulting mature OPA1 mRNA can be translated into different OPA1 proteins, thereby modulating the amount of OPA1 protein in the patient’s cells and alleviating symptoms of a condition or disease associated with OPA1 deficiency, such as an eye disease or condition, Optic atrophy type 1, autosomal dominant optic atrophy (ADOA), ADOA-plus syndrome; a mitochondrial disorder; glaucoma; normal tension glaucoma; Charcot-Marie- tooth disease; mitochondria dysfunction; diabetic retinopathy; age-related macular degeneration; retinal ganglion cell death; mitochondrial fission-mediated mitochondrial dysfunction; progressive external ophthalmoplegia; deafness; ataxia; motor neuropathy; sensory neuropathy; myopathy; Behr syndrome; brain dysfunction; encephalopathy; peripheral neuropathy; fatal infantile mitochondrial encephalomyopathy; hypertrophic cardiomyopathy; spastic ataxic syndrome; sensory motor peripheral neuropathy; hypotonia; gastrointestinal dysmotility and dysphagia; optic atrophy; optic atrophy plus syndrome; Mitochondrial DNA depletion syndrome 14; late-onset cardiomyopathy; diabetic cardiomyopathy; Alzheimer’s Disease; focal segmental glomerulosclerosis; kidney disease; Huntington’s Disease; cognitive function decline in healthy aging; Prion diseases; late onset dementia and parkinsonism; mitochondrial myopathy; Leigh syndrome; Friedreich’s ataxia; Parkinson’s disease;

MELAS (Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes); pyruvate dehydrogenase complex deficiency; chronic kidney disease; Leber’s hereditary optic neuropathy; obesity; age-related systemic neurodegeneration; skeletal muscle atrophy; heart and brain ischemic damage; or massive liver apoptosis.

[0155] In some embodiments, the diseases or conditions that can be treated or ameliorated using the method or composition disclosed herein are not directly associated with the target protein (gene) that the therapeutic agent targets. In some embodiments, a therapeutic agent provided herein can target a protein (gene) that is not directly associated with a disease or condition, but the modulation of expression of the target protein (gene) can treat or ameliorate the disease or condition.

[0156] In some embodiments, the antisense oligomer modulates splicing of a nonsense-mediated RNA decay-inducing exon (NMD exon) from a pre-mRNA in a cell of the subject, wherein the pre-mRNA encodes the OPA1 protein and comprises the NMD exon, thereby modulating a level of processed mRNA that is processed from the pre-mRNA, and modulating expression of the OPA1 protein in the cell.

[0157] In some embodiments, the antisense oligomer: (a) binds to a targeted portion of the pre-mRNA; (b) modulates binding of a factor involved in splicing of the NMD exon; or (c) a combination of (a) and (b).

[0158] In some embodiments, the targeted portion of the pre-mRNA is proximal to the NMD exon. [0159] In various embodiments, the present disclosure provides a therapeutic agent which can target OPA1 mRNA transcripts to modulate splicing or protein expression level. The therapeutic agent can be a small molecule, polynucleotide, or polypeptide. In some embodiments, the therapeutic agent is an ASO. Various regions or sequences on the OP Al pre-mRNA can be targeted by a therapeutic agent, such as an ASO. In some embodiments, the ASO targets an OPA1 pre-mRNA transcript containing an NMD exon. In some embodiments, the ASO targets a sequence within an NMD exon of an OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence upstream (or 5’) from the 5’ end of an NMD exon (3’ss) of an OP Al pre-mRNA transcript. In some embodiments, the ASO targets a sequence downstream (or 3’) from the 3’ end of an NMD exon (5’ss) of an OP Al pre-mRNA transcript. In some embodiments, the ASO targets a sequence that is within an intron flanking on the 5’ end of the NMD exon of an OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence that is within an intron flanking the 3’ end of the NMD exon of an OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence comprising an NMD exon-intron boundary of an OP Al pre-mRNA transcript. An NMD exon-intron boundary can refer to the junction of an intron sequence and an NMD exon region. The intron sequence can flank the 5’ end of the NMD exon, or the 3’ end of the NMD exon. In some embodiments, the ASO targets a sequence within an exon of an OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron of an OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence comprising both a portion of an intron and a portion of an exon of an OP Al pre-mRNA transcript.

[0160] In some embodiments, the ASO targets a sequence about 4 to about 300 nucleotides upstream (or 5’) from the 5’ end of the NMD exon. In some embodiments, the ASO targets a sequence about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides upstream (or 5’) from the 5’ end of the NMD exon region. In some embodiments, the ASO may target a sequence more than 300 nucleotides upstream from the 5’ end of the NMD exon. In some embodiments, the ASO targets a sequence about 4 to about 300 nucleotides downstream (or 3’) from the 3’ end of the NMD exon. In some embodiments, the ASO targets a sequence about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides downstream from the 3’ end of the NMD exon. In some embodiments, the ASO targets a sequence more than 300 nucleotides downstream from the 3’ end of the NMD exon. [0161] In some embodiments, the OPA1 NMD exon-containing pre-mRNA transcript is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 1. In some embodiments, the OP Al NMD exon pre-mRNA transcript comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 2-5.

[0162] In some embodiments, the OPA1 NMD exon-containing pre-mRNA transcript (or NMD exon mRNA) comprises a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOS: 2-5. In some embodiments, OP Al NMD exon-containing pre-mRNA transcript (or NMD exon mRNA) is encoded by a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOS: 2-5. In some embodiments, the targeted portion of the NMD exon mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleotides of any one of SEQ ID NOS: 2-5.

[0163] In some embodiments, the ASO targets exon 6x of an OPA1 NMD exon-containing pre-mRNA comprising NIE exon 6, exon 7x of an OPA1 NMD exon-containing pre-mRNA comprising NIE exon 7, or exon 28x of an OP Al NMD exon-containing pre-mRNA comprising NIE exon 28. In some embodiments, the ASO targets exon (GRCh38/ hg38: chr3 193628509 193628616) of OP Al pre-mRNA; or exon (GRCh38/ hg38: chr3 193603500 193603557) of OPAl. In some embodiments, the ASO targets an NMD exon of OPAl pre-mRNA other than NMD exon (GRCh38/hg38: chr3 193628509 193628616). [0164] In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream (or 5’) from the 5’ end of exon 6x of OPAl, exon 7x of OPAl, or exon 28x of OPAl. In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream (or 5’) from GRCh38/ hg38: chr3 193628509 of OPAP, or GRCh38/ hg38: chr3 193603500 of OPAl .

[0165] In some embodiments, the ASO targets a sequence at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream (or 5’) from the 5’ end of exon 6x of OPAl, exon 7x of OPAl, or exon 28x of OPAl. In some embodiments, the ASO targets a sequence at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream (or 5’) from GRCh38/ hg38: chr3 193628509 of OPAL, or GRCh38/ hg38: chr3 193603500 of OPAL

[0166] In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream (or 3’) from the 3’ end of exon 6x of OPAL exon 7x of OPAL or exon 28x of OPAL In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream (or 3’) from GRCh38/ hg38: chr3 193628616 of OPAL, or GRCh38/ hg38: chr3 193603557 of OPAL

[0167] In some embodiments, the ASO targets a sequence at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream (or 3’) from the 3’ end of exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPAL In some embodiments, the ASO targets a sequence at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream (or 3’) from GRCh38/ hg38: chr3 193628616 of OPAL, or GRCh38/ hg38: chr3 193603557 of OPAL

[0168] In some embodiments, the ASO has a sequence complementary to the targeted portion of the NMD exon mRNA according to any one of SEQ ID NOS: 2-5 or 279.

[0169] In some embodiments, the ASO targets a sequence upstream from the 5’ end of an NMD exon. For example, ASOs targeting a sequence upstream from the 5’ end of an NMD exon (exon 6x of OP Al, exon 7x of OPA1, or exon 28x of OPA1) comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least 8 contiguous nucleobases of SEQ ID NO: 2 or 3. For example, ASOs targeting a sequence upstream from the 5’ end of an NMD exon (e.g., exon (GRCh38/ hg38: chr3 193628509 to 193628616) of OT V; or exon (GRCh38/ hg38: chr3 193603500 193603557) of OPAL) can comprise a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 4 or 5.

[0170] In some embodiments, the ASOs target a sequence containing an exon-intron boundary (or junction). For example, ASOs targeting a sequence containing an exon-intron boundary can comprise a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least 8 contiguous nucleobases of any one of SEQ ID NOS: 2-5. In some embodiments, the ASOs target a sequence downstream from the 3’ end of an NMD exon. For example, ASOs targeting a sequence downstream from the 3’ end of an NMD exon (e.g., exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1) can comprise a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 2 or 3, or at least 8 contiguous nucleobases of SEQ ID NO: 2 or 3. For example, ASOs targeting a sequence downstream from the 3’ end of an NMD exon (e.g., exon (GRCh38/ hg38: chr3 193628509 to 193628616) of OPAL, or exon (GRCh38/ hg38: chr3 193603500 to 193603557) of OPAL) can comprise a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 4 or 5, or at least 8 contiguous nucleobases of SEQ ID NO: 4 or 5. In some embodiments, ASOs target a sequence within an NMD exon.

[0171] In some embodiments, the ASO targets exon 6x of an OP Al NMD exon-containing pre-mRNA comprising NIE exon 6, exon 7x of an OPA1 NMD exon-containing pre-mRNA comprising NIE exon 7, or exon 28x of an OP Al NMD exon-containing pre-mRNA comprising NIE exon 28. In some embodiments, the ASO targets a sequence downstream (or 3’) from the 5’ end of exon 6x, exon 7x, or exon 28x of an OP Al pre-mRNA. In some embodiments, the ASO targets a sequence upstream (or 5’) from the 3’ end of exon 6x, exon 7x, or exon 28x of an OP Al pre-mRNA.

[0172] In some embodiments, the targeted portion of the OPA1 NMD exon-containing pre-mRNA is in intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, hybridization of an ASO to the targeted portion of the NMD exon pre-mRNA results in exon skipping of at least one of NMD exon within intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and subsequently increases OPA1 protein production. In some embodiments, the targeted portion of the OP Al NMD exon-containing pre-mRNA is in intron 6 of OP Al, or intron 28 of OP Al. In some embodiments, the targeted portion of the OPA1 NMD exon-containing pre-mRNA is intron (GRCh38/ hg38: chr3 193626203 to 193631611) of OPAL, or intron (GRCh38/ hg38: chr3 193593374 to 193614710) of OPAL

[0173] In some embodiments, the methods and compositions of the present disclosure are used to increase the expression of OPA1 by inducing exon skipping of a pseudo-exon of an OP Al NMD exoncontaining pre-mRNA. In some embodiments, the pseudo-exon is a sequence within any of introns 1-50. In some embodiments, the pseudo-exon is a sequence within any of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the pseudo-exon can be an OPA1 intron or a portion thereof. In some embodiments, the pseudo-exon is within intron 6 of OP Al, or intron 28 of OPAL In some embodiments, the pseudo-exon is within intron (GRCh38/ hg38: chr3 193626203 to 193631611) of OPAL, or intron (GRCh38/ hg38: chr3 193593374 to 193614710) of OPA1.

[0174] In some embodiments, the ASO targets an OP Al pre-mRNA transcript to induce exon skipping of a coding exon, e.g., an alternatively spliced exon. In some embodiments, the ASO targets a sequence within a coding exon, e.g., an alternatively spliced exon, of an OP Al pre-mRNA transcript. In some embodiments, the ASO targets a sequence upstream (or 5’) from the 5’ end of a coding exon (3’ss) of an OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence downstream (or 3’) from the 3’ end of a coding exon (5’ss) of an OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence that is within an intron flanking on the 5’ end of the coding exon of an OP Al pre- mRNA transcript. In some embodiments, the ASO targets a sequence that is within an intron flanking the 3’ end of the coding exon of an OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence comprising an exon-intron boundary of an OPA1 pre-mRNA transcript. An exon-intron boundary can refer to the junction of an intron sequence and an exon sequence. The intron sequence can flank the 5’ end of the coding exon, or the 3’ end of the coding exon. In some embodiments, the ASO targets a sequence within an exon of an OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron of an OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence comprising both a portion of an intron and a portion of an exon of an OPA1 pre-mRNA transcript.

[0175] In some embodiments, the ASO targets a sequence about 4 to about 300 nucleotides upstream (or 5’) from the 5’ end of the coding exon, e.g., alternatively spliced exon. In some embodiments, the ASO targets a sequence about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides upstream (or 5’) from the 5’ end of the coding exon region. In some embodiments, the ASO may target a sequence more than 300 nucleotides upstream from the 5’ end of the coding exon. In some embodiments, the ASO targets a sequence about 4 to about 300 nucleotides downstream (or 3’) from the 3’ end of the coding exon. In some embodiments, the ASO targets a sequence about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides downstream from the 3’ end of the coding exon. In some embodiments, the ASO targets a sequence more than 300 nucleotides downstream from the 3’ end of the coding exon.

[0176] In some embodiments, the OPA1 pre-mRNA transcript is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 1. In some embodiments, the OPA1 pre-mRNA transcript comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 2-5.

[0177] In some embodiments, the OPA1 pre-mRNA transcript (or NMD exon mRNA) comprises a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOS: 2-5. In some embodiments, OPA1 pre-mRNA transcript (or NMD exon mRNA) is encoded by a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOS: 2-5. In some embodiments, the targeted portion of the OP Al pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleotides of any one of SEQ ID NOS: 2-5.

[0178] In some embodiments, the ASO targets exon 7 of an OP Al pre-mRNA, i.e., the ASO targets exon (GRCh38/ hg38: chr3 193626092 to 193626202) of OPA1 pre-mRNA.

[0179] In some embodiments, the ASO targets a coding exon of an OP Al pre-mRNA other than exon 7, i.e., the ASO targets an exon of OPA1 pre-mRNA other than exon defined by (GRCh38/ hg38: chr3 193626092 to 193626202).

[0180] In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream (or 5’) from the 5’ end of exon 7 of OP Al. In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream (or 5’) from GRCh38/ hg38: chr3 193626092 of OPAL

[0181] In some embodiments, the ASO targets a sequence at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream (or 5’) from the 5’ end of exon 7 of OP Al. In some embodiments, the ASO targets a sequence at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream (or 5’) from GRCh38/ hg38: 193626092 of OPAL

[0182] In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream (or 3’) from the 3’ end of exon 7 of OPAL In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream (or 3’) from GRCh38/ hg38: chr3 193626202 of OPAL

[0183] In some embodiments, the ASO targets a sequence at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream (or 3’) from the 3’ end of exon 7 of OPA1 . In some embodiments, the ASO targets a sequence at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream (or 3’) from GRCh38/ hg38: chr3 193626202 of OPAL

[0184] In some embodiments, the ASO has a sequence complementary to the targeted portion of the NMD exon mRNA according to any one of SEQ ID NOS: 2-5 or 277.

[0185] In some embodiments, the ASO targets a sequence upstream from the 5 ’ end of a coding exon, e.g., an alternatively spliced exon. For example, ASOs targeting a sequence upstream from the 5’ end of a coding exon (e.g., exon 7 of OP Al) comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least 8 contiguous nucleobases of SEQ ID NO: 2 or 3. For example, ASOs targeting a sequence upstream from the 5’ end of a coding exon (e.g., exon (GRCh38/ hg38: 193626092 to 193626202) of OPAl) can comprise a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 4 or 5.

[0186] In some embodiments, the ASOs target a sequence containing an exon-intron boundary (or junction). For example, ASOs targeting a sequence containing an exon-intron boundary can comprise a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least 8 nucleobases nucleotides of any one of SEQ ID NOS: 2-5. In some embodiments, the ASOs target a sequence downstream from the 3’ end of a coding exon, e.g., an alternatively spliced exon. For example, ASOs targeting a sequence downstream from the 3’ end of a coding exon (e.g., exon 7 of OPAl) can comprise a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 2 or 3, or at least 8 contiguous nucleobases of SEQ ID NO: 2 or 3. For example, ASOs targeting a sequence downstream from the 3’ end of a coding exon (e.g., exon 7 of OPAl) can comprise a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 4 or 5, or at least 8 contiguous nucleobases of SEQ ID NO: 4 or 5. In some embodiments, ASOs target a sequence within a coding exon, e.g., an alternatively spliced exon.

Protein Expression

[0187] In some embodiments, the methods described herein are used to increase the production of a functional OPAl protein or RNA. As used herein, the term “functional” refers to the amount of activity or function of an OPAl protein or RNA that is necessary to eliminate any one or more symptoms of a treated condition or disease, e.g., Optic atrophy type 1. In some embodiments, the methods are used to increase the production of a partially functional OPAl protein or RNA. As used herein, the term “partially functional” refers to any amount of activity or function of the OPAl protein or RNA that is less than the amount of activity or function that is necessary to eliminate or prevent any one or more symptoms of a disease or condition. In some embodiments, a partially functional protein or RNA will have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% less activity relative to the fully functional protein or RNA.

[0188] In some embodiments, the method is a method of increasing the expression of the OPAl, protein by cells of a subject having an OPAl pre-mRNA, wherein the subject has a disease or condition, e.g., Optic atrophy type 1, caused by a deficient amount of activity of OPAl protein, and wherein the deficient amount of the OPAl protein is caused by haploinsufficiency of the OPAl protein. In such an embodiment, the subject has a first allele encoding a functional OPAl protein, and a second allele from which the OPAl protein is not produced. In another such embodiment, the subject has a first allele encoding a functional OPA1 protein, and a second allele encoding a nonfunctional OPA1 protein. In another such embodiment, the subject has a first allele encoding a functional OPA1 protein, and a second allele encoding a partially functional OPA1 protein. In any of these embodiments, the antisense oligomer binds to a targeted portion of the OP Al pre-mRNA transcribed from the second allele, thereby inducing exon skipping of the pseudo-exon from the pre-mRNA, and causing an increase in the level of mature mRNA encoding functional OPA1 protein, and an increase in the expression of the OPA1 protein in the cells of the subject.

[0189] In some embodiments, the method is a method of increasing the expression of the OPA1 protein by cells of a subject having an OPA1 pre-mRNA, wherein the subject has a disease or condition caused by a deficient amount of activity of OPA1 protein, and wherein the deficient amount of the OPA1 protein is caused by autosomal recessive inheritance.

[0190] In some embodiments, the method is a method of increasing the expression of the OPA1 protein by cells of a subject having an OPA1 pre-mRNA, wherein the subject has a disease or condition, e.g., Optic atrophy type 1, caused by a deficient amount of activity of OPA1, protein, and wherein the deficient amount of the OPA1 protein is caused by autosomal dominant inheritance.

[0191] In related embodiments, the method is a method of using an ASO to increase the expression of a protein or functional RNA. In some embodiments, an ASO may be used to increase the expression of OPA1 protein in cells of a subject having an OPA1 pre-mRNA, wherein the subject has a deficiency, e.g., Optic atrophy type 1; in the amount or function of an OPA1 protein.

[0192] In some embodiments, the pre-mRNA transcript that encodes the protein that is causative of the disease or condition is targeted by the agent, e.g., the oligonucleotides, described herein. In some cases, it is the NMD exon-containing pre-mRNA transcript targeted by the agent, e.g., the oligonucleotides, described herein. In some cases, the agent, e.g., the oligonucleotides, described herein, are designed to target a coding exon of the pre-mRNA. In some cases, the agent, e.g., the oligonucleotides, described herein can induce skipping of the NMD exon, a coding exon, or both. In some embodiments, an nmd exon-containing pre-mRNA transcript that encodes a protein that is not causative of the disease is targeted by the ASOs. For example, a disease that is the result of a mutation or deficiency of a first protein in a particular pathway may be ameliorated by targeting a pre-mRNA that encodes a second protein, thereby increasing production of the second protein. In some embodiments, the function of the second protein is able to compensate for the mutation or deficiency of the first protein (which is causative of the disease or condition).

[0193] In some embodiments, the subject has:

(a) a first mutant allele from which

(i) the OPA1 protein is produced at a reduced level compared to production from a wild-type allele,

(ii) the OPA1 protein is produced in a form having reduced function compared to an equivalent wildtype protein, or

(iii) the OPA1 protein or functional RNA is not produced; and

(b) a second mutant allele from which (i) the 0PA1 protein is produced at a reduced level compared to production from a wild-type allele,

(iii) the OPA1 protein is not produced, and wherein the NMD exon-containing pre-mRNA is transcribed from the first allele and/or the second allele. In these embodiments, the ASO binds to a targeted portion of the NMD exon-containing pre-mRNA transcribed from the first allele or the second allele, thereby inducing exon skipping of the pseudo-exon from the NMD exon-containing pre-mRNA, and causing an increase in the level of mRNA encoding OPA1 protein and an increase in the expression of the target protein or functional RNA in the cells of the subject. In these embodiments, the target protein or functional RNA having an increase in expression level resulting from the exon skipping of the pseudo-exon from the NMD exon-containing pre-mRNA may be either in a form having reduced function compared to the equivalent wild-type protein (partially functional), or having full function compared to the equivalent wild-type protein (fully functional).

[0194] In some embodiments, the subject has:

(a) a first mutant allele from which

(iii) the OPA1 protein or functional RNA is not produced; and

(b) a second mutant allele from which

(iii) the OPA1 protein is not produced, and wherein the OP Al pre-mRNA is transcribed from the first allele and/or the second allele. In these embodiments, the ASO binds to a targeted portion of the OP Al pre-mRNA transcribed from the first allele or the second allele, thereby inducing exon skipping of a coding exon from the OP Al pre-mRNA, and causing an increase in the expression of the target OPA1 protein in the cells of the subject. In these embodiments, the target OPA1 protein having an increase in expression level resulting from the exon skipping of the coding exon from the OPA1 pre-mRNA may be either in a form having reduced function compared to the equivalent full-length wild-type protein (partially functional), or having full function compared to the equivalent full-length wild-type protein (fully functional).

[0195] In some embodiments, the level of mRNA encoding OPA1 protein is increased 1. 1- to 10-fold, when compared to the amount of mRNA encoding OPA1 protein that is produced in a control cell, e.g., one that is not treated with the antisense oligomer or one that is treated with an antisense oligomer that does not bind to the targeted portion of the OP Al pre-mRNA.

[0196] In some embodiments, a subject treated using the methods of the present disclosure expresses a partially functional OPA1 protein from one allele, wherein the partially functional OPA1 protein may be caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion. In some embodiments, a subject treated using the methods of the disclosure expresses a nonfunctional OPA1 protein from one allele, wherein the nonfunctional OPA1 protein may be caused by a frameshift mutation, a nonsense mutation, a missense mutation, a partial gene deletion, in one allele. In some embodiments, a subject treated using the methods of the disclosure has an OPA1 whole gene deletion, in one allele.

Exon Inclusion

[0197] As used herein, a “NMD exon-containing pre-mRNA” is a pre-mRNA transcript that contains at least one pseudo-exon. Alternative or aberrant splicing can result in inclusion of the at least one pseudoexon in the mature mRNA transcripts. The terms “mature mRNA,” and “fully spliced mRNA,” are used interchangeably herein to describe a fully processed mRNA. Inclusion of the at least one pseudo-exon can be non-productive mRNA and lead to NMD of the mature mRNA. NMD exon-containing mature mRNA may sometimes lead to aberrant protein expression.

[0198] In some embodiments, the included pseudo-exon is the most abundant pseudo-exon in a population of NMD exon-containing pre-mRNAs transcribed from the gene encoding the target protein in a cell. In some embodiments, the included pseudo-exon is the most abundant pseudo-exon in a population of NMD exon-containing pre-mRNAs transcribed from the gene encoding the target protein in a cell, wherein the population of NMD exon-containing pre-mRNAs comprises two or more included pseudoexons. In some embodiments, an antisense oligomer targeted to the most abundant pseudo-exon in the population of NMD exon-containing pre-mRNAs encoding the target protein induces exon skipping of one or two or more pseudo-exons in the population, including the pseudo-exon to which the antisense oligomer is targeted or binds. In some embodiments, the targeted region is in a pseudo-exon that is the most abundant pseudo-exon in an NMD exon-containing pre-mRNA encoding the OPA1 protein.

[0199] The degree of exon inclusion can be expressed as percent exon inclusion, e.g., the percentage of transcripts in which a given pseudo-exon is included. In brief, percent exon inclusion can be calculated as the percentage of the amount of RNA transcripts with the exon inclusion, over the sum of the average of the amount of RNA transcripts with exon inclusion plus the average of the amount of RNA transcripts with exon exclusion.

[0200] In some embodiments, an included pseudo-exon is an exon that is identified as an included pseudo-exon based on a determination of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% inclusion. In embodiments, a included pseudo-exon is an exon that is identified as a included pseudo-exon based on a determination of about 5% to about 100%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 10% to about 100%, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 100%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 70%, about 15% to about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 100%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 25% to about 100%, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, or about 25% to about 35%, inclusion. ENCODE data (described by, e.g., Tilgner, et al. , 2012, “Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co- transcriptional in the human genome but inefficient for IncRNAs,” Genome Research 22(9): 1616-25) can be used to aid in identifying exon inclusion.

[0201] In some embodiments, contacting cells with an ASO that is complementary to a targeted portion of an OP Al pre-mRNA transcript results in an increase in the amount of OPA1 protein produced by at least 10%, 20%, 30%, 40%, 50%, 60%, 80%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or 1000%, compared to the amount of the protein produced by a cell in the absence of the ASO/absence of treatment. In some embodiments, the total amount of OPA1 protein produced by the cell to which the antisense oligomer is contacted is increased about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%, compared to the amount of target protein produced by a control compound. In some embodiments, the total amount of OPA1 protein produced by the cell to which the antisense oligomer is contacted is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5 -fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5 -fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4- fold, at least about 5 -fold, or at least about 10-fold, compared to the amount of target protein produced by a control compound. A control compound can be, for example, an oligonucleotide that is not complementary to a targeted portion of the pre-mRNA.

[0202] In some embodiments, contacting cells with an ASO that is complementary to a targeted portion of an OP Al pre-mRNA transcript results in an increase in the amount of mRNA encoding OPA1, including the mature mRNA encoding the target protein. In some embodiments, the amount of mRNA encoding OPA1 protein, or the mature mRNA encoding the OPA1 protein, is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 80%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or 1000%, compared to the amount of the protein produced by a cell in the absence of the ASO/absence of treatment. In some embodiments, the total amount of the mRNA encoding OPA1 protein, or the mature mRNA encoding OPA1 protein produced in the cell to which the antisense oligomer is contacted is increased about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%, compared to the amount of mature RNA produced in an untreated cell, e.g., an untreated cell or a cell treated with a control compound. In some embodiments, the total amount of the mRNA encoding OPA1 protein, or the mature mRNA encoding OPA1 protein produced in the cell to which the antisense oligomer is contacted is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6- fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5-fold, at least about 2- fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5 -fold, or at least about 10-fold compared to the amount of mature RNA produced in an untreated cell, e.g., an untreated cell or a cell treated with a control compound. A control compound can be, for example, an oligonucleotide that is not complementary to a targeted portion of the OPA1 NMD exoncontaining pre-mRNA.

[0203] The NMD exon can be in any length. In some embodiments, the NMD exon comprises a full sequence of an intron, in which case, it can be referred to as intron retention. In some embodiments, the NMD exon can be a portion of the intron. In some embodiments, the NMD exon can be a 5’ end portion of an intron including a 5’ss sequence. In some embodiments, the NMD exon can be a 3’ end portion of an intron including a 3’ss sequence. In some embodiments, the NMD exon can be a portion within an intron without inclusion of a 5’ss sequence. In some embodiments, the NMD exon can be a portion within an intron without inclusion of a 3’ss sequence. In some embodiments, the NMD exon can be a portion within an intron without inclusion of either a 5 ’ss or a 3’ss sequence. In some embodiments, the NMD exon can be from 5 nucleotides to 10 nucleotides in length, from 10 nucleotides to 15 nucleotides in length, from 15 nucleotides to 20 nucleotides in length, from 20 nucleotides to 25 nucleotides in length, from 25 nucleotides to 30 nucleotides in length, from 30 nucleotides to 35 nucleotides in length, from 35 nucleotides to 40 nucleotides in length, from 40 nucleotides to 45 nucleotides in length, from 45 nucleotides to 50 nucleotides in length, from 50 nucleotides to 55 nucleotides in length, from 55 nucleotides to 60 nucleotides in length, from 60 nucleotides to 65 nucleotides in length, from 65 nucleotides to 70 nucleotides in length, from 70 nucleotides to 75 nucleotides in length, from 75 nucleotides to 80 nucleotides in length, from 80 nucleotides to 85 nucleotides in length, from 85 nucleotides to 90 nucleotides in length, from 90 nucleotides to 95 nucleotides in length, or from 95 nucleotides to 100 nucleotides in length. In some embodiments, the NMD exon can be at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleoids, at least 70 nucleotides, at least 80 nucleotides in length, at least 90 nucleotides, or at least 100 nucleotides in length. In some embodiments, the NMD exon can be from 100 to 200 nucleotides in length, from 200 to 300 nucleotides in length, from 300 to 400 nucleotides in length, from 400 to 500 nucleotides in length, from 500 to 600 nucleotides in length, from 600 to 700 nucleotides in length, from 700 to 800 nucleotides in length, from 800 to 900 nucleotides in length, or from 900 to 1,000 nucleotides in length. In some embodiments, the NMD exon may be longer than 1,000 nucleotides in length.

[0204] Inclusion of a pseudo-exon can lead to a frameshift and the introduction of a premature termination codon (PTC) in the mature mRNA transcript rendering the transcript a target of NMD. Mature mRNA transcript containing NMD exon can be non-productive mRNA transcript which does not lead to protein expression. The PTC can be present in any position downstream of an NMD exon. In some embodiments, the PTC can be present in any exon downstream of an NMD exon. In some embodiments, the PTC can be present within the NMD exon. For example, inclusion of exon 6x of OP Al, exon 7x of OPA1, or exon 28x of OPA1, in an mRNA transcript encoded by the OPA1 gene can induce a PTC in the mRNA transcript. For example, inclusion of exon (GRCh38/ hg38: chr3 193628509 193628616) of OPA , or exon (GRCh38/ hg38: chr3 193603500 193603557) of OPA1 in an mRNA transcript encoded by the OPAL

[0205] In some aspects, provided herein is a method of modulating expression of an OPA1 protein by promoting inclusion of a coding exon. The method can comprise contacting an agent to a cell having an OPA1 pre-mRNA, wherein the agent comprises an oligonucleotide that binds to: (a) a targeted portion of the pre-mRNA within an intronic region immediately upstream of a 5’ end of the coding exon of the pre- mRNA; or (b) a targeted portion of the pre-mRNA within an intronic region immediately downstream of a 3’ end of the coding exon of the pre-mRNA; whereby the agent increases a level of a processed mRNA that is processed from the pre-mRNA and that contains the coding exon in the cell. In some cases, the coding exon to be included is an alternatively spliced exon. In some cases, the method promotes inclusion of the coding exon in the processed mRNA during splicing of the pre-mRNA in the cell. [0206] In some of these embodiments for inclusion of coding exon, the target portion of the pre-mRNA is within a region spanning from 100 to 50, from 100 to 60, from 100 to 70, from 100 to 80, or from 100 to 90 nucleotides upstream of a 5’ end of the coding exon. In some cases, the target portion of the pre- mRNA is within a region spanning from 40 to 100, from 50 to 100, from 60 to 100, from 70 to 100, from 80 to 100, or from 90 to 100 nucleotides downstream of a 3’ end of the coding exon. In some cases, the coding exon is exon 7 of OP Al . In some cases, the coding exon comprises a sequence with at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO: 277. In some cases, the coding exon comprises SEQ ID NO: 277. The targeted portion of the pre-mRNA can be within a region spanning from 100 to 50, from 100 to 60, from 100 to 70, from 100 to 80, or from 100 to 90 nucleotides upstream of genomic site GRCh38/ hg38: chr3 193626092. In some cases, the targeted portion of the pre-mRNA is within a region spanning from 40 to 100, from 50 to 100, from 60 to 100, from 70 to 100, from 80 to 100, or from 90 to 100 nucleotides downstream of genomic site GRCh38/ hg38: chr3 193626202.

[0207] In some cases, the inclusion of the coding exon in the processed mRNA in the cell contacted with the agent is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5 -fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5 -fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4- fold, at least about 5 -fold, or at least about 10-fold, compared to in the absence of the agent.

Exclusion of Both NMD Exon and Coding Exon

[0208] In some embodiments, provided herein is a method of modulating expression of a target protein by targeting a pre-mRNA and modulating exclusion of both a coding exon and a nonsense-mediated RNA decay-inducing exon (NMD exon) from the pre-mRNA. In some cases, the method comprises contacting an agent to the cell, and the agent promotes exclusion of both the coding exon and the NMD exon from the pre-mRNA, thereby increasing level of a processed mRNA that is processed from the pre-mRNA and lacks both the coding exon and the NMD exon. In some cases, the agent binds to a targeted portion of the pre-mRNA, or modulates binding of a factor involved in splicing of the coding exon, the NMD exon, or both. In some cases, the agent interferes with binding of the factor involved in splicing of the coding exon, the NMD exon, or both, to a region of the targeted portion. In some cases, the NMD exon is within an intronic region adjacent to the coding exon. In some cases, the NMD exon is within an intronic region immediately upstream of the coding exon. In some cases, the NMD exon is within an intronic region immediately downstream of the coding exon. In some cases, the coding exon is an alternatively spliced exon.

[0209] In some cases, the targeted portion of the pre-mRNA is proximal to the coding exon. The targeted portion of the pre-mRNA can be located in an intronic region immediately upstream of the coding exon. The targeted portion of the pre-mRNA can be located in an intronic region immediately downstream of the coding exon. In some cases, the targeted portion of the pre-mRNA can be located within the coding exon. In some cases, the targeted portion of the pre-mRNA is within a region spanning from 49 to 1, from 39 to 1, from 29 to 1, or from 19 to 1 nucleotide(s) upstream of 5’ end of the coding exon. In some cases, the targeted portion of the pre-mRNA is within a region spanning from 100 nucleotides upstream of the coding exon to 100 nucleotides downstream of the coding exon. In some cases, the targeted portion comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides of the coding exon.

[0210] In some cases, the targeted portion of the pre-mRNA is proximal to the NMD exon. In some cases, the targeted portion of the pre-mRNA is located in an intronic region immediately upstream of the NMD exon. In some cases, the targeted portion of the pre-mRNA is located in an intronic region immediately downstream of the NMD exon. In some cases, the targeted portion of the pre-mRNA is located within the NMD exon. In some cases, the targeted portion of the pre-mRNA is within a region spanning from 100 nucleotides upstream of the NMD exon to 100 nucleotides downstream of the NMD exon.

[0211] In some embodiments, the method described herein is applicable to modulation of expression of OP Al protein by modulating exclusion of both exon 7 and an NMD exon (e.g., exon 7x) of OP Al pre- mRNA that contains both exon 7 and exon 7x. In some cases, the coding exon comprises a sequence with at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO: 277. In some cases, the coding exon comprises SEQ ID NO: 277. In some cases, the targeted portion of the pre-mRNA is immediately upstream of the coding exon GRCh38/ hg38: chr3 193626092 to 193626202. In some cases, the targeted portion of the pre-mRNA is immediately downstream of the coding exon GRCh38/ hg38: chr3 193626092 to 193626202. In some cases, the targeted portion of the pre-mRNA is within a region spanning from 49 to 1, from 39 to 1, from 29 to 1, or from 19 to 1 nucleotide(s) upstream of GRCh38/ hg38: chr3 193626092. In some cases, the targeted portion of the pre-mRNA is within a region spanning from 100 nucleotides upstream of genomic site GRCh38/ hg38: chr3 193626092 to 100 nucleotides downstream of genomic site GRCh38/ hg38: chr3 193626202. In some cases, the targeted portion of the pre-mRNA is within the coding exon GRCh38/ hg38: chr3 193626092 to 193626202. In some cases, the targeted portion of the pre-mRNA comprises an exon-intron junction of the coding exon GRCh38/ hg38: chr3 193626092 to 193626202. In some cases, the NMD exon comprises a sequence with at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO: 279. In some cases, the NMD exon comprises SEQ ID NO: 279. In some cases, the targeted portion of the pre-mRNA is immediately upstream of the NMD exon GRCh38/ hg38: chr3 193628509 to 193628616. In some cases, the targeted portion of the pre- mRNA is immediately downstream of the NMD exon GRCh38/ hg38: chr3 193628509 to 193628616. In some cases, the targeted portion of the pre-mRNA is within a region spanning from 100 nucleotides upstream of genomic site GRCh38/ hg38: chr3 193628509 to 100 nucleotides downstream of genomic site GRCh38/ hg38: chr3 193628616.

[0212] In some cases, the targeted portion of the pre-mRNA is within the NMD exon GRCh38/ hg38: chr3 193628509 to 193628616. In some cases, the targeted portion of the pre-mRNA comprises an exon- intron junction of the NMD exon GRCh38/ hg38: chr3 193628509 to 193628616. In some cases, the targeted portion comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides of the NMD exon.

[0213] In some cases, the exclusion of the coding exon from the pre-mRNA in the cell contacted with the agent is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5 -fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5 -fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4- fold, at least about 5 -fold, or at least about 10-fold, compared to in the absence of contacting with the agent. In some cases, the exclusion of the NMD exon from the pre-mRNA in the cell contacted with the agent is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5 -fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5 -fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4- fold, at least about 5 -fold, or at least about 10-fold, compared to in the absence of contacting with the agent. In some cases, the method results in an increase in the level of the processed mRNA in the cell. The level of the processed mRNA in the cell contacted with the agent can be increased by about 1. 1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5 -fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1. 1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold, compared to in the absence of contacting with the agent.

[0214] In some cases, the method results in an increase in expression of the OPA1 protein in the cell. A level of the OPA1 protein expressed from the processed mRNA in the cell contacted with the agent can be increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5 -fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5-fold, at least about 2- fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5 -fold, or at least about 10-fold, compared to in the absence of contacting with the agent.

[0215] In some cases, a level of the OPA1 protein expressed from the processed mRNA in the cell contacted with the agent is increased by at least about 1.5-fold compared to in the absence of contacting with the agent.

[0216] In some cases, the OPA1 protein expressed from the processed mRNA that lacks exon 7 and exon 7x is a functional OPA1 protein. The OPA1 protein expressed from the processed mRNA that lacks exon 7 and exon 7x can be at least partially functional as compared to a wild-type OPA1 protein. The OPA1 protein expressed from the processed mRNA that lacks exon 7 and exon 7x can be at least partially functional as compared to a full-length wild-type OPA1 protein.

Therapeutic Agents

[0217] In various embodiments of the present disclosure, compositions and methods comprising a therapeutic agent are provided to modulate protein expression level of OPA1. In some embodiments, provided herein are compositions and methods to modulate alternative splicing of OPA1 pre-mRNA. In some embodiments, provided herein are compositions and methods to induce exon skipping in the splicing of OP Al pre-mRNA, e.g., to induce skipping of a pseudo-exon during splicing of OP Al pre- mRNA. In other embodiments, therapeutic agents may be used to induce the inclusion of an exon in order to decrease the protein expression level.

[0218] A therapeutic agent disclosed herein can be a NIE repressor agent. A therapeutic agent may comprise a polynucleic acid polymer.

[0219] According to one aspect of the present disclosure, provided herein is a method of treatment or prevention of a condition or disease associated with a functional OPA1 protein deficiency, comprising administering a NIE repressor agent to a subject to increase levels of functional OPA1 protein, wherein the agent binds to a region of the pre-mRNA transcript to decrease inclusion of the NMD exon in the mature transcript. For example, provided herein is a method of treatment or prevention of a condition associated with a functional OPA1 protein deficiency, comprising administering a NIE repressor agent to a subject to increase levels of functional OPA1 protein, wherein the agent binds to a region of an intron containing an NMD exon (e.g., exon 6x of OPA P exon 7x of OPA 1. or exon 28x of OPA1) of the pre- mRNA transcript or to an NMD exon-activating regulatory sequence in the same intron. For example, provided herein is a method of treatment or prevention of a condition associated with a functional OPA1 protein deficiency, comprising administering a NIE repressor agent to a subject to increase levels of functional OPA1 protein, wherein the agent binds to a region of an intron containing an NMD exon (e.g., exon (GRCh38/ hg38: chr3 193628509 193628616) of OPAP, or exon (GRCh38/ hg38: chr3 193603500 193603557) of OPAP) of the pre-mRNA transcript or to an NMD exon-activating regulatory sequence in the same intron. In some embodiments, the method comprises administering a NIE repressor agent to a subject to increase levels of functional OPA1 protein, wherein the agent binds to a region of an intron containing an NMD exon (e.g., exon of OPA1 other than exon 7x defined by (GRCh38/ hg38: chr3 193628509 193628616) or exon defined by (GRCh38/ hg38: chr3 193603500 193603557)) of the pre - mRNA transcript or to an NMD exon-activating regulatory sequence in the same intron. In some embodiments, the therapeutic agent promotes exclusion of an NMD exon of OPA1 pre-mRNA other than exon 7x defined by (GRCh38/ hg38: chr3 193628509 193628616) or exon defined by (GRCh38/ hg38: chr3 193603500 193603557). In some embodiments, the composition disclosed herein includes an agent that promotes exclusion of an NMD exon of OPA1 pre-mRNA other than exon 7x defined by (GRCh38/ hg38: chr3 193628509 193628616) or exon defined by (GRCh38/ hg38: chr3 193603500 193603557). [0220] Where reference is made to reducing NMD exon inclusion in the mature mRNA, the reduction may be complete, e.g., 100%, or may be partial. The reduction may be clinically significant. The reduction/correction may be relative to the level of NMD exon inclusion in the subject without treatment, or relative to the amount of NMD exon inclusion in a population of similar subjects. The reduction/correction may be at least 10% less NMD exon inclusion relative to the average subject, or the subject prior to treatment. The reduction may be at least 20% less NMD exon inclusion relative to an average subject, or the subject prior to treatment. The reduction may be at least 40% less NMD exon inclusion relative to an average subject, or the subject prior to treatment. The reduction may be at least 50% less NMD exon inclusion relative to an average subject, or the subject prior to treatment. The reduction may be at least 60% less NMD exon inclusion relative to an average subject, or the subject prior to treatment. The reduction may be at least 80% less NMD exon inclusion relative to an average subject, or the subject prior to treatment. The reduction may be at least 90% less NMD exon inclusion relative to an average subject, or the subject prior to treatment.

[0221] According to one aspect of the present disclosure, provided herein is a method of treatment or prevention of a condition or disease associated with a functional OPA1 protein deficiency, comprising administering an agent to a subject to increase levels of functional OPA1 protein, wherein the agent binds to a region of the pre-mRNA transcript to decrease inclusion of a coding exon (e.g., exon 7) in the mature transcript. For example, provided herein is a method of treatment or prevention of a condition associated with a functional OPA1 protein deficiency, comprising administering an agent to a subject to increase levels of functional OPA1 protein, wherein the agent binds to a region containing a coding exon (e.g., exon 7 of OP Al) of the pre-mRNA transcript. For example, provided herein is a method of treatment or prevention of a condition associated with a functional OPA1 protein deficiency, comprising administering an agent to a subject to increase levels of functional OPA1 protein, wherein the agent binds to a region containing a coding exon (e.g., exon (GRCh38/ hg38: chr3 193626092 to 193626202) of OPA1) of the pre-mRNA transcript. In some embodiments, the method comprises administering an agent to a subject to increase levels of functional OPA1 protein, wherein the agent binds to a region containing a coding exon (e.g., exon of OP Al other than exon 7 defined by (GRCh38/ hg38: chr3 193626092 to 193626202)) of the pre-mRNA transcript. In some embodiments, the therapeutic agent promotes exclusion of a coding exon of OPA1 pre-mRNA other than exon 7 defined by (GRCh38/ hg38: chr3 193626092 to 193626202). In some embodiments, the composition disclosed herein includes an agent that promotes exclusion of a coding exon of OP Al pre-mRNA other than exon 7 defined by (GRCh38/ hg38: chr3 193626092 to 193626202).

[0222] Where reference is made to increasing active OPA 1 protein levels, the increase may be clinically significant. The increase may be relative to the level of active OPA1 protein in the subject without treatment, or relative to the amount of active OPA1 protein in a population of similar subjects. The increase may be at least 10% more active OPA1 protein relative to the average subject, or the subject prior to treatment. The increase may be at least 20% more active OPA1 protein relative to the average subject, or the subject prior to treatment. The increase may be at least 40% more active OPA 1 protein relative to the average subject, or the subject prior to treatment. The increase may be at least 50% more active OPA1 protein relative to the average subject, or the subject prior to treatment. The increase may be at least 80% more active OPA1 protein relative to the average subject, or the subject prior to treatment. The increase may be at least 100% more active OPA1 protein relative to the average subject, or the subject prior to treatment. The increase may be at least 200% more active OPA1 protein relative to the average subject, or the subject prior to treatment The increase may be at least 500% more active OPA1 protein relative to the average subject, or the subject prior to treatment.

[0223] In embodiments wherein the NIE repressor agent comprises a polynucleic acid polymer, the polynucleic acid polymer may be about 50 nucleotides in length. The polynucleic acid polymer may be about 45 nucleotides in length. The polynucleic acid polymer may be about 40 nucleotides in length. The polynucleic acid polymer may be about 35 nucleotides in length. The polynucleic acid polymer may be about 30 nucleotides in length. The polynucleic acid polymer may be about 24 nucleotides in length. The polynucleic acid polymer may be about 25 nucleotides in length. The polynucleic acid polymer may be about 20 nucleotides in length. The polynucleic acid polymer may be about 19 nucleotides in length. The polynucleic acid polymer may be about 18 nucleotides in length. The polynucleic acid polymer may be about 17 nucleotides in length. The polynucleic acid polymer may be about 16 nucleotides in length. The polynucleic acid polymer may be about 15 nucleotides in length. The polynucleic acid polymer may be about 14 nucleotides in length. The polynucleic acid polymer may be about 13 nucleotides in length. The polynucleic acid polymer may be about 12 nucleotides in length. The polynucleic acid polymer may be about 11 nucleotides in length. The polynucleic acid polymer may be about 10 nucleotides in length. The polynucleic acid polymer may be between about 10 and about 50 nucleotides in length. The polynucleic acid polymer may be between about 10 and about 45 nucleotides in length. The polynucleic acid polymer may be between about 10 and about 40 nucleotides in length. The polynucleic acid polymer may be between about 10 and about 35 nucleotides in length. The polynucleic acid polymer may be between about 10 and about 30 nucleotides in length. The polynucleic acid polymer may be between about 10 and about 25 nucleotides in length. The polynucleic acid polymer may be between about 10 and about 20 nucleotides in length. The polynucleic acid polymer may be between about 15 and about 25 nucleotides in length. The polynucleic acid polymer may be between about 15 and about 30 nucleotides in length. The polynucleic acid polymer may be between about 12 and about 30 nucleotides in length.

[0224] The sequence of the polynucleic acid polymer may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% complementary to a target sequence of an mRNA transcript, e.g., a partially processed mRNA transcript. The sequence of the polynucleic acid polymer may be 100% complementary to a target sequence of a pre-mRNA transcript. [0225] The sequence of the polynucleic acid polymer may have 4 or fewer mismatches to a target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may have 3 or fewer mismatches to a target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may have 2 or fewer mismatches to a target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may have 1 or fewer mismatches to a target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may have no mismatches to a target sequence of the pre-mRNA transcript.

[0226] The polynucleic acid polymer may specifically hybridize to a target sequence of the pre-mRNA transcript. For example, the polynucleic acid polymer may have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence complementarity to a target sequence of the pre-mRNA transcript. The hybridization may be under high stringent hybridization conditions.

[0227] The polynucleic acid polymer comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 2-5. The polynucleic acid polymer may comprise a sequence with 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 2-5.

[0228] Where reference is made to a polynucleic acid polymer sequence, the skilled person will understand that one or more substitutions may be tolerated, optionally two substitutions may be tolerated in the sequence, such that it maintains the ability to hybridize to the target sequence; or where the substitution is in a target sequence, the ability to be recognized as the target sequence. References to sequence identity may be determined by BLAST sequence alignment using standard/default parameters. For example, the sequence may have 99% identity and still function according to the present disclosure. In other embodiments, the sequence may have 98% identity and still function according to the present disclosure. In another embodiment, the sequence may have 95% identity and still function according to the present disclosure. In another embodiment, the sequence may have 90% identity and still function according to the present disclosure.

Antisense Oligomers

[0229] Provided herein is a composition comprising an antisense oligomer that induces exon skipping by binding to a targeted portion of an OP Al pre-mRNA, e.g., an OP Al NMD exon-containing pre-mRNA. As used herein, the terms “ASO” and “antisense oligomer” are used interchangeably and refer to an oligomer such as a polynucleotide, comprising nucleobases that hybridizes to a target nucleic acid (e.g., an OP Al pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (G-U). The ASO may have exact sequence complementary to the target sequence or near complementarity (e.g., sufficient complementarity to bind the target sequence and enhancing splicing at a splice site). ASOs are designed so that they bind (hybridize) to a target nucleic acid (e.g., a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, if they hybridize to a site other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not a target nucleic acid (to a few sites other than a target nucleic acid). Design of an ASO can take into consideration the occurrence of the nucleic acid sequence of the targeted portion of the pre-mRNA transcript or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular pre-mRNA or transcriptome, such that the likelihood the ASO will bind other sites and cause “off-target” effects is limited. Any antisense oligomers known in the art (for example, in PCT Application No. PCT/US2014/054151, published as WO 2015/035091, titled “Reducing Nonsense-Mediated mRNA Decay,” incorporated by reference herein), can be used to practice the methods described herein.

[0230] In some embodiments, ASOs “specifically hybridize” to or are “specific” to a target nucleic acid or a targeted portion of an OP Al pre-mRNA, e.g., an NMD exon-containing pre-mRNA. Typically, such hybridization occurs with a T_m substantially greater than 37 °C, preferably at least 50 °C, and typically between 60 °C to approximately 90 °C. Such hybridization preferably corresponds to stringent hybridization conditions. At a given ionic strength and pH, the T_m is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.

[0231] Oligomers, such as oligonucleotides, are “complementary” to one another when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides. A double -stranded polynucleotide can be “complementary” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. Complementarity (the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion (e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules. The sequence of an antisense oligomer (ASO) need not be 100% complementary to that of its target nucleic acid to hybridize. In certain embodiments, ASOs can comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, an ASO in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non- complementary nucleobases may be clustered together or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. Percent complementarity of an ASO with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul, et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0232] An ASO need not hybridize to all nucleobases in a target sequence and the nucleobases to which it does hybridize may be contiguous or noncontiguous. ASOs may hybridize over one or more segments of a pre-mRNA transcript, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure may be formed). In certain embodiments, an ASO hybridizes to noncontiguous nucleobases in a target pre-mRNA transcript. For example, an ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobase(s) to which the ASO does not hybridize.

[0233] The ASOs described herein comprise nucleobases that are complementary to nucleobases present in a target portion of an OPA1 pre-mRNA, e.g., an NMD exon-containing pre-mRNA. The term ASO embodies oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridizing to a complementary nucleobase on a target mRNA but does not comprise a sugar moiety, such as a peptide nucleic acid (PNA). The ASOs may comprise naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding. The term “naturally occurring nucleotides” includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” includes nucleotides with modified or substituted sugar groups and/or having a modified backbone. In some embodiments, all of the nucleotides of the ASO are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described herein will be evident to one of skill in the art and can be found, for example, in U.S. Patent No. 8,258,109 B2, U.S. Patent No. 5,656,612, U.S. Patent Publication No. 2012/0190728, and Dias and Stein, Mol. Cancer Ther. 2002, 347-355, herein incorporated by reference in their entirety.

[0234] One or more nucleobases of an ASO may be any naturally occurring, unmodified nucleobase such as adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA. Examples of modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5, 6-dihydrouracil, 5 -methylcytosine, and 5- hydroxymethoylcytosine .

[0235] The ASOs described herein also comprise a backbone structure that connects the components of an oligomer. The term “backbone structure” and “oligomer linkages” may be used interchangeably and refer to the connection between monomers of the ASO. In naturally occurring oligonucleotides, the backbone comprises a 3 ’-5’ phosphodiester linkage connecting sugar moieties of the oligomer. The backbone structure or oligomer linkages of the ASOs described herein may include (but are not limited to) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the like. See, e.g., EaPlanche, et al.. Nucleic Acids Res. 14:9081 (1986); Stec, et al., J. Am. Chem. Soc. 106:6077 (1984), Stein, et al., Nucleic Acids Res. 16:3209 (1988), Zon, et al., Anti-Cancer Drug Design 6:539 (1991); Zon, et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec, et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990). In some embodiments, the backbone structure of the ASO does not contain phosphorous but rather contains peptide bonds, for example, in a peptide nucleic acid (PNA), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups. In some embodiments, the backbone modification is a phosphorothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.

[0236] In some embodiments, the stereochemistry at each of the phosphorus intemucleotide linkages of the ASO backbone is random. In some embodiments, the stereochemistry at each of the phosphorus intemucleotide linkages of the ASO backbone is controlled and is not random. For example, U.S. Pat. App. Pub. No. 2014/0194610, “Methods for the Synthesis of Functionalized Nucleic Acids,” incorporated herein by reference, describes methods for independently selecting the handedness of chirality at each phosphorous atom in a nucleic acid oligomer. In some embodiments, an ASO used in the methods of the disclosure, including, but not limited to, any of the ASOs set forth herein in Tables 5 and 6, comprises an ASO having phosphorus intemucleotide linkages that are not random. In some embodiments, a composition used in the methods of the disclosure comprises a pure diastereomeric ASO. In some embodiments, a composition used in the methods of the disclosure comprises an ASO that has diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

[0237] In some embodiments, the ASO has a nonrandom mixture of Rp and Sp configurations at its phosphorus intemucleotide linkages. For example, it has been suggested that a mix of Rp and Sp is required in antisense oligonucleotides to achieve a balance between good activity and nuclease stability (Wan, et al., 2014, “Synthesis, biophysical properties and biological activity of second-generation antisense oligonucleotides containing chiral phosphorothioate linkages,” Nucleic Acids Research 42(22): 13456-13468, incorporated herein by reference). In some embodiments, an ASO used in the methods of the disclosure, including, but not limited to, any of the ASOs set forth herein in SEQ ID NOS: 2-5, comprises about 5-100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, with the remainder Sp, or about 100% Rp. In some embodiments, an ASO used in the methods of the disclosure, including, but not limited to, any of the ASOs set forth herein comprise a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleobases of any one of SEQ ID NOS: 2- 5, comprises about 10% to about 100% Rp, about 15% to about 100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp, about 40% to about 100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about 60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, or about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp, with the remainder Sp.

[0238] In some embodiments, an ASO used in the methods of the disclosure, including, but not limited to, any of the ASOs set forth herein, comprises a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleobases of any one of SEQ ID NOS: 2-5, comprises about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, with the remainder Rp, or about 100% Sp. In embodiments, an ASO used in the methods of the disclosure, including, but not limited to, any of the ASOs set forth herein, comprises a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleobases of any one of SEQ ID NOS: 2-5, comprises about 10% to about 100% Sp, about 15% to about 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, about 40% to about 100% Sp, about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55% Sp, with the remainder Rp.

[0239] Any of the ASOs described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog, including a morpholine ring. Non-limiting examples of modified sugar moieties include 2’ substitutions such as 2’-O-methyl (2’-0-Me), 2’-O-methoxyethyl (2’MOE), 2’-O-aminoethyl, 2’F; N3’->P5’ phosphoramidate, 2’dimethylaminooxyethoxy, 2 ’dimethylaminoethoxy ethoxy, 2’-guanidinidium, 2’-O- guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. In some embodiments, the sugar moiety modification is selected from 2’-0-Me, 2’F, and 2’MOE. In some embodiments, the sugar moiety modification is an extra bridge bond, such as in a locked nucleic acid (LNA). In some embodiments the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises a ribofuransyl or 2’deoxyribofuransyl modification. In some embodiments, the sugar moiety comprises 2 ’4 ’-constrained 2’O-methyloxyethyl (cMOE) modifications. In some embodiments, the sugar moiety comprises cEt 2’, 4’ constrained 2’-0 ethyl BNA modifications. In some embodiments, the sugar moiety comprises tricycloDNA (tcDNA) modifications. In some embodiments, the sugar moiety comprises ethylene nucleic acid (ENA) modifications. In some embodiments, the sugar moiety comprises MCE modifications. Modifications are known in the art and described in the literature, e.g., by Jarver, et al., 2014, “A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications,” Nucleic Acid Therapeutics 24(1): 37-47, incorporated by reference for this purpose herein.

[0240] In some embodiments, each monomer of the ASO is modified in the same way, for example each linkage of the backbone of the ASO comprises a phosphorothioate linkage or each ribose sugar moiety comprises a 2’0-methyl modification. Such modifications that are present on each of the monomer components of an ASO are referred to as “uniform modifications.” In some examples, a combination of different modifications may be desired, for example, an ASO may comprise a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholines).

Combinations of different modifications to an ASO are referred to as “mixed modifications” or “mixed chemistries.”

[0241] In some embodiments, the ASO comprises one or more backbone modifications. In some embodiments, the ASO comprises one or more sugar moiety modifications. In some embodiments, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, the ASO comprises a 2’MOE modification and a phosphorothioate backbone. In some embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs or any component of an ASO (e.g., a nucleobase, sugar moiety, backbone) described herein may be modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO. For example, an ASO or one or more components of any ASO may be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (i.e., RNase H); improve uptake of the ASO into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the ASO; and/or modulate the half- life of the ASO.

[0242] In some embodiments, the ASOs are comprised of 2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides. ASOs comprised of such nucleotides are especially well-suited to the methods disclosed herein; oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable, for example, for oral delivery in some embodiments described herein. See e.g., Geary, et al., J Pharmacol Exp Ther. 2001; 296(3):890-7; Geary, et al., J Pharmacol Exp Ther. 2001; 296(3):898-904.

[0243] In some embodiments, the ASOs comprise 5 ’-methylcytosine (5’-MeC) nucleotides. In some embodiments, the ASOs comprise of at least one 5 ’-methylcytosine (5’-MeC) nucleotide. In some embodiments, each cytosine of the ASO is a 5 ’-methylcytosine (5’-MeC). In some embodiments, the ASOs comprise a 5 ’-methyluracil (5’-MeU). In some embodiments, the ASOs comprise at least one 5’- methyluracil (5’-MeU). In some embodiments, at least one cytosine or thymidine of the antisense oligomer is a 5 ’-methyluracil (5’-MeU). In some embodiments, each cytosine or thymidine of the antisense oligomer is a 5 ’-methyluracil (5’-MeU).

[0244] In some embodiments, the ASO has the following structure:

or a pharmaceutically acceptable salt thereof.

[0245] In some embodiments, the ASO has the following structure: or a pharmaceutically acceptable salt thereof. [0246] In some embodiments, the ASO has the following structure: or a pharmaceutically acceptable salt thereof.

[0247] In some embodiments, the ASO has the following structure: or a pharmaceutically acceptable salt thereof. [0248] In some embodiments, the ASO has the following structure: or a pharmaceutically acceptable salt thereof.

[0249] In some embodiments, the ASO has any one of the following structures:

[0250] In some embodiments, the ASO is in a salt form. In some embodiments, the salt form is sodium salt. In some embodiments, the sodium salt form of the ASO has the following structure:

[0251] In some embodiments, the ASO is in a salt form. In some embodiments, the salt form is sodium salt. In some embodiments, the sodium salt form of the ASO has the following structure:

[0252] In some embodiments, the ASO is in a salt form. In some embodiments, the salt form is sodium salt. In some embodiments, the sodium salt form of the ASO has the following structure:

[0253] In some embodiments, the ASO is in a salt form. In some embodiments, the salt form is sodium salt. In some embodiments, the sodium salt form of the ASO has the following structure: [0254] In some embodiments, the ASO is in a salt form. In some embodiments, the salt form is sodium salt. In some embodiments, the sodium salt form of the ASO has the following structure:

[0255] In some embodiments, the ASO has any one of the following structures:

or a pharmaceutically acceptable salt thereof.

[0256] In some embodiments, the ASO has the following structure: [0257] In some embodiments, the ASO has the following structure:

[0258] In some embodiments, the ASO has the following structure:

[0259] In some embodiments, the ASO has the following structure:

[0260] In some embodiments, the ASO has the following structure: [0261] Methods of synthesizing ASOs will be known to one of skill in the art. Alternatively or in addition, ASOs may be obtained from a commercial source.

[0262] Unless specified otherwise, the left-hand end of single-stranded nucleic acid (e.g., pre-mRNA transcript, oligonucleotide, ASO, etc.) sequences is the 5’ end and the left-hand direction of single or double-stranded nucleic acid sequences is referred to as the 5’ direction. Similarly, the right-hand end or direction of a nucleic acid sequence (single or double stranded) is the 3’ end or direction. Generally, a region or sequence that is 5’ to a reference point in a nucleic acid is referred to as “upstream,” and a region or sequence that is 3’ to a reference point in a nucleic acid is referred to as “downstream.” Generally, the 5 ’ direction or end of an mRNA is where the initiation or start codon is located, while the 3’ end or direction is where the termination codon is located. In some aspects, nucleotides that are upstream of a reference point in a nucleic acid may be designated by a negative number, while nucleotides that are downstream of a reference point may be designated by a positive number. For example, a reference point (e.g., an exon-exon junction in mRNA) may be designated as the “zero” site, and a nucleotide that is directly adjacent and upstream of the reference point is designated “minus one,” e.g., “-1,” while a nucleotide that is directly adjacent and downstream of the reference point is designated “plus one,” e.g., “+1 ”

[0263] In some embodiments, the ASOs are complementary to (and bind to) a targeted portion of an OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA, that is downstream (in the 3’ direction) of the 5’ splice site (or 3’ end of the NMD exon) of the included exon in an OP Al pre-mRNA (e.g., the direction designated by positive numbers relative to the 5’ splice site). In some embodiments, the ASOs are complementary to a targeted portion of the OPA1 pre-mRNA, e.g., the OPA1 NMD exoncontaining pre-mRNA that is within the region about +1 to about +500 relative to the 5’ splice site (or 3’ end) of the included exon. In some embodiments, the ASOs may be complementary to a targeted portion of an OP Al pre-mRNA, e.g., an OP Al NMD exon-containing pre-mRNA, that is within the region between nucleotides +6 and +40,000 relative to the 5’ splice site (or 3’ end) of the included exon. In some aspects, the ASOs are complementary to a targeted portion that is within the region about +1 to about +40,000, about +1 to about +30,000, about +1 to about +20,000, about +1 to about +15,000, about +1 to about +10,000, about +1 to about +5,000, about +1 to about +4,000, about +1 to about +3,000, about +1 to about +2,000, about +1 to about +1,000, about +1 to about +500, about +1 to about +490, about +1 to about +480, about +1 to about +470, about +1 to about +460, about +1 to about +450, about +1 to about +440, about +1 to about +430, about +1 to about +420, about +1 to about +410, about +1 to about +400, about +1 to about +390, about +1 to about +380, about +1 to about +370, about +1 to about +360, about +1 to about +350, about +1 to about +340, about +1 to about +330, about +1 to about +320, about +1 to about +310, about +1 to about +300, about +1 to about +290, about +1 to about +280, about +1 to about +270, about +1 to about +260, about +1 to about +250, about +1 to about +240, about +1 to about +230, about +1 to about +220, about +1 to about +210, about +1 to about +200, about +1 to about +190, about +1 to about +180, about +1 to about +170, about +1 to about +160, about +1 to about +150, about +1 to about +140, about +1 to about +130, about +1 to about +120, about +1 to about +110, about +1 to about +100, about +1 to about +90, about +1 to about +80, about +1 to about +70, about +1 to about +60, about +1 to about +50, about +1 to about +40, about +1 to about +30, or about +1 to about +20 relative to 5’ splice site (or 3’ end) of the included exon. In some aspects, the ASOs are complementary to a targeted portion that is within the region from about +1 to about +100, from about +100 to about +200, from about +200 to about +300, from about +300 to about +400, or from about +400 to about +500 relative to 5’ splice site (or 3’ end) of the included exon.

[0264] In some embodiments, the ASOs are complementary to (and bind to) a targeted portion of an OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA, that is upstream (in the 5’ direction) of the 5’ splice site (or 3’ end) of the included exon in an OP Al pre-mRNA, e.g., an OP Al NMD exoncontaining pre-mRNA (e.g., the direction designated by negative numbers relative to the 5’ splice site). In some embodiments, the ASOs are complementary to a targeted portion of the OP Al pre-mRNA, e.g., the OP Al NMD exon-containing pre-mRNA, that is within the region about -4 to about -270 relative to the 5’ splice site (or 3 ’end) of the included exon. In some embodiments, the ASOs may be complementary to a targeted portion of an OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA, that is within the region between nucleotides -1 and -40,000 relative to the 5’ splice site (or 3’ end) of the included exon. In some aspects, the ASOs are complementary to a targeted portion that is within the region about - 1 to about -40,000, about -1 to about -30,000, about -1 to about -20,000, about -1 to about -15,000, about -1 to about -10,000, about -1 to about -5,000, about -1 to about -4,000, about -1 to about -3,000, about -1 to about -2,000, about -1 to about -1,000, about -1 to about -500, about -1 to about -490, about -1 to about -480, about -1 to about -470, about -1 to about -460, about -1 to about -450, about -1 to about -440, about -1 to about -430, about -1 to about -420, about -1 to about -410, about -1 to about -400, about -1 to about - 390, about -1 to about -380, about -1 to about -370, about -1 to about -360, about -1 to about -350, about - 1 to about -340, about -1 to about -330, about -1 to about -320, about -1 to about -310, about -1 to about - 300, about -1 to about -290, about -1 to about -280, about -1 to about -270, about -1 to about -260, about - 1 to about -250, about -1 to about -240, about -1 to about -230, about -1 to about -220, about -1 to about - 210, about -1 to about -200, about -1 to about -190, about -1 to about -180, about -1 to about -170, about - 1 to about -160, about -1 to about -150, about -1 to about -140, about -1 to about -130, about -1 to about - 120, about -1 to about -110, about -1 to about -100, about -1 to about -90, about -1 to about -80, about -1 to about -70, about -1 to about -60, about -1 to about -50, about -1 to about -40, about -1 to about -30, or about -1 to about -20 relative to 5’ splice site (or 3’ end) of the included exon.

[0265] In some embodiments, the ASOs are complementary to a targeted region of an OP Al pre-mRNA, e.g., an OP Al NMD exon-containing pre-mRNA, that is upstream (in the 5’ direction) of the 3’ splice site (or 5’ end) of the included exon in an OP Al pre-mRNA (e.g., in the direction designated by negative numbers). In some embodiments, the ASOs are complementary to a targeted portion of the OP Al pre- mRNA, e.g., the OPA1 NMD exon-containing pre-mRNA, that is within the region about -1 to about -500 relative to the 3’ splice site (or 5’ end) of the included exon. In some embodiments, the ASOs are complementary to a targeted portion of the OP Al pre-mRNA that is within the region -1 to -40,000 relative to the 3’ splice site of the included exon. In some aspects, the ASOs are complementary to a targeted portion that is within the region about -1 to about -40,000, about -1 to about -30,000, -1 to about -20,000, about -1 to about -15,000, about -1 to about -10,000, about -1 to about -5,000, about -1 to about - 4,000, about -1 to about -3,000, about -1 to about -2,000, about -1 to about -1,000, about -1 to about -500, about -1 to about -490, about -1 to about -480, about -1 to about -470, about -1 to about -460, about -1 to about -450, about -1 to about -440, about -1 to about -430, about -1 to about -420, about -1 to about -410, about -1 to about -400, about -1 to about -390, about -1 to about -380, about -1 to about -370, about -1 to about -360, about -1 to about -350, about -1 to about -340, about -1 to about -330, about -1 to about -320, about -1 to about -310, about -1 to about -300, about -1 to about -290, about -1 to about -280, about -1 to about -270, about -1 to about -260, about -1 to about -250, about -1 to about -240, about -1 to about -230, about -1 to about -220, about -1 to about -210, about -1 to about -200, about -1 to about -190, about -1 to about -180, about -1 to about -170, about -1 to about -160, about -1 to about -150, about -1 to about -140, about -1 to about -130, about -1 to about -120, about -1 to about -110, about -1 to about -100, about -1 to about -90, about -1 to about -80, about -1 to about -70, about -1 to about -60, about -1 to about -50, about -1 to about -40, about -1 to about -30, or about -1 to about -20 relative to 3’ splice site of the included exon. In some aspects, the ASOs are complementary to a targeted portion that is within the region from about -1 to about -100, from about -100 to about -200, from about -200 to about -300, from about -300 to about -400, or from about -400 to about -500 relative to 3’ splice site of the included exon.

[0266] In some embodiments, the ASOs are complementary to a targeted region of an OP Al pre-mRNA, e.g., an OP Al NMD exon-containing pre-mRNA, that is downstream (in the 3’ direction) of the 3’ splice site (5’ end) of the included exon in an OPA1 pre-mRNA, e.g., an OP Al NMD exon-containing pre- mRNA (e.g., in the direction designated by positive numbers). In some embodiments, the ASOs are complementary to a targeted portion of the OP Al pre-mRNA that is within the region of about +1 to about +40,000 relative to the 3’ splice site of the included exon. In some aspects, the ASOs are complementary to a targeted portion that is within the region about +1 to about +40,000, about +1 to about +30,000, about +1 to about +20,000, about +1 to about +15,000, about +1 to about +10,000, about +1 to about +5,000, about +1 to about +4,000, about +1 to about +3,000, about +1 to about +2,000, about +1 to about +1,000, about +1 to about +500, about +1 to about +490, about +1 to about +480, about +1 to about +470, about +1 to about +460, about +1 to about +450, about +1 to about +440, about +1 to about +430, about +1 to about +420, about +1 to about +410, about +1 to about +400, about +1 to about +390, about +1 to about +380, about +1 to about +370, about +1 to about +360, about +1 to about +350, about +1 to about +340, about +1 to about +330, about +1 to about +320, about +1 to about +310, about +1 to about +300, about +1 to about +290, about +1 to about +280, about +1 to about +270, about +1 to about +260, about +1 to about +250, about +1 to about +240, about +1 to about +230, about +1 to about +220, about +1 to about +210, about +1 to about +200, about +1 to about +190, about +1 to about +180, about +1 to about +170, about +1 to about +160, about +1 to about +150, about +1 to about +140, about +1 to about +130, about +1 to about +120, about +1 to about +110, about +1 to about +100, about +1 to about +90, about +1 to about +80, about +1 to about +70, about +1 to about +60, about +1 to about +50, about +1 to about +40, about +1 to about +30, or about +1 to about +20, or about +1 to about +10 relative to 3’ splice site of the included exon.

[0267] In some embodiments, the targeted portion of the OPA1 pre-mRNA, e.g., the OPA1 NMD exoncontaining pre-mRNA, is within the region +100 relative to the 5’ splice site (3’ end) of the included exon to -100 relative to the 3’ splice site (5’ end) of the included exon. In some embodiments, the targeted portion of the OP Al NMD exon-containing pre-mRNA is within the NMD exon. In some embodiments, the target portion of the OP Al NMD exon-containing pre-mRNA comprises a pseudo-exon and intron boundary.

[0268] The ASOs may be of any length suitable for specific binding and effective enhancement of splicing. In some embodiments, the ASOs consist of 8 to 50 nucleobases. For example, the ASO may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases in length. In some embodiments, the ASOs consist of more than 50 nucleobases. In some embodiments, the ASO is from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15 nucleobases, 13 to 50 nucleobases, 13 to 40 nucleobases, 13 to 35 nucleobases, 13 to 30 nucleobases, 13 to 25 nucleobases, 13 to 20 nucleobases, 14 to 50 nucleobases, 14 to 40 nucleobases, 14 to 35 nucleobases, 14 to 30 nucleobases, 14 to 25 nucleobases, 14 to 20 nucleobases, 15 to 50 nucleobases, 15 to 40 nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20 nucleobases, 20 to 50 nucleobases, 20 to 40 nucleobases, 20 to 35 nucleobases, 20 to 30 nucleobases, 20 to 25 nucleobases, 25 to 50 nucleobases, 25 to 40 nucleobases, 25 to 35 nucleobases, or 25 to 30 nucleobases in length. In some embodiments, the ASOs are 18 nucleotides in length. In some embodiments, the ASOs are 15 nucleotides in length. In some embodiments, the ASOs are 25 nucleotides in length.

[0269] In some embodiments, two or more ASOs with different chemistries but complementary to the same targeted portion of the pre-mRNA, e.g., NMD exon-containing pre-mRNA, are used. In some embodiments, two or more ASOs that are complementary to different targeted portions of the pre-mRNA, e.g., the NMD exon-containing pre-mRNA, are used.

[0270] In some embodiments, the antisense oligonucleotides of the disclosure are chemically linked to one or more moieties or conjugates, e.g., a targeting moiety or other conjugate that enhances the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, a lipid moiety, e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, or adamantane acetic acid. Oligonucleotides comprising lipophilic moieties and preparation methods have been described in the published literature. In embodiments, the antisense oligonucleotide is conjugated with a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N- acetylgalactosamine (GalNAc), N-Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), a lipid, or a polyhydrocarbon compound. Conjugates can be linked to one or more of any nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, e.g., using a linker. Linkers can include a bivalent or trivalent branched linker. In embodiments, the conjugate is attached to the 3’ end of the antisense oligonucleotide. Methods of preparing oligonucleotide conjugates are described, e.g., in U.S. Pat. No. 8,450,467, “Carbohydrate conjugates as delivery agents for oligonucleotides,” incorporated by reference herein.

[0271] In some embodiments, the nucleic acid to be targeted by an ASO is an OPA1 pre-mRNA, e.g., NMD exon-containing pre-mRNA expressed in a cell, such as a eukaryotic cell. In some embodiments, the term “cell” may refer to a population of cells. In some embodiments, the cell is in a subject. In some embodiments, the cell is isolated from a subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is a condition or disease-relevant cell or a cell line. In some embodiments, the cell is in vitro (e.g., in cell culture).

Pharmaceutical Compositions

[0272] Pharmaceutical compositions or formulations comprising the agent, e.g., antisense oligonucleotide, of the described compositions and for use in any of the described methods can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the published literature. In embodiments, a pharmaceutical composition or formulation for treating a subject comprises an effective amount of any antisense oligomer as described herein, or a pharmaceutically acceptable salt, solvate, hydrate or ester thereof. The pharmaceutical formulation comprising an antisense oligomer may further comprise a pharmaceutically acceptable excipient, diluent or carrier.

[0273] Pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc. , and are commensurate with a reasonable benefit/risk ratio. (See, e.g., S. M. Berge, et al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein by reference for this purpose. The salts can be prepared in situ during the final isolation and purification of the compounds, or separately by reacting the free base form with a suitable organic acid. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other documented methodologies such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy- ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

[0274] In some embodiments, the compositions are formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. In embodiments, the compositions are formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. In embodiments, a pharmaceutical formulation or composition of the present disclosure includes, but is not limited to, a solution, emulsion, microemulsion, foam or liposome-containing formulation (e.g., cationic or noncationic liposomes).

[0275] The pharmaceutical composition or formulation described herein may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients as appropriate and well known to those of skill in the art or described in the published literature. In embodiments, liposomes also include sterically stabilized liposomes, e.g., liposomes comprising one or more specialized lipids. These specialized lipids result in liposomes with enhanced circulation lifetimes. In embodiments, a sterically stabilized liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. In some embodiments, a surfactant is included in the pharmaceutical formulation or compositions. The use of surfactants in drug products, formulations and emulsions is well known in the art. In embodiments, the present disclosure employs a penetration enhancer to effect the efficient delivery of the antisense oligonucleotide, e.g., to aid diffusion across cell membranes and /or enhance the permeability of a lipophilic drug. In some embodiments, the penetration enhancers are a surfactant, fatty acid, bile salt, chelating agent, or non-chelating nonsurfactant.

[0276] In some embodiments, the pharmaceutical formulation comprises multiple antisense oligonucleotides. In embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent.

[0277] In some embodiments, the pharmaceutical composition is prepared by diluting a concentrate comprising the antisense oligomer.

[0278] In some embodiments, the antisense oligomer is present in the concentrate at a concentration of about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, or about 40 mg/ml to about 60 mg/ml.

[0279] In some embodiments, the antisense oligomer is present in the concentrate at a concentration of about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

[0280] In some embodiments, the concentrate is a phosphate-buffered solution.

[0281] In some embodiments, the pharmaceutical formulation comprises: (a) an antisense oligomer, wherein the antisense oligomer comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOS: 6-275 or 280-299; and (b) a pharmaceutically acceptable diluent; wherein the antisense oligomer is dissolved or suspended in a solution at a concentration of about 1 mg/ml to about 200 mg/ml. [0282] In some embodiments, the pharmaceutical formulation comprises (a) an antisense oligomer, wherein the antisense oligomer comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOS: 6-275 or 280-299; and (b) a pharmaceutically acceptable diluent; wherein the antisense oligomer is dissolved or suspended in a solution, and wherein the antisense oligomer has any one of the following chemical structures: [0283] In some embodiments, the pharmaceutical formulation comprises (a) an antisense oligomer, wherein the antisense oligomer comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOS: 6-275 or 280-299; and (b) a pharmaceutically acceptable diluent; wherein the antisense oligomer is dissolved or suspended in a solution, and wherein the antisense oligomer has any one of the following chemical structures:

or a pharmaceutically acceptable salt thereof.

[0284] In some embodiments, the antisense oligomer is present in the solution at a concentration of about 1 mg/ml to about 200 mg/ml.

[0285] In some embodiments, the antisense oligomer is present in the solution at a concentration of about 5 mg/ml to about 200 mg/ml, about 10 mg/ml to about 200 mg/ml, about 15 mg/ml to about 200 mg/ml, about 20 mg/ml to about 200 mg/ml, about 25 mg/ml to about 200 mg/ml, about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, 2 mg/ml to about 150 mg/ml, about 5 mg/ml to about 150 mg/ml, about 10 mg/ml to about 150 mg/ml, about 15 mg/ml to about 150 mg/ml, about 20 mg/ml to about 150 mg/ml, about 25 mg/ml to about 150 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, 2 mg/ml to about 100 mg/ml, about 5 mg/ml to about 100 mg/ml, about 10 mg/ml to about 100 mg/ml, about 15 mg/ml to about 100 mg/ml, about 20 mg/ml to about 100 mg/ml, about 25 mg/ml to about 100 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, 2 mg/ml to about 80 mg/ml, about 5 mg/ml to about 80 mg/ml, about 10 mg/ml to about 80 mg/ml, about 15 mg/ml to about 80 mg/ml, about 20 mg/ml to about 80 mg/ml, about 25 mg/ml to about 80 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, 2 mg/ml to about 60 mg/ml, about 5 mg/ml to about 60 mg/ml, about 10 mg/ml to about 60 mg/ml, about 15 mg/ml to about 60 mg/ml, about 20 mg/ml to about 60 mg/ml, about 25 mg/ml to about 60 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, about 40 mg/ml to about 60 mg/ml, 2 mg/ml to about 40 mg/ml, about 5 mg/ml to about 40 mg/ml, about 10 mg/ml to about 40 mg/ml, about 15 mg/ml to about 40 mg/ml, about 20 mg/ml to about 40 mg/ml, or about 25 mg/ml to about 40 mg/ml.

[0286] In some embodiments, the antisense oligomer is present in the solution at a concentration of about 1 mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 12 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 18 mg/ml, about 20 mg/ml, about 22 mg/ml, about 24 mg/ml, about 26 mg/ml, about 28 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 80 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

[0287] In some embodiments, the pharmaceutical composition is prepared by diluting a concentrate comprising the antisense oligomer. In some embodiments, the pharmaceutical composition is prepared by diluting a concentrate consisting of the antisense oligomer.

[0288] In some embodiments, the antisense oligomer is present in the concentrate at a concentration of about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, or about 40 mg/ml to about 60 mg/ml.

[0289] In some embodiments, the antisense oligomer is present in the concentrate at a concentration of about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

[0290] In some embodiments, the concentrate is phosphate buffered. In some embodiments, the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, or water for injection. In some embodiments, the antisense oligomer is solubilized or diluted in a solution comprising sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, and water for injection. In some embodiments, the antisense oligomer is solubilized or diluted in an isotonic solution. In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate-buffered solution with at least pH 5.8. In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate-buffered (pH 6.6 - 7.6) solution. In some embodiments, the pharmaceutical formulation does not comprise a preservative. In some embodiments, the pharmaceutical formulation is suitable for an intravitreal injection. In some embodiments, the pharmaceutical formulation is packaged in a single use vial.

[0291] In some embodiments, the pharmaceutical compositions are assembled into a kit comprising: (i) a concentrate comprising an antisense oligomer (ASO), wherein the ASO comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOS: 6-275 or 280-299; and (ii) a diluent, wherein the concentrate is miscible with the diluent; and (iii) instructions for diluting the concentrate with the diluent. [0292] In some embodiments, the pharmaceutical compositions are assembled into a kit consisting of: (i) a concentrate comprising an antisense oligomer (ASO), wherein the ASO consists of a sequence with at least 80% sequence identity to any one of SEQ ID NOS: 6-275 or 280-299; and (ii) a diluent, wherein the concentrate is miscible with the diluent; and (iii) instructions for diluting the concentrate with the diluent. [0293] In some embodiments, the antisense oligomer is present in the concentrate at a concentration of about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, or about 40 mg/ml to about 60 mg/ml.

[0294] In some embodiments, the antisense oligomer is present in the concentrate at a concentration of about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

[0295] In some embodiments, the concentrate is phosphate buffered. In some embodiments, the diluent is a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, or water for injection. In some embodiments, the diluent is a solution comprising sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, and water for injection. In some embodiments, the diluent comprises an isotonic solution. In some embodiments, the diluent comprises a phosphate-buffered solution with at least pH 5.8. In some embodiments, the diluent comprises a phosphate-buffered (pH 6.6 - 7.6) solution. In some embodiments, the concentrate or the diluent does not comprise a preservative. [0296] In some embodiments, the diluent is a solution consisting of one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, or water for injection. In some embodiments, the diluent is a solution consisting of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, and water for injection. In some embodiments, the diluent consists of an isotonic solution. In some embodiments, the diluent consists of a phosphate-buffered solution with at least pH 5.8. In some embodiments, the diluent consists of a phosphate-buffered (pH 6.6 - 7.6) solution. In some embodiments, the concentrate or the diluent does not consist of a preservative.

[0297] In some embodiments, the instructions for diluting the concentrate with the diluent comprise instructions for diluting or solubilizing the ASO to a concentration of about 2 mg/ml to 200 mg/ml in the diluent.

[0298] In some embodiments, the instructions for diluting the concentrate with the diluent comprise instructions for diluting or solubilizing the ASO to a concentration of about 5 mg/ml to about 200 mg/ml, about 10 mg/ml to about 200 mg/ml, about 15 mg/ml to about 200 mg/ml, about 20 mg/ml to about 200 mg/ml, about 25 mg/ml to about 200 mg/ml, about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, 2 mg/ml to about 150 mg/ml, about 5 mg/ml to about 150 mg/ml, about 10 mg/ml to about 150 mg/ml, about 15 mg/ml to about 150 mg/ml, about 20 mg/ml to about 150 mg/ml, about 25 mg/ml to about 150 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, 2 mg/ml to about 100 mg/ml, about 5 mg/ml to about 100 mg/ml, about 10 mg/ml to about 100 mg/ml, about 15 mg/ml to about 100 mg/ml, about 20 mg/ml to about 100 mg/ml, about 25 mg/ml to about 100 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, 2 mg/ml to about 80 mg/ml, about 5 mg/ml to about 80 mg/ml, about 10 mg/ml to about 80 mg/ml, about 15 mg/ml to about 80 mg/ml, about 20 mg/ml to about 80 mg/ml, about 25 mg/ml to about 80 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, 2 mg/ml to about 60 mg/ml, about 5 mg/ml to about 60 mg/ml, about 10 mg/ml to about 60 mg/ml, about 15 mg/ml to about 60 mg/ml, about 20 mg/ml to about 60 mg/ml, about 25 mg/ml to about 60 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, about 40 mg/ml to about 60 mg/ml, 2 mg/ml to about 40 mg/ml, about 5 mg/ml to about 40 mg/ml, about 10 mg/ml to about 40 mg/ml, about 15 mg/ml to about 40 mg/ml, about 20 mg/ml to about 40 mg/ml, or about 25 mg/ml to about 40 mg/ml in the diluent.

[0299] In some embodiments, the instructions for diluting the concentrate with the diluent comprise instructions for diluting or solubilizing the antisense oligomer to a concentration of about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 12 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 18 mg/ml, about 20 mg/ml, about 22 mg/ml, about 24 mg/ml, about 26 mg/ml, about 28 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 80 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml in the diluent.

[0300] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 6-275 or 280-299.

[0301] In some embodiments, the antisense oligomer consists of a nucleotide sequence having at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 6-275 or 280-299.

[0302] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 280-283, 288, and 290-292. In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 92-96, and 166-168. [0303] In some embodiments, the antisense oligomer consists of a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 280-283, 288, and 290-292. In some embodiments, the antisense oligomer consists of a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 92-96, and 166-168. [0304] In some embodiments, the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2’-O-methyl moiety, a 2’-Fluoro moiety, a 2’-O-N-methyl-acetamide (2’-NMA), or a 2’-O-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomer comprises a 5 ’-methylcytosine (5’-MeC). In some embodiments, each cytosine of the antisense oligomer is a 5 ’-methylcytosine (5’-MeC). In some embodiments, the antisense oligomer comprises a 5’- methyluracil (5 ’ -MeU) .

[0305] In some embodiments, the antisense oligomer consists of a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the antisense oligomer consists of a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2’-O- methyl moiety, a 2’-Fluoro moiety, a 2’-O-N-methyl-acetamide (2’-NMA), or a 2’-O-methoxyethyl moiety. In some embodiments, the antisense oligomer consists of at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomer consists of a 5 ’-methylcytosine (5’-MeC). In some embodiments, each cytosine of the antisense oligomer is a 5 ’-methylcytosine (5’-MeC). In some embodiments, the antisense oligomer consists of a 5’- methyluracil (5 ’ -MeU) .

[0306] In some embodiments, each cytosine or thymidine of the antisense oligomer is a 5 ’-methyluracil (5 ’-MeU). In some embodiments, the antisense oligomer consists of a phosphorothioate linkage. In some embodiments, each intemucleoside linkage of the ASO is a phosphorothioate linkage. In some embodiments, the antisense oligomer consists of a locked nucleic acid (LNA).

[0307] In some embodiments, the antisense oligomer comprises from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to

35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

[0308] In some embodiments, the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to

[0309] In some embodiments, the antisense oligomer has any one of the following chemical structures:

[0310] In some embodiments, the antisense oligomer has any one of the following chemical structures:

or a pharmaceutically acceptable salt thereof.

[0311] Described herein, in some aspects, are uses of an antisense oligomer for the manufacture of a medicament for treating or preventing a disease or condition characterized by a reduced expression or function of OPA1 protein in a human subject in need thereof, wherein the medicament is administered to one eye of the subject at a dose of about 0.005 mg to about 20 mg, and wherein the antisense oligomer comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOS: 6-275 or 280-299. [0312] Described herein, in some aspects, are uses of an antisense oligomer for the manufacture of a medicament for treating or preventing a disease or condition characterized by a reduced expression or function of OPA1 protein in a human subject in need thereof, wherein the medicament is administered to one eye of the subject at a dose of about 0.005 mg to about 20 mg, and wherein the antisense oligomer consists of a sequence with at least 80% sequence identity to any one of SEQ ID NOS: 6-275 or 280-299. [0313] In some embodiments, the medicament is administered to the one eye of the subject at a dose of about 0.005 mg to about 15 mg, about 0.005 mg to about 10 mg, about 0.005 mg to about 5 mg, about 0.005 mg to about 1 mg, about 0.01 mg to about 15 mg, about 0.01 mg to about 10 mg, about 0.01 mg to about 5 mg, about 0.01 mg to about 2.5 mg, about 0.01 mg to about 1.0 mg, about 0.01 mg to about 0.5 mg, about 0.01 mg to about 0.25 mg, about 0.01 mg to about 0. 1 mg, about 0.01 mg to about 0.05 mg, about 0.05 mg to about 10 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 2.5 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.25 mg, about 0.05 mg to about 0. 1 mg, about 0.1 mg to about 5 mg, about 0. 1 mg to about 2.5 mg, about 0.1 mg to about 1.5 mg, about 0. 1 mg to about 1.0 mg, about 0.1 mg to about 0.5 mg, or about 0. 1 mg to about 0.25 mg of the antisense oligomer.

[0314] In some embodiments, the medicament is administered to the one eye of the subject at a dose of about 0.005 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.75 mg, about 2.0 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, about 5.0 mg, about 5.5 mg, about 6.0 mg, about 7.0 mg, about 8.0 mg, about 9.0 mg, about 10 mg, about 12.5 mg, about 15 mg, about 17.5 mg, or about 20 mg of the antisense oligomer.

[0315] In some embodiments, the medicament is administered to the one eye of the subject at a dose of about 0. 1 mg to about 1.5 mg, about 0.1 mg to about 1.4 mg, about 0.1 mg to about 1.2 mg, about 0. 1 mg to about 1.0 mg, about 0.1 mg to about 0.8 mg, about 0.1 mg to about 0.7 mg, about 0.1 mg to about 0.5 mg, about 0.1 mg to about 0.3 mg, about 0.2 mg to about 1.5 mg, about 0.2 mg to about 1.4 mg, about 0.2 mg to about 1.2 mg, about 0.2 mg to about 1.0 mg, about 0.2 mg to about 0.8 mg, about 0.2 mg to about 0.7 mg, about 0.2 mg to about 0.5 mg, about 0.3 mg to about 1.5 mg, about 0.3 mg to about 1.4 mg, about 0.3 mg to about 1.2 mg, about 0.3 mg to about 1.0 mg, about 0.3 mg to about 0.8 mg, about 0.3 mg to about 0.7 mg, about 0.3 mg to about 0.5 mg, about 0.5 mg to about 1.5 mg, about 0.5 mg to about 1.4 mg, about 0.5 mg to about 1.2 mg, about 0.5 mg to about 1.0 mg, about 0.5 mg to about 0.8 mg, about 0.5 mg to about 0.7 mg, about 0.7 mg to about 1.5 mg, about 0.7 mg to about 1.4 mg, about 0.7 mg to about 1.2 mg, about 0.7 mg to about 1.0 mg, about 0.8 mg to about 1.5 mg, about 0.8 mg to about 1.4 mg, about 0.8 mg to about 1.2 mg, about 0.8 mg to about 1.0 mg, about 1.0 mg to about 1.5 mg, about 1.0 mg to about 1.4 mg, about 1.0 mg to about 1.2 mg, about 1.2 mg to about 1.5 mg, or about 1.2 mg to about 1.4 mg of the antisense oligomer.

[0316] In some embodiments, the medicament is administered to the one eye of the subject at a dose of about 0. 1 mg, about 0.2 mg, about 0.3 mg, 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, or about 1.5 mg of the antisense oligomer.

Combination Therapies

[0317] In some embodiments, the ASOs disclosed in the present disclosure can be used in combination with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents can comprise a small molecule. For example, the one or more additional therapeutic agents can comprise a small molecule described in WO2016128343A1, WO2017053982A1, WO2016196386A 1 , WO201428459A 1 , WO201524876A2, WO2013119916A2, and WO2014209841A2, which are incorporated by reference herein in their entirety. In some embodiments, the one or more additional therapeutic agents comprise an ASO that can be used to correct intron retention.

Treatment of Subjects

[0318] Any of the compositions provided herein may be administered to an individual. “Individual” may be used interchangeably with “subject” or “patient.” An individual may be a mammal, for example, a human or animal such as a non-human primate, a rodent, a rabbit, a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a pig, or a sheep. In embodiments, the individual is a human. In embodiments, the individual is a fetus, an embryo, or a child. In other embodiments, the individual may be another eukaryotic organism, such as a plant. In some embodiments, the compositions provided herein are administered to a cell ex vivo.

[0319] In some embodiments, the compositions provided herein are administered to an individual as a method of treating a disease or disorder. In some embodiments, the individual has a genetic disease, such as any of the diseases described herein. In some embodiments, the individual is at risk of having a disease, such as any of the diseases described herein. In some embodiments, the individual is at increased risk of having a disease or disorder caused by insufficient amount of a protein or insufficient activity of a protein. If an individual is “at an increased risk” of having a disease or disorder caused insufficient amount of a protein or insufficient activity of a protein, the method involves preventative or prophylactic treatment. For example, an individual may be at an increased risk of having such a disease or disorder because of family history of the disease. Typically, individuals at an increased risk of having such a disease or disorder benefit from prophylactic treatment (e.g., by preventing or delaying the onset or progression of the disease or disorder). In embodiments, a fetus is treated in utero, e.g., by administering the ASO composition to the fetus directly or indirectly (e.g., via the mother).

[0320] In some cases, the subject pharmaceutical composition and method are applicable for treatment of a condition or disease associated with OPA1 deficiency. In some cases, the subject pharmaceutical composition and method are applicable for treatment of an eye disease or condition. In some cases, the subject pharmaceutical composition and method are applicable for treatment of Optic atrophy type 1, autosomal dominant optic atrophy (ADOA), ADOA-plus syndrome; a mitochondrial disorder; glaucoma; normal tension glaucoma; Charcot-Marie-Tooth disease; mitochondria dysfunction; diabetic retinopathy; age-related macular degeneration; retinal ganglion cell death; mitochondrial fission-mediated mitochondrial dysfunction; progressive external ophthalmoplegia; deafness; ataxia; motor neuropathy; sensory neuropathy; myopathy; Behr syndrome; brain dysfunction; encephalopathy; peripheral neuropathy; fatal infantile mitochondrial encephalomyopathy; hypertrophic cardiomyopathy; spastic ataxic syndrome; sensory motor peripheral neuropathy; hypotonia; gastrointestinal dysmotility and dysphagia; optic atrophy; optic atrophy plus syndrome; Mitochondrial DNA depletion syndrome 14; late- onset cardiomyopathy; diabetic cardiomyopathy; Alzheimer’s Disease; focal segmental glomerulosclerosis; kidney disease; Huntington’s Disease; cognitive function decline in healthy aging; Prion diseases; late onset dementia and parkinsonism; mitochondrial myopathy; Leigh syndrome;

Friedreich’s ataxia; Parkinson’s disease; MELAS (Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes); pyruvate dehydrogenase complex deficiency; chronic kidney disease; Leber’s hereditary optic neuropathy; obesity; age-related systemic neurodegeneration; skeletal muscle atrophy; heart and brain ischemic damage; or massive liver apoptosis.

[0321] In some embodiments, the disease or condition comprises Optic atrophy type 1. In some embodiments, the disease or condition comprises autosomal dominant optic atrophy (ADOA).

[0322] In some embodiments, the subject is characterized by having: (i) a heterozygous OPA1 gene variant; (ii) clear ocular media to allow for adequate visualization of the vitreous and fundus and to achieve appropriate quality of all ophthalmic assessments; (iii) an AREDS Clinical Lens Standard of <1 for posterior subcapsular (PSC) opacity; (iv) a BCVA EDTRS letter score of >35 and <70 with each eye individually that is administered with the pharmaceutical composition; or (v) any combination of (i)-(iv) . [0323] In some embodiments, the subject is additionally characterized by: (1) not having a gain-of- function variant, or compound heterozygous or homozygous pathogenic or likely pathogenic variant in OPA1 gene; (2) not having only benign or likely benign variants in the OPA1 gene; (3) not having extraocular phenotypic manifestations of (syndromic) ADOA (ADOA-plus); (4) not having been diagnosed with Behr syndrome; (5) not having a known pathogenic mutation in another gene implicated in optic atrophy or retinal diseases; (6) not having diabetic retinopathy with potential for development of proliferative diabetic retinopathy, diabetic macular edema, or optic neuropathy; (7) not having or having a history of any ocular condition in either eye; (8) not having a history of intraocular surgery or comeal surgery including refractive surgery in either eye within 12 weeks prior to the administering; (9) not having a history of retinal photocoagulation; (10) not having a history or presence of retinal vein occlusion; (11) not being considered to be at risk for uveitis or ocular infection because of having any of the following: an active flare of non-infectious uveitis or an episode of infectious uveitis or other ocular infection in either eye within 12 months prior to the administering; (12) not having dry age-related macular degeneration in either eye; (13) not having high myopia (>6 diopters); (14) not having a history of cancer (except a diagnosis of basal cell, squamous cell skin cancer, or carcinoma in situ of the cervix that has been successfully treated); (15) not taking, or having taken at any time, any medication or treatment that can or might cause an optic neuropathy; (16) not having any history of nutritional deficiency (including B12 and/or folate deficiencies), or not having a known deficiency in serum B12 or folate, not having had bariatric surgery; (17) any combination of (1)-(16).

[0324] In some embodiments, the method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition and comprises administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NO: 6-275 or 280-299, and wherein the method comprises administering to one eye of the subject the pharmaceutical composition at a dose of about 0.005 to about 20 mg of the antisense oligomer, and wherein the method comprises administering to one eye of the subject the pharmaceutical composition at a dose of about 0.005 to about 20 mg of the antisense oligomer.

[0325] In some embodiments, the method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition and comprises administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 90% sequence identity to the sequence set forth in any one of SEQ ID NO: 6-275 or 280-299, and wherein the method comprises administering to one eye of the subject the pharmaceutical composition at a dose of about 0.005 to about 20 mg of the antisense oligomer. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1, 0.3, 0.5, or 0.7 mg of the antisense oligomer, with an option to add two subsequent doses, each at about 0.1, 0.3, 0.5, or 0.7 mg of the antisense oligomer, capped at a maximum total dosage of about 1.2 mg. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1, 0.3, 0.5, or 0.7 mg of the antisense oligomer, with no additional subsequent doses. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1, 0.3, 0.5, or 0.7 mg of the antisense oligomer, followed by one subsequent dose of about 0.1, 0.3, 0.5, or 0.7 mg of the antisense oligomer. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0. 1 mg of the antisense oligomer, followed by two subsequent doses each at about 0.1, 0.3, 0.5, or 0.7 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0. 1 mg of the antisense oligomer, followed by two subsequent doses of about 0.1 mg of the antisense oligomer. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0. 1 mg of the antisense oligomer, followed by two subsequent doses of about 0.3 mg of the antisense oligomer. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0. 1 mg of the antisense oligomer, followed by two subsequent doses of about 0.5 mg of the antisense oligomer. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.3 mg of the antisense oligomer, followed by two subsequent doses of about 0.1 mg of the antisense oligomer. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.3 mg of the antisense oligomer, followed by two subsequent doses of about 0.3 mg of the antisense oligomer. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.3 mg of the antisense oligomer, followed by two subsequent doses, each about 0.1, 0.3, 0.5, or 0.7 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.5 mg of the antisense oligomer, followed by two subsequent doses, each at about 0. 1, 0.3, or 0.5 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.5 mg of the antisense oligomer, followed by one subsequent dose of about 0.7 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by two subsequent doses, each at about 0.1 or 0.3 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by one subsequent dose of about 0.5 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg.

[0326] In some embodiments, the method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition and comprises administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 100% sequence identity to the sequence set forth in any one of SEQ ID NO: 6-275 or 280-299, and wherein the method comprises administering to one eye of the subject the pharmaceutical composition at a dose of about 0.005 to about 20 mg of the antisense oligomer, and wherein the method comprises administering to one eye of the subject the pharmaceutical composition at a dose of about 0.005 to about 20 mg of the antisense oligomer.

[0327] In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0.005 mg to about 15 mg, about 0.005 mg to about 10 mg, about 0.005 mg to about 5 mg, about 0.005 mg to about 1 mg, about 0.01 mg to about 15 mg, about 0.01 mg to about 10 mg, about 0.01 mg to about 5 mg, about 0.01 mg to about 2.5 mg, about 0.01 mg to about 1.0 mg, about 0.01 mg to about 0.5 mg, about 0.01 mg to about 0.25 mg, about 0.01 mg to about 0. 1 mg, about 0.01 mg to about 0.05 mg, about 0.05 mg to about 10 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 2.5 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.25 mg, about 0.05 mg to about 0.1 mg, about 0. 1 mg to about 5 mg, about 0.1 mg to about 2.5 mg, about 0.1 mg to about 1.5 mg, about 0.1 mg to about 1.0 mg, about 0. 1 mg to about 0.5 mg, or about 0. 1 mg to about 0.25 mg of the antisense oligomer.

[0328] In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0.005 mg, about 0.01 mg, about 0.05 mg, about 0. 1 mg, about 0.2 mg, about 0.3 mg, 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.75 mg, about 2.0 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, about 5.0 mg, about 5.5 mg, about 6.0 mg, about 7.0 mg, about 8.0 mg, about 9.0 mg, about 10 mg, about 12.5 mg, about 15 mg, about 17.5 mg, or about 20 mg of the antisense oligomer.

[0329] In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0. 1 mg to about 1.5 mg, about 0.1 mg to about 1.4 mg, about 0. 1 mg to about 1.2 mg, about 0.1 mg to about 1.0 mg, about 0.1 mg to about 0.8 mg, about 0. 1 mg to about 0.7 mg, about 0.1 mg to about 0.5 mg, about 0.1 mg to about 0.3 mg, about 0.2 mg to about 1.5 mg, about 0.2 mg to about 1.4 mg, about 0.2 mg to about 1.2 mg, about 0.2 mg to about 1.0 mg, about 0.2 mg to about 0.8 mg, about 0.2 mg to about 0.7 mg, about 0.2 mg to about 0.5 mg, about 0.3 mg to about 1.5 mg, about 0.3 mg to about 1.4 mg, about 0.3 mg to about 1.2 mg, about 0.3 mg to about 1.0 mg, about 0.3 mg to about 0.8 mg, about 0.3 mg to about 0.7 mg, about 0.3 mg to about 0.5 mg, about 0.5 mg to about 1.5 mg, about 0.5 mg to about 1.4 mg, about 0.5 mg to about 1.2 mg, about 0.5 mg to about 1.0 mg, about 0.5 mg to about 0.8 mg, about 0.5 mg to about 0.7 mg, about 0.7 mg to about 1.5 mg, about 0.7 mg to about 1.4 mg, about 0.7 mg to about 1.2 mg, about 0.7 mg to about 1.0 mg, about 0.8 mg to about 1.5 mg, about 0.8 mg to about 1.4 mg, about 0.8 mg to about 1.2 mg, about 0.8 mg to about 1.0 mg, about 1.0 mg to about 1.5 mg, about 1.0 mg to about 1.4 mg, about 1.0 mg to about 1.2 mg, about 1.2 mg to about 1.5 mg, or about 1.2 mg to about 1.4 mg of the antisense oligomer.

[0330] In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0. 1 mg, about 0.2 mg, about 0.3 mg, 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, or about 1.5 mg of the antisense oligomer.

[0331] In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition in a volume of about 5 pl to about 250 pl, about 10 pl to about 250 pl, about 20 pl to about 250 pl, about 30 pl to about 250 pl, about 40 pl to about 250 pl, about 50 pl to about 250 pl, about 60 pl to about 250 pl, about 70 pl to about 250 pl, about 80 pl to about 250 pl, about 100 pl to about 250 pl, about 120 pl to about 250 pl, about 150 pl to about 250 pl, about 160 pl to about 250 pl, about 180 pl to about 500 pl, about 200 pl to about 250 pl, about 220 pl to about 250 pl, about 5 pl to about 220 pl, about 10 pl to about 220 pl, about 20 pl to about 220 pl, about 30 pl to about 220 pl, about 40 pl to about 220 pl, about 50 pl to about 220 pl, about 60 pl to about 220 pl, about 70 pl to about 220 pl, about 80 pl to about 220 pl, about 100 pl to about 220 pl, about 120 pl to about 220 pl, about 150 pl to about 220 pl, about 160 pl to about 220 pl, about 180 pl to about 220 pl, about 5 pl to about 200 pl, about 10 pl to about 200 pl, about 20 pl to about 200 pl, about 30 pl to about 200 pl, about 40 pl to about 200 pl, about 50 pl to about 200 pl, about 60 pl to about 200 pl, about 70 pl to about 200 pl, about 80 pl to about 200 pl, about 100 pl to about 200 pl, about 120 pl to about 200 pl, about 150 pl to about 200 pl, about 160 pl to about 200 pl, about 180 pl to about 200 pl, about 5 pl to about 180 pl, about 10 pl to about 180 pl, about 20 pl to about 180 pl, about 30 pl to about 180 pl, about 40 pl to about 180 pl, about 50 pl to about 180 pl, about 60 pl to about 180 pl, about 70 pl to about 180 pl, about 80 pl to about 180 pl, about 100 pl to about 180 pl, about 120 pl to about 180 pl, about 150 pl to about 180 pl, about 5 pl to about 150 pl, about 10 pl to about 150 pl, about 20 pl to about 150 pl, about 30 pl to about 150 pl, about 40 pl to about 150 pl, about 50 pl to about 150 pl, about 60 pl to about 150 pl, about 70 pl to about 150 pl, about 80 pl to about 150 pl, about 100 pl to about 150 pl, about 120 pl to about 150 pl, about 5 pl to about 150 pl, about 10 pl to about 120 pl, about 20 pl to about 120 pl, about 30 pl to about 120 pl, about 40 pl to about 120 pl, about 50 pl to about 120 pl, about 60 pl to about 120 pl, about 70 pl to about 120 pl, about 80 pl to about 120 pl, about 100 pl to about 120 pl, about 5 pl to about 100 pl, about 10 pl to about 100 pl, about 20 pl to about 100 pl, about 30 pl to about 100 pl, about 40 pl to about 100 pl, about 50 pl to about 100 pl, about 60 pl to about 100 pl, about 70 pl to about 100 pl, about 80 pl to about 100 pl, about 5 pl to about 80 pl, about 10 pl to about 80 pl, about 20 pl to about 80 pl, about 30 pl to about 80 pl, about 40 pl to about 80 pl, about 50 pl to about 80 pl, about 60 pl to about 80 pl, about 5 pl to about 60 pl, about 10 pl to about 60 pl, about 20 pl to about 60 pl, about 30 pl to about 60 pl, about 40 pl to about 60 pl, or about 50 pl to about 60 pl.

[0332] In some embodiments, the method comprises administering to the one eye of the subject the pharmaceutical composition in a volume of about 5 pl, about 8 pl, about 10 pl, about 12 pl, about 15 pl, about 18 pl, about 20 pl, about 25 pl, about 28 pl, about 30 pl, about 35 pl, about 40 pl, about 45 pl, about 48 pl, about 50 pl, about 55 pl, about 60 pl, about 65 pl, about 70 pl, about 75 pl, about 80 pl, about 90 pl, about 100 pl, about 120 pl, about 150 pl, about 160 pl, about 180 pl, about 200 pl, about 220 pl, or about 250 pl.

[0333] In some embodiments, the method comprises administering the pharmaceutical composition to both left eye and right eye of the subject. In some embodiments, the method comprises administering the pharmaceutical composition at the same dose to both the left eye and the right eye of the subject. In some embodiments, the method comprises administering the pharmaceutical composition at different doses to the left eye and the right eye of the subject. In some embodiments, the method comprises administering the pharmaceutical composition to at least one eye of the subject.

[0334] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 6-275 or 280-299. In some embodiments, the antisense oligomer consists of a nucleotide sequence having at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 6-275 or 280-299.

[0335] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 280-283, 288, and 290-292. In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 92-96, and 166-168. In some embodiments, the antisense oligomer consists of a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 280-283, 288, and 290-292. In some embodiments, the antisense oligomer consists of a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID Nos: 36, 236, 242, 250, 92-96, and 166-168.

[0336] In some embodiments, administering comprises administering multiple doses of the pharmaceutical composition to the human subject. In some embodiments, administering comprises administering a first dose of the pharmaceutical composition to the human subject and a subsequent dose of the pharmaceutical composition to the human subject. In some embodiments, the subsequent dose is lower than the previous dose following an indication that administration of the previous dose is not tolerated. In some embodiments, the subsequent dose is the same as the previous dose following an indication that administration of the previous dose is tolerated. In some embodiments, the subsequent dose is higher than the previous dose following an indication that administration of the previous dose is tolerated. In some embodiments, the subsequent dose is the same as the previous dose following an indication that administration of the previous dose is effective. In some embodiments, the subsequent dose is lower than the previous dose following an indication that administration of the previous dose is effective. In some embodiments, the subsequent dose is higher than the previous dose following an indication that administration of the previous dose is not effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0. 1 mg, 0.3 mg, 0.5 mg, or 0.7 mg of the antisense oligomer, with an option to add two subsequent doses, each at about 0.1, 0.3, 0.5, or 0.7 mg of the antisense oligomer, capped at a maximum total dosage of about 1.2 mg, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1 mg, 0.3 mg, 0.5 mg, or 0.7 mg of the antisense oligomer, with an option to add two subsequent doses, each at about 0.1 mg, 0.3 mg, 0.5 mg, or 0.7 mg the antisense oligomer, capped at a maximum total dosage of about 1.2 mg, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1 mg, 0.3 mg, 0.5 mg, or 0.7 mg of the antisense oligomer, with no additional subsequent doses, following an indication that administration of the first dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1 mg, 0.3 mg, 0.5 mg, or 0.7 mg of the antisense oligomer, with no additional subsequent doses, following an indication that administration of the first dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1 mg, 0.3 mg, 0.5 mg, or 0.7 mg of the antisense oligomer, with no additional subsequent doses, following an indication that administration of the previous dose is not tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1 mg, 0.3 mg, 0.5 mg, or 0.7 mg of the antisense oligomer, with no additional subsequent doses, following an indication that administration of the previous dose is not effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1 mg, 0.3 mg, 0.5 mg, or 0.7 mg of the antisense oligomer, followed by one subsequent dose of about 0.1 mg, 0.3 mg, 0.5 mg, or 0.7 mg of the antisense oligomer, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1, 0.3, 0.5, or 0.7 mg of the antisense oligomer, followed by one subsequent dose of about 0.1 mg, 0.3 mg, 0.5 mg, or 0.7 mg of the antisense oligomer, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1 mg of the antisense oligomer, followed by two subsequent doses each at about 0. 1 mg, 0.3 mg, 0.5 mg, or 0.7 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1 mg of the antisense oligomer, followed by two subsequent doses each at about 0.1 mg, 0.3 mg, 0.5 mg, or 0.7 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1 mg of the antisense oligomer, followed by two subsequent doses of about 0.1 mg of the antisense oligomer, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0. 1 mg of the antisense oligomer, followed by two subsequent doses of about 0. 1 mg of the antisense oligomer, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1 mg of the antisense oligomer, followed by two subsequent doses of about 0.3 mg of the antisense oligomer, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0. 1 mg of the antisense oligomer, followed by two subsequent doses of about 0.3 mg of the antisense oligomer, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.1 mg of the antisense oligomer, followed by two subsequent doses of about 0.5 mg of the antisense oligomer, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of

I l l about 0. 1 mg of the antisense oligomer, followed by two subsequent doses of about 0.5 mg of the antisense oligomer, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.3 mg of the antisense oligomer, followed by two subsequent doses of about 0.1 mg of the antisense oligomer, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.3 mg of the antisense oligomer, followed by two subsequent doses of about 0.1 mg of the antisense oligomer, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.3 mg of the antisense oligomer, followed by two subsequent doses of about 0.1 mg of the antisense oligomer, following an indication that administration of the first dose is not tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.3 mg of the antisense oligomer, followed by two subsequent doses of about 0.3 mg of the antisense oligomer, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.3 mg of the antisense oligomer, followed by two subsequent doses of about 0.3 mg of the antisense oligomer, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.3 mg of the antisense oligomer, followed by two subsequent doses, each about 0.1 mg, 0.3 mg, 0.5 mg, or 0.7 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.3 mg of the antisense oligomer, followed by two subsequent doses, each about 0.1 mg, 0.3 mg, 0.5 mg, or 0.7 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.5 mg of the antisense oligomer, followed by two subsequent doses, each at about 0. 1 mg, 0.3 mg, or 0.5 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.5 mg of the antisense oligomer, followed by two subsequent doses, each at about 0. 1 mg, 0.3 mg, or 0.5 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.5 mg of the antisense oligomer, followed by two subsequent doses, each at about 0. 1 mg or 0.3 mg of the antisense oligomer, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.5 mg of the antisense oligomer, followed by two subsequent doses, each at about 0. 1 mg or 0.3 mg of the antisense oligomer, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.5 mg of the antisense oligomer, followed by two subsequent doses, each at about 0. 1 mg or 0.3 mg of the antisense oligomer, following an indication that administration of the previous dose is not tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.5 mg of the antisense oligomer, followed by one subsequent dose of about 0.7 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.5 mg of the antisense oligomer, followed by one subsequent dose of about 0.7 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by two subsequent doses, each at about 0. 1 mg or 0.3 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by two subsequent doses, each at about 0. 1 mg or 0.3 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by two subsequent doses, each at about 0. 1 mg or 0.3 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is not tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by two subsequent doses, each at about 0.1, 0.3, or 0.5 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by two subsequent doses, each at about 0. 1 mg, 0.3 mg, or 0.5 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by two subsequent doses, each at about 0. 1 mg, 0.3 mg, or 0.5 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is not effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by one subsequent dose of about 0.1 mg, 0.3 mg, or 0.5 mg of the antisense oligomer, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by one subsequent dose of about 0.1 mg, 0.3 mg, or 0.5 mg of the antisense oligomer, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by one subsequent dose of about 0.1 mg, 0.3 mg, or 0.5 mg of the antisense oligomer, following an indication that administration of the previous dose is not tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by one subsequent dose of about 0.5 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is tolerated. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by one subsequent dose of about 0.5 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is effective. In some embodiments, the pharmaceutical composition is administered at a first dose of about 0.7 mg of the antisense oligomer, followed by one subsequent dose of about 0.5 mg of the antisense oligomer, such that the maximum total dosage is about 1.2 mg, following an indication that administration of the previous dose is not tolerated.

[0337] In some embodiments, the pharmaceutical composition is a liquid composition. In some embodiments, the method comprises administering the pharmaceutical composition as a bolus injection over 1 to 60 minutes, 1 to 50 minutes, 1 to 40 minutes, 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes, 1 to 5 minutes, or 1 to 3 minutes.

[0338] In some embodiments, the method comprises administering the pharmaceutical composition as a bolus injection. In some embodiments, the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, or water for injection. In some embodiments, the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, and water for injection. In some embodiments, the antisense oligomer is solubilized or diluted in an isotonic solution. In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate- buffered solution with at least pH 5.8. In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate-buffered (pH 6.6 - 7.6) solution. In some embodiments, the pharmaceutical formulation does not comprise a preservative.

[0339] In some embodiments, the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml to about 200 mg/ml.

[0340] In some embodiments, the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml to about 200 mg/ml, about 5 mg/ml to about 200 mg/ml, about 10 mg/ml to about 200 mg/ml, about 15 mg/ml to about 200 mg/ml, about 20 mg/ml to about 200 mg/ml, about 25 mg/ml to about 200 mg/ml, about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, 2 mg/ml to about 150 mg/ml, about 5 mg/ml to about 150 mg/ml, about 10 mg/ml to about 150 mg/ml, about 15 mg/ml to about 150 mg/ml, about 20 mg/ml to about 150 mg/ml, about 25 mg/ml to about 150 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, 2 mg/ml to about 100 mg/ml, about 5 mg/ml to about 100 mg/ml, about 10 mg/ml to about 100 mg/ml, about 15 mg/ml to about 100 mg/ml, about 20 mg/ml to about 100 mg/ml, about 25 mg/ml to about 100 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, 2 mg/ml to about 80 mg/ml, about 5 mg/ml to about 80 mg/ml, about 10 mg/ml to about 80 mg/ml, about 15 mg/ml to about 80 mg/ml, about 20 mg/ml to about 80 mg/ml, about 25 mg/ml to about 80 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, 2 mg/ml to about 60 mg/ml, about 5 mg/ml to about 60 mg/ml, about 10 mg/ml to about 60 mg/ml, about 15 mg/ml to about 60 mg/ml, about 20 mg/ml to about 60 mg/ml, about 25 mg/ml to about 60 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, about 40 mg/ml to about 60 mg/ml, 2 mg/ml to about 40 mg/ml, about 5 mg/ml to about 40 mg/ml, about 10 mg/ml to about 40 mg/ml, about 15 mg/ml to about 40 mg/ml, about 20 mg/ml to about 40 mg/ml, or about 25 mg/ml to about 40 mg/ml. [0341] In some embodiments, the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 12 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 18 mg/ml, about 20 mg/ml, about 22 mg/ml, about 24 mg/ml, about 26 mg/ml, about 28 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 80 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml. Autosomal dominant optic atrophy (ADOA) is the most common inherited optic nerve disorder and is characterized by retinal ganglion cell loss. In some cases, 65-90% of ADOA cases are caused by mutations in one allele of the OPA1 gene. OPA1 gene encodes an OPA1 protein that is a mitochondrial GTPase, which can have a critical maintenance role in mitochondria structure and function. Most OPA1 mutations can lead to a haploinsufficiency, resulting in about a 50% decrease of normal OPA1 protein levels. Approximately 1 out of 30,000 people are affected globally with a higher incidence of ~1 out of 10,000 in Denmark due to a founder effect. ADOA can present within the first decade of life. 80% of ADOA patients are symptomatic before 10 years of age. The disease can cause progressive and irreversible vision loss and up to 46% of patients are registered as legally blind. [0342] In some embodiments, the method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition comprises administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NO: 6-275 or 280-299, wherein the disease or condition comprises autosomal dominant optic atrophy (ADOA), and wherein the subject is characterized by having: (i) a heterozygous OPA1 gene variant; (ii) clear ocular media to allow for adequate visualization of the vitreous and fundus and to achieve appropriate quality of all ophthalmic assessments; (iii) an AREDS Clinical Lens Standard of <1 for posterior subcapsular (PSC) opacity; (iv) a BCVA EDTRS letter score of >35 and <70 with each eye individually that is administered with the pharmaceutical composition; or (v) any combination of (i)-(iv) .

[0343] In some embodiments, the method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition comprises administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 90% sequence identity to the sequence set forth in any one of SEQ ID NO: 6-275 or 280-299, wherein the disease or condition comprises autosomal dominant optic atrophy (ADOA), and wherein the subject is characterized by having: (i) a heterozygous OPA1 gene variant; (ii) clear ocular media to allow for adequate visualization of the vitreous and fundus and to achieve appropriate quality of all ophthalmic assessments; (iii) an AREDS Clinical Lens Standard of <1 for posterior subcapsular (PSC) opacity; (iv) a BCVA EDTRS letter score of >35 and <70 with each eye individually that is administered with the pharmaceutical composition; or (v) any combination of (i)-(iv) .

[0344] In some embodiments, the method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition comprises administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 100% sequence identity to the sequence set forth in any one of SEQ ID NO: 6- 275 or 280-299, wherein the disease or condition comprises autosomal dominant optic atrophy (ADOA), and wherein the subject is characterized by having: (i) a heterozygous OPA1 gene variant; (ii) clear ocular media to allow for adequate visualization of the vitreous and fundus and to achieve appropriate quality of all ophthalmic assessments; (iii) an AREDS Clinical Lens Standard of <1 for posterior subcapsular (PSC) opacity; (iv) a BCVA EDTRS letter score of >35 and <70 with each eye individually that is administered with the pharmaceutical composition; or (v) any combination of (i)-(iv) .

[0345] In some embodiments, the method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition comprises administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NO: 6-275 or 280-299, wherein the disease or condition comprises autosomal dominant optic atrophy (ADOA), and wherein the subject is characterized by: (1) not having a gain-of-function variant, or compound heterozygous or homozygous pathogenic or likely pathogenic variant in OP Al gene; (2) not having only benign or likely benign variants in the OPA1 gene; (3) not having extraocular phenotypic manifestations of (syndromic) ADOA (ADOA-plus); (4) not having been diagnosed with Behr syndrome; (5) not having a known pathogenic mutation in another gene implicated in optic atrophy or retinal diseases; (6) not having diabetic retinopathy with potential for development of proliferative diabetic retinopathy, diabetic macular edema, or optic neuropathy; (7) not having or having a history of any ocular condition in either eye; (8) not having a history of intraocular surgery or corneal surgery including refractive surgery in either eye within 12 weeks prior to the administering; (9) not having a history of retinal photocoagulation; (10) not having a history or presence of retinal vein occlusion; (11) not being considered to be at risk for uveitis or ocular infection because of having any of the following: an active flare of non-infectious uveitis or an episode of infectious uveitis or other ocular infection in either eye within 12 months prior to the administering; (12) not having dry age-related macular degeneration in either eye; (13) not having high myopia (>6 diopters); (14) not having a history of cancer (except a diagnosis of basal cell, squamous cell skin cancer, or carcinoma in situ of the cervix that has been successfully treated); (15) not taking, or having taken at any time, any medication or treatment that can or might cause an optic neuropathy; (16) not having any history of nutritional deficiency (including B12 and/or folate deficiencies), or not having a known deficiency in serum B12 or folate, not having had bariatric surgery; or (17) any combination of (1)-(16).

[0346] In some embodiments, the method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition comprises administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 90% sequence identity to the sequence set forth in any one of SEQ ID NO: 6-275 or 280-299, wherein the disease or condition comprises autosomal dominant optic atrophy (ADOA), and wherein the subject is characterized by: (1) not having a gain-of-function variant, or compound heterozygous or homozygous pathogenic or likely pathogenic variant in OP Al gene; (2) not having only benign or likely benign variants in the OPA1 gene; (3) not having extraocular phenotypic manifestations of (syndromic) ADOA (ADOA-plus); (4) not having been diagnosed with Behr syndrome; (5) not having a known pathogenic mutation in another gene implicated in optic atrophy or retinal diseases; (6) not having diabetic retinopathy with potential for development of proliferative diabetic retinopathy, diabetic macular edema, or optic neuropathy; (7) not having or having a history of any ocular condition in either eye; (8) not having a history of intraocular surgery or corneal surgery including refractive surgery in either eye within 12 weeks prior to the administering; (9) not having a history of retinal photocoagulation; (10) not having a history or presence of retinal vein occlusion; (11) not being considered to be at risk for uveitis or ocular infection because of having any of the following: an active flare of non-infectious uveitis or an episode of infectious uveitis or other ocular infection in either eye within 12 months prior to the administering; (12) not having dry age-related macular degeneration in either eye; (13) not having high myopia (>6 diopters); (14) not having a history of cancer (except a diagnosis of basal cell, squamous cell skin cancer, or carcinoma in situ of the cervix that has been successfully treated); (15) not taking, or having taken at any time, any medication or treatment that can or might cause an optic neuropathy; (16) not having any history of nutritional deficiency (including B12 and/or folate deficiencies), or not having a known deficiency in serum B12 or folate, not having had bariatric surgery; or (17) any combination of (1)-(16).

[0347] In some embodiments, the method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition comprises administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 100% sequence identity to the sequence set forth in any one of SEQ ID NO: 6- 275 or 280-299, wherein the disease or condition comprises autosomal dominant optic atrophy (ADOA), and wherein the subject is characterized by: (1) not having a gain-of-function variant, or compound heterozygous or homozygous pathogenic or likely pathogenic variant in OP Al gene; (2) not having only benign or likely benign variants in the OPA1 gene; (3) not having extraocular phenotypic manifestations of (syndromic) ADOA (ADOA-plus); (4) not having been diagnosed with Behr syndrome; (5) not having a known pathogenic mutation in another gene implicated in optic atrophy or retinal diseases; (6) not having diabetic retinopathy with potential for development of proliferative diabetic retinopathy, diabetic macular edema, or optic neuropathy; (7) not having or having a history of any ocular condition in either eye; (8) not having a history of intraocular surgery or corneal surgery including refractive surgery in either eye within 12 weeks prior to the administering; (9) not having a history of retinal photocoagulation; (10) not having a history or presence of retinal vein occlusion; (11) not being considered to be at risk for uveitis or ocular infection because of having any of the following: an active flare of non-infectious uveitis or an episode of infectious uveitis or other ocular infection in either eye within 12 months prior to the administering; (12) not having dry age-related macular degeneration in either eye; (13) not having high myopia (>6 diopters); (14) not having a history of cancer (except a diagnosis of basal cell, squamous cell skin cancer, or carcinoma in situ of the cervix that has been successfully treated); (15) not taking, or having taken at any time, any medication or treatment that can or might cause an optic neuropathy; (16) not having any history of nutritional deficiency (including B12 and/or folate deficiencies), or not having a known deficiency in serum B12 or folate, not having had bariatric surgery; or (17) any combination of (1)-(16).

[0348] In some cases, a therapeutic agent comprises an oligonucleotide. In some cases, a therapeutic agent comprises a vector, e.g., a viral vector, expressing a oligonucleotide that binds to the targeted region of a pre-mRNA the encodes the target peptide sequence. The methods provided herein can be adapted to contacting a vector that encodes an agent, e.g., an oligonucleotide, to a cell, so that the agent binds to a pre-mRNA in the cell and modulates the processing of the pre-mRNA. In some cases, the viral vector comprises an adenoviral vector, adeno-associated viral (AAV) vector, lentiviral vector, Herpes Simplex Virus (HSV) viral vector, retroviral vector, or any applicable viral vector. In some cases, a therapeutic agent comprises a gene editing tool that is configured to modify a gene encoding the target peptide sequence such that a gene region that encodes the inefficient translation region is deleted. In some cases, a gene editing tool comprises vector, e.g., viral vector, for gene editing based on CRISPR-Cas9, TALEN, Zinc Finger, or other applicable technologies.

[0349] Suitable routes for administration of ASOs of the present disclosure may vary depending on cell type to which delivery of the ASOs is desired. Multiple tissues and organs are affected by ADOA, with the eye being the most significantly affected tissue. The ASOs of the present disclosure may be administered to patients parenterally, for example, by intravitreal injection, intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

[0350] In some embodiments, the pharmaceutical composition is administered via intravitreal injection. In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a small molecule. In some embodiments, the additional therapeutic agent comprises an antisense oligomer. In some embodiments, the additional therapeutic agent comprises an ophthalmologic drug. In some embodiments, the additional therapeutic agent consists of an antisense oligomer. In some embodiments, the additional therapeutic agent consists of an ophthalmologic drug.

[0351] In embodiments, the antisense oligonucleotide is administered with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier by any method known in the art. For example, delivery of agents by administration of an adenovirus vector to motor neurons in muscle tissue is described in U.S. Pat. No. 6,632,427, “Adenoviral-vector-mediated gene transfer into medullary motor neurons,” incorporated herein by reference. Delivery of vectors directly to the brain, e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra, is described, e.g., in U.S. Pat. No. 6,756,523, “Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain,” incorporated herein by reference.

[0352] In some embodiments, the antisense oligonucleotides are linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. In embodiments, the antisense oligonucleotide is coupled to a substance, known in the art to promote penetration or transport across the blood-brain barrier, e.g., an antibody to the transferrin receptor. In embodiments, the antisense oligonucleotide is linked with a viral vector, e.g., to render the antisense compound more effective or increase transport across the blood-brain barrier. In embodiments, osmotic blood brain barrier disruption is assisted by infusion of sugars, e.g., meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, U(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, U(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol, D(+) arabitol, U(-) arabitol, D(+) fucose, U(-) fucose, D(-) lyxose, U(+) lyxose, and U(-) lyxose, or amino acids, e.g., glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine. Methods and materials for enhancing blood brain barrier penetration are described, e.g., in U.S. Pat. No. 9,193,969, “Compositions and methods for selective delivery of oligonucleotide molecules to specific neuron types,” U.S. Pat. No. 4,866,042, “Method for the delivery of genetic material across the blood brain barrier,” U.S. Pat. No. 6,294,520, “Material for passage through the blood-brain barrier,” and U.S. Pat. No. 6,936,589, “Parenteral delivery systems,” each incorporated herein by reference.

[0353] In some embodiments, subjects treated using the methods and compositions are evaluated for improvement in condition using any methods known and described in the art.

Methods of Identifying Additional ASOs that Induce Exon Skipping

[0354] Also within the scope of the present disclosure are methods for identifying or determining ASOs that induce exon skipping of an OP Al NMD exon-containing pre-mRNA. For example, a method can comprise identifying or determining ASOs that induce pseudo-exon skipping of an OPA1 NMD exoncontaining pre-mRNA. ASOs that specifically hybridize to different nucleotides within the target region of the pre-mRNA may be screened to identify or determine ASOs that improve the rate and/or extent of splicing of the target intron. In some embodiments, the ASO may block or interfere with the binding site(s) of a splicing repressor(s)/silencer. Any method known in the art may be used to identify (determine) an ASO that when hybridized to the target region of the exon results in the desired effect (e.g., pseudo-exon skipping, protein or functional RNA production). These methods also can be used for identifying ASOs that induce exon skipping of the included exon by binding to a targeted region in an intron flanking the included exon, or in a non-included exon. An example of a method that may be used is provided below.

[0355] A round of screening, referred to as an ASO “walk” may be performed using ASOs that have been designed to hybridize to a target region of a pre-mRNA. For example, the ASOs used in the ASO walk can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 3’ splice site of the included exon (e.g., a portion of sequence of the exon located upstream of the target/included exon) to approximately 100 nucleotides downstream of the 3’ splice site of the target/included exon and/or from approximately 100 nucleotides upstream of the 5’ splice site of the included exon to approximately 100 nucleotides downstream of the 5’ splice site of the target/included exon (e.g., a portion of sequence of the exon located downstream of the target/included exon). For example, a first ASO of 15 nucleotides in length may be designed to specifically hybridize to nucleotides +6 to +20 relative to the 3 ’ splice site of the target/included exon. A second ASO may be designed to specifically hybridize to nucleotides +11 to +25 relative to the 3’ splice site of the target/included exon. ASOs are designed as such spanning the target region of the pre-mRNA. In embodiments, the ASOs can be tiled more closely, e.g., every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled from 100 nucleotides downstream of the 5’ splice site, to 100 nucleotides upstream of the 3’ splice site. In some embodiments, the ASOs can be tiled from about 1,160 nucleotides upstream of the 3’ splice site, to about 500 nucleotides downstream of the 5’ splice site. In some embodiments, the ASOs can be tiled from about 500 nucleotides upstream of the 3’ splice site, to about 1,920 nucleotides downstream of the 3’ splice site.

[0356] One or more ASOs, or a control ASO (an ASO with a scrambled sequence, sequence that is not expected to hybridize to the target region) are delivered, for example by transfection, into a diseaserelevant cell line that expresses the target pre-mRNA (e.g., an NMD exon-containing pre-mRNA described herein). The exon skipping effects of each of the ASOs may be assessed by any method known in the art, for example, by reverse transcriptase (RT)-PCR using primers that span the splice junction, as described in Example 4. A reduction or absence of a longer RT-PCR product produced using the primers spanning the region containing the included exon (e.g., including the flanking exons of the NMD exon) in ASO-treated cells as compared to in control ASO-treated cells indicates that splicing of the target NMD exon has been enhanced. In some embodiments, the exon skipping efficiency (or the splicing efficiency to splice the intron containing the NMD exon), the ratio of spliced to unspliced pre-mRNA, the rate of splicing, or the extent of splicing may be improved using the ASOs described herein. The amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced functional protein production). Any method known in the art for assessing and/or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.

[0357] A second round of screening, referred to as an ASO “micro-walk” may be performed using ASOs that have been designed to hybridize to a target region of a pre-mRNA. The ASOs used in the ASO micro-walk are tiled every 1 nucleotide to further refine the nucleotide acid sequence of the pre-mRNA that when hybridized with an ASO results in exon skipping (or enhanced splicing of NMD exon).

[0358] Regions defined by ASOs that promote splicing of the target intron are explored in greater detail by means of an ASO “micro-walk,” involving ASOs spaced in 1-nt steps, as well as longer ASOs, typically 18-25 nt.

[0359] As described for the ASO walk above, the ASO micro-walk is performed by delivering one or more ASOs, or a control ASO (an ASO with a scrambled sequence, sequence that is not expected to hybridize to the target region), for example, by transfection, into a disease-relevant cell line that expresses the target pre-mRNA. The splicing-inducing effects of each of the ASOs may be assessed by any method known in the art, for example, by reverse transcriptase (RT)-PCR using primers that span the NMD exon, as described herein (see, e.g, Example 4). A reduction or absence of a longer RT-PCR product produced using the primers spanning the NMD exon in ASO-treated cells as compared to in control ASO-treated cells indicates that exon skipping (or splicing of the target intron containing an NMD exon) has been enhanced. In some embodiments, the exon skipping efficiency (or the splicing efficiency to splice the intron containing the NMD exon), the ratio of spliced to unspliced pre-mRNA, the rate of splicing, or the extent of splicing may be improved using the ASOs described herein. The amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced functional protein production). Any method known in the art for assessing and/or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.

[0360] ASOs that when hybridized to a region of a pre-mRNA result in exon skipping (or enhanced splicing of the intron containing an NMD exon) and increased protein production may be tested in vivo using animal models, for example, transgenic mouse models in which the full-length human gene has been knocked-in or in humanized mouse models of disease. Suitable routes for administration of ASOs may vary depending on the disease and/or the cell types to which delivery of the ASOs is desired. ASOs may be administered, for example, by intravitreal injection, intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. Following administration, the cells, tissues, and/or organs of the model animals may be assessed to determine the effect of the ASO treatment by, for example, evaluating splicing (e.g., efficiency, rate, extent) and protein production by methods known in the art and described herein. The animal models may also be any phenotypic or behavioral indication of the disease or disease severity. [0361] Also within the scope of the present disclosure is a method to identify or validate an NMD- inducing exon in the presence of an NMD inhibitor, for example, cycloheximide. An exemplary method is provided in Example 2.

Flavoprotein Fluorescence Analysis, Processing, and Correlation with ADOA

[0362] Also within the scope of the present disclosure are methods for correlating mitochondrial flavoprotein fluorescence, which is measured as average flavoprotein fluorescence (FPF) intensity in one eye, with eye condition in ADOA. Also within the scope of the present disclosure are methods for correlating mitochondrial flavoprotein fluorescence, which is measured as average flavoprotein fluorescence (FPF) intensity in one eye, with mitochondrial dysfunction in ADOA. Also within the scope of the present disclosure are methods for correlating mitochondrial flavoprotein fluorescence, which is output as an flavoprotein fluorescence (FPF) intensity score in one eye, with mitochondrial dysfunction in ADOA. In some embodiments, mitochondrial flavoprotein fluorescence is output as an FPF score. In some embodiments, mitochondrial flavoprotein fluorescence is produced by proteins in the mitochondria that utilize prosthetic groups or cofactors such as flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD). In some embodiments, mitochondrial flavoprotein fluorescence is produced by flavoproteins such as, but not limited to, adrenodoxin reductase, cytochrome P450 reductase, epidermin biosynthesis protein (EpiD), the B chain of dipicolinate synthase, dipicolinate synthase, phenylacrylic acid decarboxylase, phototrophin, cryptochrome, lactate oxidase (1-lactate: oxygen oxidoreductase), pyruvate oxidase (pyruvate: oxygen oxidoreductase (phosphorylating)), xanthine oxidaseelectron transfer flavoprotein (ETF), or ETF-ubiquinone oxidoreductase (ETF-QO), or any combination thereof.

[0363] In some embodiments, the flavoprotein fluorescence (FPF) intensity score is determined from at least a measurement of FPF intensity. In some embodiments, the flavoprotein fluorescence (FPF) intensity score is further determined from at least one parameter selected from the group consisting of Best Corrected Visual Acuity (BCVA) letter score; Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score; high-contrast Best Corrected Visual Acuity (HC BCVA) letter score; low-contrast Best Corrected Visual Acuity (LC BCVA) letter score; Humphrey 10-2 Visual Field test Mean Deviation (MD); Humphrey 10-2 Visual Field test Pattern Standard Deviation (PSD); Visual Acuity Score (VAS); Quality of life questionnaires (NEI-VFQ-25, IVI-C, EQ- 5D, EQ-5D-Y); Refraction test result; Minnesota Reading (MNRead) acuity chart score; Pelli-Robson chart score; slit lamp examination result; intraocular pressure using a Tonopen; perimetry; dilated fundoscopy result; retinal nerve fiber layer (RNFL) measurement; optical coherence tomography (OCT) result; and macular ganglion cell layer/inner plexiform layer (GCL/IPL) thickness measurements; Curve Width (CW) measurement; Electroretinogram (ERG) result; and fundus photography result. In some embodiments, the measurement of FPF intensity is an average pixel intensity over a Region of Interest (ROI) of a detectable signal emitted in response to exposure to at least one excitation flash for an amount of exposure time. In some embodiments, the detectable signal is a fluorescent signal. In some embodiments, the fluorescent signal comprises green light comprising a wavelength or combination of wavelengths between about 520 nm and about 540 nm. In some embodiments, the excitation flash comprises blue light comprising a wavelength or combination of wavelengths between about 430 nm and about 470 nm. In some embodiments, the excitatory flash comprises blue light comprising a wavelength or a combination of wavelengths of about 430, 435, 440, 445, 450, 455, 460, 465, or 470 nm. In some embodiments, the excitatory flash comprises blue light consisting of a wavelength or a combination of wavelengths of about 430, 435, 440, 445, 450, 455, 460, 465, or 470 nm. In some embodiments, the excitatory flash comprises blue light comprising a wavelength of about 465 nm. In some embodiments, the excitatory flash comprises blue light consisting of a wavelength of about 465 nm. In some embodiments, the fluorescent signal comprising green light comprises a wavelength or a combination of wavelengths of about 520, 525, 530, 535, or 540 nm. In some embodiments, the fluorescent signal comprising green light consists of a wavelength or a combination of wavelengths of about 520, 525, 530, 535, or 540 nm.

[0364] Flavoproteins in mitochondria have been found to emit green light when stimulated with cobalt blue light. Flavoprotein fluorescence (FPF) intensity can be read by any instrument capable of producing excitatory blue light comprising a wavelength or a combination of wavelengths between about 430 nm and about 470 nm, and detecting light emitted from a sample (a readout) at a wavelength or combination of wavelengths between about 520 nm and 540 nm resulting from excitation by the blue light stimulation. In some embodiments, the excitatory blue light comprises a wavelength or a combination of wavelengths of about 430, 435, 440, 445, 450, 455, 460, 465, or 470 nm. In some embodiments, the excitatory blue light consists of a wavelength or a combination of wavelengths of about 430, 435, 440, 445, 450, 455, 460, 465, or 470 nm. In some embodiments, the excitatory blue light comprises a wavelength of about 465 nm. In some embodiments, the excitatory blue light consists of a wavelength of about 465 nm. In some embodiments, the emitted green light comprises a wavelength or a combination of wavelengths of about 520, 525, 530, 535, or 540 nm. In some embodiments, the emitted green light consists of a wavelength or a combination of wavelengths of about 520, 525, 530, 535, or 540 nm. The instrument can be referred to as the FPF instrument. In some embodiments, the instrument can be an ophthalmoscope, a modified camera, a confocal infrared scanner, or a metabolic detector. In some embodiments, the instrument is the OcuMet Beacon (OcuSciences, Ann Arbor, MI) device. In some embodiments, the FPF instrument comprises an excitation band-pass filter tuned to 465 nm and an emission band-pass filter adjusted to enhance retinal FPF and minimize contaminating signals from other ocular fluorophores. In some embodiments, the ocular fluorophores may be retinal fluorophores. In some embodiments, retinal fluorophores can comprise lipofuscin. In some embodiments, the FPF instrument comprises optical filters that maximize the signal-to-noise ratio and minimize background or confounding fluorescence from other ocular fluorophores.

[0365] In some embodiments, the measurement of FPF intensity is an average pixel intensity over a Region of Interest (ROI) of a detectable signal emitted in response to exposure to at least one excitation flash for an amount of exposure time. In some embodiments, a region of interest (ROI) in an eye of a subject is stimulated with an excitation flash for an amount of exposure time. In some embodiments, a detectable signal produced by the ROI in the eye of the subject in response to the excitation flash is measured. In some embodiments, the detectable signal is a fluorescent signal. In some embodiments, the detectable signal is emitted green light. In some embodiments, an instrument stimulates a region of interest (ROI) in an eye of a subject with an excitation flash for an amount of exposure time. In some embodiments, an instrument measures a detectable signal produced by the ROI in the eye of the subject in response to the excitation flash. In some embodiments, the instrument stimulates a region of interest (ROI) in an eye of a subject with an excitation flash for an amount of exposure time and measures a detectable signal produced by the ROI in the eye of the subject in response to the excitation flash. In some embodiments, the detectable signal is a fluorescent signal. In some embodiments the fluorescent signal is green light comprising a wavelength or a combination of wavelengths between about 520 nm and about 540 nm. In some embodiments, the excitation flash comprises blue light comprising a wavelength or combination of wavelengths between about 430 nm and about 470 nm. In some embodiments, the excitation flash comprises blue light comprising a wavelength of about 465 nm. In some embodiments, the excitation flash consists of blue light comprising a wavelength of about 465 nm. In some embodiments, the region of interest comprises at least one region from the group consisting of a macular- papillary (Mac) retinal nerve fiber layer (RNFL); an ocular global macula; an ocular Superior Nasal sector; an ocular Inferior Nasal sector; an ocular Nasal sector; an ocular Superior Temporal sector; an ocular Inferior Temporal sector; an ocular Temporal sector; and an ocular peripapillary retinal nerve fiber layer (pRNFL). In some embodiments, the region of interest comprises the global macula, the ocular Temporal Inferior sector, the ocular Temporal Superior sector, or the ocular Temporal sector, or any combination thereof. In some embodiments, the region of interest comprises the ocular Temporal Inferior sector. In some embodiments, the region of interest comprises the ocular Temporal Superior sector. In some embodiments, the region of interest comprises the ocular Temporal sector. In some embodiments, the region of interest comprises the global macula, the ocular Nasal Inferior sector, the ocular Nasal Superior sector, or the ocular Nasal sector, or any combination thereof. In some embodiments, the region of interest comprises the global macula, the ocular Nasal Inferior sector, or the ocular Nasal sector, or any combination thereof. In some embodiments, the region of interest comprises the ocular Nasal Inferior sector. In some embodiments, the region of interest comprises the ocular Nasal Superior sector. In some embodiments, the region of interest comprises the ocular Nasal sector. In some embodiments, the instrument comprises a confocal infrared scanner that captures an infrared image in a region of interest (ROI) in an eye, and a metabolic detector that enhances wavelengths near the emission peak of FPF (between about 520 nm and about 540 nm) upon stimulation of the eye with the excitatory wavelengths (between about 430 nm and about 470 nm) and produces a corresponding FPF heatmap that colors areas of increased mitochondrial dysfunction in the eye. In some embodiments, the instrument can at least measure the FPF intensity of an eye of an ADOA patient upon stimulation of the eye with the excitatory wavelengths (between about 430 nm and about 470 nm). In some embodiments, the instrument measures FPF intensity using grayscale units (GSU). In some embodiments, the instrument calculates FPF intensity using decibel grayscale units (dB GSU). In some embodiments, the instrument can measure the FPF intensity of an eye of an ADOA patient upon stimulation of the eye with the excitatory wavelengths (between about 430 nm and about 470 nm) and produce at least a colorized image of the FPF intensity of an eye of the ADOA patient. In some embodiments, the instrument that measures FPF intensity upon stimulation of the eye with the excitatory wavelengths (between about 430 nm and about 470 nm) can also calculate an FPF score based on the average pixel intensity of the FPF readout. In some embodiments, the instrument produces an FPF score based on the FPF intensity measured in decibel grayscale units (dB GSU). In some embodiments, the instrument produces an FPF score based on the FPF intensity measured in grayscale units (GSU).

[0366] In some embodiments, the measurement of FPF intensity is derived from contacting an eye of the subject for an amount of exposure time with an excitation flash. In some embodiments, a region of the eye is exposed to an amount of time of excitation flash (wavelength between about 430 nm and about 470 run). In some embodiments, the amount of time is an amount of exposure time. In some embodiments, a region of the eye is exposed to between about 1 and 100 ms of excitation flash (wavelength between about 430 nm and about 470 nm). In some embodiments, a region of the eye is exposed to at least between about 1 and 100 ms of excitation flash (wavelength between about 430 nm and about 470 nm). In some embodiments, a region of the eye is exposed to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, or 100 ms of excitation flash (wavelength between about 430 nm and about 470 nm). In some embodiments, a region of the eye is exposed to about 10 ms of excitation flash (wavelength between about 430 nm and about 470 nm). In some embodiments, a region of the eye is exposed to about 20 ms of excitation flash (wavelength between about 430 nm and about 470 nm). In some embodiments, a region of the eye is exposed to about 30 ms of excitation flash (wavelength between about 430 nm and about 470 nm). In some embodiments, a region of the eye is exposed to about 40 ms of excitation flash (wavelength between about 430 nm and about 470 nm). In some embodiments, a region of the eye is exposed to about 50 ms of excitation flash (wavelength between about 430 nm and about 470 nm). In some embodiments, a region of the eye is exposed to about 60 ms of excitation flash (wavelength between about 430 nm and about 470 nm). In some embodiments, a region of the eye is exposed to about 100 ms of excitation flash (wavelength between about 430 nm and about 470 nm). In some embodiments, a region of the eye is exposed to at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, or 100 ms of excitation flash (wavelength between at least about 430 nm and at least about 470 nm). In some embodiments, a region of the eye is exposed to at least about 10 ms of excitation flash (wavelength between at least about 430 nm and at least about 470 nm). In some embodiments, a region of the eye is exposed to at least about 30 ms of excitation flash (wavelength between at least about 430 nm and at least about 470 nm). In some embodiments, a region of the eye is exposed to at least about 60 ms of excitation flash (wavelength between at least about 430 nm and at least about 470 nm). In some embodiments, a region of the eye is exposed to at least about 100 ms of excitation flash (wavelength between at least about 430 nm and at least about 470 nm). In some embodiments, a region of the eye is exposed to at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, or 100 ms of excitation flash (wavelength between at most about 430 nm and at most about 470 nm). In some embodiments, a region of the eye is exposed to at most about 10 ms of excitation flash (wavelength between at most about 430 nm and at most about 470 nm). In some embodiments, a region of the eye is exposed to at most about 30 ms of excitation flash (wavelength between at most about 430 nm and at most about 470 nm). In some embodiments, a region of the eye is exposed to at most about 60 ms of excitation flash (wavelength between at most about 430 nm and at most about 470 nm). In some embodiments, a region of the eye is exposed to at most about 100 ms of excitation flash (wavelength between at most about 430 nm and at most about 470 nm).

[0367] In some embodiments, a region of the eye is exposed to at least one instance of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time. In some embodiments, a region of the eye is exposed to one instance of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time. In some embodiments, a region of the eye is exposed to more than one instance of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced a valid FPF readout. In some embodiments, a region of the eye is exposed to more than one instance of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced an invalid FPF readout. In some embodiments, a region of the eye is exposed to at least two instances of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced a valid FPF readout. In some embodiments, a region of the eye is exposed to at least two instances of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced an invalid FPF readout. In some embodiments, a region of the eye is exposed to at most two instances of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced a valid FPF readout. In some embodiments, a region of the eye is exposed to at most two instances of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced an invalid FPF readout. In some embodiments, a region of the eye is exposed to at least three instances of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced a valid FPF readout. In some embodiments, a region of the eye is exposed to at least three instances of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced an invalid FPF readout. In some embodiments, a region of the eye is exposed to at most three instances of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced a valid FPF readout. In some embodiments, a region of the eye is exposed to at most three instances of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced an invalid FPF readout. In some embodiments, a region of the eye is exposed to at least four instances of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced a valid FPF readout. In some embodiments, a region of the eye is exposed to at least four instances of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced an invalid FPF readout. In some embodiments, a region of the eye is exposed to at most four instances of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced a valid FPF readout. In some embodiments, a region of the eye is exposed to at most four instances of excitation flash (wavelength between about 430 nm and about 470 nm) for an amount of time, following an indication that the previous instance of excitation flash produced an invalid FPF readout. A valid FPF readout comprises minimally noisy FPF readouts, or many non-negative FPF intensity values. An invalid FPF readout comprises no FPF readout, noisy FPF readout, or many negative FPF intensity values. In some embodiments, an eye is exposed to at least two sequential exposures of excitation flash (wavelength between about 430 nm and about 470 nm) in one examination session. In some embodiments, an eye is exposed to at least three sequential exposures of excitation flash (wavelength between about 430 nm and about 470 nm) in one examination session. In some embodiments, an eye is exposed to at least four sequential exposures of excitation flash (wavelength between about 430 nm and about 470 nm) in one examination session.

[0368] In some embodiments, an eye of an ADOA patient may be in a worse disease state than the other. In some embodiments, an eye of an ADOA patient may be in a better disease state than the other In some embodiments, both eyes of an ADOA patient may have the same disease state. In some embodiments, both eyes of an ADOA patient may have a similar disease state.

[0369] In some embodiments, the FPF score for an eye is calculated from one FPF readout. In some embodiments, the FPF score for an eye is calculated from at least one FPF readout. In some embodiments, the FPF score for an eye is calculated from at least two FPF readouts. In some embodiments, the FPF score for an eye is calculated from at least three FPF readouts. In some embodiments, the FPF score for an eye is calculated from at least four FPF readouts.

Correlations Between FPF Intensity Score and Eye Condition or Disease in ADOA

[0370] In some embodiments, an FPF intensity score of between about 20 and 35 for a subject between the ages of 21 and about 30 corresponds to a normal eye state in a healthy control subject. In some embodiments, an FPF intensity score of between about 35 and about 45 for a subject between the ages of about 31 and 40 corresponds to a normal eye state in a healthy control subject. In some embodiments, an FPF intensity score of between about 46 and about 55 for a subject between the ages of about 41 and 50 corresponds to a normal eye state in a healthy control subject. In some embodiments, an FPF intensity score of between about 56 and about 69 for a subject between the ages of about 51 and 60 corresponds to a normal eye state in a healthy control subject.

[0371] In some embodiments, an eye condition or disease is ADOA. In some embodiments, the eye condition of a subject with ADOA is an ADOA disease state. In some embodiments, an FPF intensity score is correlated with age. In some embodiments, an FPF intensity score is positively correlated with age. In some embodiments, an FPF intensity score is negatively correlated with age. In some embodiments, a high FPF intensity score is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state older than about 40.

[0372] In some embodiments, an eye condition or disease is ADOA. In some embodiments, the eye condition of a subject with ADOA is an ADOA disease state. In some embodiments, the FPF intensity score in the global optic disc of a subject is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the global optic disc of a subject is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the global optic disc of a subject is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the global optic disc of a subject is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the global optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the global optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the global optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the global optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the global optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the global optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the global optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the global optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state older than about 40.

[0373] In some embodiments, an eye condition or disease is ADOA. In some embodiments, the eye condition of a subject with ADOA is an ADOA disease state. In some embodiments, the FPF intensity score in the Temporal sector of a subject is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal sector of a subject is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal sector of a subject is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state older than about 40.

[0374] In some embodiments, an eye condition or disease is ADOA. In some embodiments, the eye condition of a subject with ADOA is an ADOA disease state. In some embodiments, the FPF intensity score in the Temporal Inferior sector of a subject is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal Inferior sector of a subject is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal Inferior sector of a subject is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state older than about 40.

[0375] In some embodiments, the FPF intensity score in the Temporal Superior sector of a subject is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal Superior sector of a subject is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal Superior sector of a subject is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal Superior sector of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal Superior sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal Superior sector of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Temporal Superior sector of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Temporal Superior sector of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Temporal Superior sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal Superior sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Temporal Superior sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Temporal Superior sector of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Temporal Superior sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Superior sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Superior sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Superior sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Temporal Superior sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Temporal Superior sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state older than about 40.

[0376] In some embodiments, an eye condition or disease is ADOA. In some embodiments, the eye condition of a subject with ADOA is an ADOA disease state. In some embodiments, the FPF intensity score in the Macula of a subject is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Macula of a subject is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Macula of a subject is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Macula of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Macula of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Macula of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Macula of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Macula of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Macula of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Macula of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Macula of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Macula of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Macula of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Macula of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Macula of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Macula of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Macula of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Macula of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state older than about 40.

[0377] In some embodiments, an eye condition or disease is ADOA. In some embodiments, the eye condition of a subject with ADOA is an ADOA disease state. In some embodiments, the FPF intensity score in the Nasal sector of the optic disc of a subject is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Nasal sector of the optic disc of a subject is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Nasal sector of the optic disc of a subject is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Nasal sector of the optic disc of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state older than about 40.

[0378] In some embodiments, the FPF intensity score in the Nasal Inferior sector of the optic disc of a subject is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Nasal Inferior sector of the optic disc of a subject is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Nasal Inferior sector of the optic disc of a subject is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Nasal Inferior sector of the optic disc of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state older than about 40.

[0379] In some embodiments, the FPF intensity score in the Nasal Superior sector of the optic disc of a subject is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Nasal Superior sector of the optic disc of a subject is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Nasal Superior sector of the optic disc of a subject is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Nasal Superior sector of the optic disc of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU is indicative of ADOA eye disease state older than about 40.

Correlation of FPF Intensity Score Derived from FPF Intensity Measurements and Results of Other Ophthalmological Exams with Eye Conditions and Diseases

[0380] In some embodiments, FPF intensity score, derived from FPF intensity and at least one other parameter or ophthalmological exam, is correlated with an eye condition or disease. In some embodiments, FPF intensity score derived from FPF intensity and at least one other ophthalmological exam is correlated with ADOA. In some embodiments, FPF intensity score derived from FPF intensity and at least one other ophthalmological exam is correlated with an ADOA disease state. In some embodiments, the opthamological exam is selected from the group consisting of Best Corrected Visual Acuity (BCVA) letter score; Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score; high-contrast Best Corrected Visual Acuity (HC BCVA) letter score; low- contrast Best Corrected Visual Acuity (LC BCVA) letter score; Humphrey 10-2 Visual Field test Mean Deviation (MD); Humphrey 10-2 Visual Field test Pattern Standard Deviation (PSD); Visual Acuity Score (VAS); Quality of life questionnaires (NEI-VFQ-25, IVI-C, EQ-5D, EQ-5D-Y); Refraction test result; Minnesota Reading (MNRead) acuity chart score; Pelli -Robson chart score; slit lamp examination result; intraocular pressure using a Tonopen; perimetry; dilated fimdoscopy result; retinal nerve fiber layer (RNFL) measurement; optical coherence tomography (OCT) result; and macular ganglion cell layer/inner plexiform layer (GCL/IPL) thickness measurements; Curve Width (CW) measurement; Electroretinogram (ERG) result; and fundus photography result.

[0381] In some embodiments, FPF intensity score is correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests. In some embodiments, FPF intensity score is correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with normal contrast. In some embodiments, FPF intensity score is correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with high contrast. In some embodiments, FPF intensity score is correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with low contrast. In some embodiments, FPF intensity score is positively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests. In some embodiments, FPF intensity score is positively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with normal contrast. In some embodiments, FPF intensity score is positively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with high contrast. In some embodiments, FPF intensity score is positively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with low contrast. In some embodiments, FPF intensity score is negatively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests. In some embodiments, FPF intensity score is negatively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with normal contrast. In some embodiments, FPF intensity score is negatively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with high contrast. In some embodiments, FPF intensity score is negatively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with low contrast.

[0382] In some embodiments, FPF intensity score in the global optic disc is correlated with Mean Deviation (MD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, FPF intensity score in the global optic disc is correlated with Mean Deviation (MD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, FPF intensity score in the global optic disc is positively correlated with Mean Deviation (MD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, FPF intensity score in the global optic disc is negatively correlated with Mean Deviation (MD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, a Humphrey 10-2 Visual Field test Mean Deviation of between about 20 and 40 is correlated with FPF intensity of between about 0 and 10 dB GSU in the global optic disc of an eye of an ADOA subject. In some embodiments, FPF intensity score in the macula is correlated with Mean Deviation (MD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, FPF intensity score in the macula is correlated with Mean Deviation (MD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, FPF intensity score in the macula is positively correlated with Mean Deviation (MD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, FPF intensity score in the macula is negatively correlated with Mean Deviation (MD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, a Humphrey 10-2 Visual Field test Mean Deviation of between about 5 and 40 is correlated with FPF intensity of between about 15 and 30 dB GSU in the macula of an eye of an ADOA subject.

[0383] In some embodiments, FPF intensity score in the global optic disc is correlated with Pattern Standard Deviation (MD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, FPF intensity score in the global optic disc is correlated with Pattern Standard Deviation (PSD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, FPF intensity score in the global optic disc is positively correlated with Pattern Standard Deviation (PSD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, FPF intensity score in the macula is correlated with Pattern Standard Deviation (PSD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, FPF intensity score in the macula is correlated with Pattern Standard Deviation (PSD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, FPF intensity score in the macula is positively correlated with Pattern Standard Deviation (PSD) resulting from Humphrey 10-2 Visual Field tests. In some embodiments, FPF intensity score in the macula is negatively correlated with Pattern Standard Deviation (PSD) resulting from Humphrey 10-2 Visual Field tests.

[0384] In some embodiments, FPF intensity score is correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests and further correlated with the likelihood of ADOA disease state. In some embodiments, FPF intensity score is correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with normal contrast and further correlated with the likelihood of ADOA disease state. In some embodiments, FPF intensity score is correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with high contrast and further correlated with the likelihood of ADOA disease state. In some embodiments, FPF intensity score is correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with low contrast and further correlated with the likelihood of ADOA disease state. In some embodiments, FPF intensity score is positively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests and further correlated with the likelihood of ADOA disease state. In some embodiments, FPF Intensity score is positively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with normal contrast and further correlated with the likelihood of ADOA disease state. In some embodiments, FPF intensity score is positively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with high contrast and further correlated with the likelihood of ADOA disease state. In some embodiments, FPF intensity score is positively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with low contrast and further correlated with the likelihood of ADOA disease state. In some embodiments, FPF intensity score is negatively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests and further correlated with the likelihood of ADOA disease state. In some embodiments, FPF intensity score is negatively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with normal contrast and further correlated with the likelihood of ADOA disease state. In some embodiments, FPF intensity score is negatively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with high contrast and further correlated with the likelihood of ADOA disease state. In some embodiments, FPF intensity score is negatively correlated with Letter Scores resulting from Best Corrected Visual Acuity (BCVA) tests with low contrast and further correlated with the likelihood of ADOA disease state.

[0385] In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual acuity (BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subj ect of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subj ect of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is Indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is indicative of ADOA eye disease state older than about 40.

[0386] In some embodiments, the FPF intensity score in the Temporal Inferior sector, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal Inferior sector, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal Inferior sector, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual acuity (BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity (BCVA) letter score of a subject, is indicative of ADOA eye disease state older than about 40.

[0387] In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High- Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5. 1 dB GSU, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is indicative of ADOA eye disease state older than about 40.

[0388] In some embodiments, the FPF intensity score in the Temporal Inferior sector, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal Inferior sector, derived from a measurement of FPF intensity and a High- Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal Inferior sector, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a High- Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5. 1 dB GSU, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-Contrast Best Corrected Visual Acuity (HC BCVA) letter score of a subject, is indicative of ADOA eye disease state older than about 40.

[0389] In some embodiments, the FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a High- contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a High-contrast Low- Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High -contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-contrast Low- Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is indicative of ADOA eye disease state older than about 40. [0390] In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a High- contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-contrast Low- Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5. 1 dB GSU, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a High-contrast Low-Contrast Best Corrected Visual Acuity (LC BCVA) letter score of a subject, is indicative of ADOA eye disease state older than about 40. [0391] In some embodiments, the FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state older than about 40.

[0392] In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5. 1 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state older than about 40.

[0393] In some embodiments, the FPF intensity score in the Temporal Inferior sector, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal Inferior sector, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal Inferior sector, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score of a subject, is indicative of ADOA eye disease state older than about 40. [0394] In some embodiments, the FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Humphrey 10- 2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the global optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlative of ADOA eye disease state. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the global optic disc of a subj ect of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the global optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state older than about 40. [0395] In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal sector, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score In the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Temporal sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 1 .3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5. 1 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Temporal sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10- 2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state older than about 40.

[0396] In some embodiments, the FPF intensity score in the Temporal Inferior sector, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal Inferior sector, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is positively correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Temporal Inferior sector, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is negatively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low FPF intensity score in the Temporal Inferior sector of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 1.3 dB GSU and 5.1 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state in subjects under 18 years of age. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 3 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state in subjects between the ages of about 18 and about 40. In some embodiments, an FPF intensity score in the Temporal Inferior sector of the optic disc of a subject of between at least about 6 dB GSU and 10 dB GSU, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Mean Deviation (MD) of a subject, is indicative of ADOA eye disease state older than about 40.

[0397] In some embodiments, the FPF intensity score in the Nasal, Nasal Inferior, Nasal Superior, Temporal, Temporal Inferior, or Temporal Superior sectors, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Patten Standard Deviation (PSD) of a subject, is correlated with the likelihood of ADOA disease state. In some embodiments, the FPF intensity score in the Nasal, Nasal Inferior, Nasal Superior, Temporal, Temporal Inferior, or Temporal Superior sectors, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Patten Standard Deviation (PSD) of a subject, is positively correlated with the likelihood of ADOA disease state. In some embodiments, a high FPF intensity score in the Nasal, Nasal Inferior, Nasal Superior, Temporal, Temporal Inferior, or Temporal Superior sectors of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Patten Standard Deviation (PSD) of a subject, is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low FPF intensity score in the Nasal, Nasal Inferior, Nasal Superior, Temporal, Temporal Inferior, or Temporal Superior sectors of the optic disc, derived from a measurement of FPF intensity and a Humphrey 10-2 Visual Field test Patten Standard Deviation (PSD) of a subject, is correlated with a lower likelihood of ADOA disease state.

Ophthalmological Parameters, Analysis, Processing, and Correlation with ADOA

[0398] Also within the scope of the present disclosure are methods for correlating a vision test score with eye condition in ADOA.

[0399] A vision test score described herein can be determined based on the results of the anatomical conditions of an eye or the functional conditions of an eye. Anatomical conditions can comprise GCU or RNFU thickness. Functional conditions can comprise Best Corrected Visual Acuity (BCVA), Humphrey 10-2 Visual Field Mean Deviation (MD), Humphrey 10-2 Visual Field test Pattern Standard Deviation (PSD), Visual Acuity Score (VAS), or Minnesota Reading Speed, or a combination thereof.

[0400] A vision test score described herein can be determined based at least on a result of a visual acuity test score. In some embodiments, a visual acuity test score is determined based at least on a Best Corrected Visual Acuity (BCVA) test. In some cases, the BCVA test comprises testing in a defined sequence for (1) ocular refraction from a fixed distance, and (2) visual acuity from a fixed distance. For instance, the BCVA test can use at least one Original Sloan Early Treatment Diabetic Retinopathy Study (ETDRS) chart, such as Chart R (Precision Vision 2110) to measure refraction, Chart 1 (Pression Vision 2111) to test the right eye (OD), and/or Chart 2 (Precision Vision 2112) to test the left eye (OS). The defined sequence can be testing for ocular refraction with Chart R before testing for visual acuity with either Chart 1 or Chart 2. The fixed distance used for the BCVA test can be a distance of about 4 meters or about 1 meter from the eyes of the subject to the front of the chart. The BCVA test can further involve calculating a letter score comprising a sum of a total number of letters correctly identified by the subject at 4 meters, plus 30; or calculating a letter score comprising a sum of a total number of letters correctly identified by the subject at 1 meter.

[0401] In some embodiments, a Best Corrected Visual Acuity (BCVA) test can comprise a Best Corrected Visual Acuity test, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS), a high- contrast Best Corrected Visual Acuity (HC BCVA) test, or a low-contrast Best Corrected Visual Acuity (LC BCVA) test. In some embodiments, the result of a Best Corrected Visual Acuity (BCVA) test, such as a Best Corrected Visual Acuity test, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS), high-contrast Best Corrected Visual Acuity (HC BCVA) test; or low-contrast Best Corrected Visual Acuity (LC BCVA) test, is recorded as Letter Scores. Letter scores can be composed of the total number of letters correctly identified by a subject from a visual acuity chart. Letter scores can be composed of the total number of letters correctly identified by a subject from a visual acuity chart from a fixed distance of either 4 meters or 1 meter. The visual acuity charts can be a chart selected from the group consisting of Chart R (Precision Vision 2110) to measure refraction, Chart 1 (Pression Vision 2111) to test the right eye (OD), and Chart 2 (Precision Vision 2112) to test the left eye (OS). The visual acuity charts can be a logMAR chart or Snellen chart. The visual acuity charts can be presented in standard contrast, high- contrast for HC BCVA, or low-contrast for LC BCVA. Similar measurement of low constrat visual acuity has been reported in multiple sclerosis patients, as described in Balcer LJ et al. (2017) Mult Scler. Apr; 23(5):734-747.

[0402] A vision test score described herein can be determined based at least on results of at least one LC BCVA test and one HC BCVA test. Contrast sensitivity is important in daily visual tasks. In some embodiments, a Delta Letter Score can be computed by subtracting a 2.5% low-contrast BCVA letter score from a high-contrast BCVA letter score. In some embodiments, a Delta Letter Score is indicative of eye condition in ADOA. In some embodiments, a high Delta Letter Score is indicative of impaired eye condition or low vision. In some embodiments, a high Delta Letter Score is indicative of impaired eye condition or low vision in ADOA. In some embodiments, a low Delta Letter Score is indicative of normal, or unimpaired eye condition. In some embodiments, a low Delta Letter Score is indicative of unimpaired eye condition in ADOA. In some embodiments, a Delta Letter Score 10-15 letters higher than a reference value, based on controls comprising subjects with normal eyes, is correlated with impaired eye condition. In some embodiments, a Delta Letter Score 10-15 letters higher than a reference value, based on controls comprising subjects with normal eyes, is correlated with the presence of eye disease. In some embodiments, a Delta Letter Score 10-15 letters higher than a reference value, based on controls comprising subjects with normal eyes, is correlated with impaired eye condition or low vision in ADOA. In some embodiments, a low Delta Letter Score is a Delta Letter Score less than 25 letters. In some embodiments, a high Delta Letter Score is a Delta Letter Score greater than 25 letters. In some embodiments, a low logMAR (<0.3) and a high Delta Letter Score (>25) is correlated with impaired eye condition or low vision. In some embodiments, a low logMAR (<0.3) and a high Delta Letter Score (>25) is correlated with impaired eye condition in ADOA. In some embodiments, a logMAR between about 0.3 and 0.6 and a high Delta Letter Score (>25) is correlated with impaired eye condition or low vision. In some embodiments, a logMAR between about 0.3 and 0.6 and a high Delta Letter Score (>25) is correlated with impaired eye condition in ADOA. In some embodiments, a logMAR between about 0.6 and 0.9 and a high Delta Letter Score (>25) is correlated with impaired eye condition or low vision. In some embodiments, a logMAR between about 0.6 and 0.9 and a high Delta Letter Score (>25) is correlated with impaired eye condition or low vision in ADOA. In some embodiments, the vision test score is determined based at least in part on a low-contrast Best Corrected Visual Acuity (LC BCVA) letter score.

[0403] LogMAR may be used as a readout of the BCVA test described herein. In some cases, low logMAR is indicative of good or better vision, and high logMAR is indicative of bad or worse vision. In some embodiments, a high letter score from a BCVA corresponds to a low logMAR, and a low letter score from a BCVA corresponds to a high logMAR. In some embodiments, a logMAR less than 0.3 is correlated with good or normal vision, a logMAR between about 0.3 and 0.6 is correlated with low vision or decreased vision, a logMAR between about 0.6 to 0.9 is correlated with poor vision, and a logMAR above 0.9 is correlated with blindness. In some embodiments, logMAR is correlated with thickness of eye structures. In some embodiments, logMAR is correlated with the thickness of the Ganglion Cell Layer- Inner Plexiform Layer (GCL/IPL). In some embodiments, logMAR is correlated with the thickness of the retinal nerve fiber layer (RNFL). In some embodiments, logMAR is correlated with Humphrey 10-2 Visual Field (VF) Median Deviation (MD). In some embodiments, logMAR is negatively correlated with thickness of eye structures. In some embodiments, logMAR is negatively correlated with the thickness of the Ganglion Cell Layer-Inner Plexiform Layer (GCL/IPL), which can comprise the global GCL/IPL, the Nasal GCL/IPL, or the Temporal GCL/IPL. In some embodiments, logMAR is negatively correlated with the thickness of the retinal nerve fiber layer (RNFL), which can comprise the global RNFL, the Nasal RNFL, or the Temporal RNFL. In some embodiments, logMAR is negatively correlated with Humphrey 10-2 Visual Field (VF) Median Deviation (MD).

[0404] In some embodiments, an eye condition or disease is ADOA. In some embodiments, the eye condition of a subject with ADOA is an ADOA disease state. In some embodiments, a vision test score is correlated with age. In some embodiments, a vision test score is negatively correlated with age. In some embodiments, a high vision test score is correlated with a higher likelihood of ADOA disease state. In some embodiments, a low vision test score is correlated with a lower likelihood of ADOA disease state. In some embodiments, a high vision test score is correlated with a higher likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a high vision test score is correlated with a higher likelihood of ADOA disease state in subjects between about 18 and about 40 years of age. In some embodiments, a high vision test score is correlated with a higher likelihood of ADOA disease state in subjects older than about 40. In some embodiments, a low vision test score is correlated with a lower likelihood of ADOA disease state. In some embodiments, a low vision test score is correlated with a lower likelihood of ADOA disease state in subjects younger than 18 years of age. In some embodiments, a low vision test score is correlated with a lower likelihood of ADOA disease state in subjects younger between about 18 and about 40 years of age. In some embodiments, a low vision test score is correlated with a lower likelihood of ADOA disease state in subjects older than about 40. [0405] A vision test score described herein can be determined based at least on results of Optical Coherence Tomography or measurements of Ganglion Cell Layer-Inner Plexiform Layer (GCL/IPL) thickness. In some embodiments, GCL/IPL thickness comprises a measurement selected from any of the following: global GCL/IPL thickness, Nasal GCL/IPL thickness, and Temporal GCL/IPL thickness. In some embodiments, logMAR from a 2.5% low-contrast BCVA is correlated with GCL/IPL thickness. In some embodiments, logMAR from a 2.5% low-contrast BCVA is negatively correlated with GCL/IPL thickness. In some embodiments, logMAR from a 2.5% low-contrast BCVA is negatively correlated with global GCL/IPL thickness. In some embodiments, logMAR from a 2.5% low-contrast BCVA is negatively correlated with Nasal GCL/IPL thickness. In some embodiments, logMAR from a 2.5% low- contrast BCVA is negatively correlated with Temporal GCL/IPL thickness. In some embodiments, logMAR from a 5% contrast BCVA is correlated with GCL/IPL thickness. In some embodiments, logMAR from a 5% contrast BCVA is negatively correlated with GCL/IPL thickness. In some embodiments, logMAR from a 5% contrast BCVA is negatively correlated with global GCL/IPL thickness. In some embodiments, logMAR from a 5% contrast BCVA is negatively correlated with Nasal GCL/IPL thickness. In some embodiments, logMAR from a 5% contrast BCVA is negatively correlated with Temporal GCL/IPL thickness. In some embodiments, logMAR from a 25% contrast BCVA is correlated with GCL/IPL thickness. In some embodiments, logMAR from a 25% contrast BCVA is negatively correlated with GCL/IPL thickness. In some embodiments, logMAR from a 25% contrast BCVA is negatively correlated with global GCL/IPL thickness. In some embodiments, logMAR from a 25% contrast BCVA is negatively correlated with Nasal GCL/IPL thickness. In some embodiments, logMAR from a 25% contrast BCVA is negatively correlated with Temporal GCL/IPL thickness. In some embodiments, logMAR from a standard contrast BCVA is correlated with GCL/IPL thickness. In some embodiments, logMAR from a standard contrast BCVA is negatively correlated with GCL/IPL thickness. In some embodiments, logMAR from a standard contrast BCVA is negatively correlated with global GCL/IPL thickness. In some embodiments, logMAR from a standard contrast BCVA is negatively correlated with Nasal GCL/IPL thickness. In some embodiments, logMAR from a standard contrast BCVA is negatively correlated with Temporal GCL/IPL thickness. In some embodiments, logMAR from a high-contrast BCVA is correlated with GCL/IPL thickness. In some embodiments, logMAR from a high -contrast BCVA is negatively correlated with GCL/IPL thickness. In some embodiments, logMAR from a high-contrast BCVA is negatively correlated with global GCL/IPL thickness. In some embodiments, logMAR from a high-contrast BCVA is negatively correlated with Nasal GCL/IPL thickness. In some embodiments, logMAR from a high-contrast BCVA is negatively correlated with Temporal GCL/IPL thickness. In some embodiments, the GCL/IPL thickness is correlated with visual acuity. In some embodiments, a GCL/IPL thickness of 53 pm or less is correlated with high logMAR. In some embodiments, a GCL/IPL thickness of 53 pm or less is correlated with poor or impaired vision in an eye condition or disease. In some embodiments, a GCL/IPL thickness of 53 pm or less is correlated with poor or impaired vision in ADOA. In some embodiments, a GCL/IPL thickness of 53 pm or more is correlated with low logMAR. In some embodiments, a GCL/IPL thickness of 53 pm or more is correlated with good or better vision. In some embodiments, the Delta Letter Score is not correlated with GCL/IPL thickness. In some embodiments, GCL/IPL thickness is weakly correlated or not correlated with the age of a subject. In some embodiments, global GCL/IPL thickness is weakly correlated or not correlated with the age of a subject. In some embodiments, Nasal GCL/IPL thickness is weakly correlated or not correlated with the age of a subject.

[0406] A vision test score described herein can be determined based at least on results of Optical Coherence Tomography or measurements of retinal nerve fiber layer (RNFL) thickness. In some embodiments, RNFL thickness comprises a measurement selected from any of the following: global RNFL thickness, Nasal RNFL thickness, and Temporal RNFL thickness. In some embodiments, logMAR from a 2.5% low-contrast BCVA is correlated with RNFL thickness. In some embodiments, logMAR from a 2.5% low-contrast BCVA is negatively correlated with RNFL thickness. In some embodiments, logMAR from a 2.5% low-contrast BCVA is negatively correlated with global RNFL thickness. In some embodiments, logMAR from a 2.5% low-contrast BCVA is negatively correlated with Nasal RNFL thickness. In some embodiments, logMAR from a 2.5% low-contrast BCVA is negatively correlated with Temporal RNFL thickness. In some embodiments, logMAR from a 5% contrast BCVA is correlated with RNFL thickness. In some embodiments, logMAR from a 5% contrast BCVA is negatively correlated with RNFL thickness. In some embodiments, logMAR from a 5% contrast BCVA is negatively correlated with global RNFL thickness. In some embodiments, logMAR from a 5% contrast BCVA is negatively correlated with Nasal RNFL thickness. In some embodiments, logMAR from a 5% contrast BCVA is negatively correlated with Temporal RNFL thickness. In some embodiments, logMAR from a 25% contrast BCVA is correlated with RNFL thickness. In some embodiments, logMAR from a 25% contrast BCVA is negatively correlated with RNFL thickness. In some embodiments, logMAR from a 25% contrast BCVA is negatively correlated with global RNFL thickness. In some embodiments, logMAR from a 25% contrast BCVA is negatively correlated with Nasal RNFL thickness. In some embodiments, logMAR from a 25% contrast BCVA is negatively correlated with Temporal RNFL thickness. In some embodiments, logMAR from a standard contrast BCVA is correlated with RNFL thickness. In some embodiments, logMAR from a standard contrast BCVA is negatively correlated with RNFL thickness. In some embodiments, logMAR from a standard contrast BCVA is negatively correlated with global RNFL thickness. In some embodiments, logMAR from a standard contrast BCVA is negatively correlated with Nasal RNFL thickness. In some embodiments, logMAR from a standard contrast BCVA is positively correlated with Temporal RNFL thickness. In some embodiments, logMAR from a standard contrast BCVA is negatively correlated with Temporal RNFL thickness. In some embodiments, logMAR from a high -contrast BCVA is correlated with RNFL thickness. In some embodiments, logMAR from a high- contrast BCVA is negatively correlated with RNFL thickness. In some embodiments, logMAR from a high -contrast BCVA is negatively correlated with global RNFL thickness. In some embodiments, logMAR from a high-contrast BCVA is negatively correlated with Nasal RNFL thickness. In some embodiments, logMAR from a high-contrast BCVA is negatively correlated with Temporal RNFL thickness. In some embodiments, the RNFL thickness is correlated with visual acuity. In some embodiments, RNFL thickness is weakly correlated or not correlated with the age of a subject. In some embodiments, global RNFL thickness is weakly correlated or not correlated with the age of a subject. In some embodiments, Nasal RNFL thickness is weakly correlated or not correlated with the age of a subject.

[0407] A vision test score described herein can be determined based at least on results of a Humphrey 10-2 Visual Field Test. In some embodiments, a Humphrey 10-2 Visual Field Test result or score is reported as Mean Deviation (MD). A “median deviation” on a Humphrey 10-2 Visual Field Test refers to the average difference from the normal expected value for the central visual field. In some embodiments, a Humphrey 10-2 Visual Field Test Mean Deviation of 0 corresponds to normal vision. In some embodiments, a more strongly negative Mean Deviation value corresponds to worse vision. In some embodiments, a less negative Mean Deviation value corresponds to vision closer to normal vision. For example, a Mean Deviation of 0 corresponds to normal vision, a Mean Deviation of -1 corresponds to low vision, and a Mean Deviation of -10 corresponds to poor vision. In some embodiments, a Humphrey 10-2 Visual Field Test Mean Deviation is highly correlated with a standard-contrast or high-contrast BCVA result. In some embodiments, a Humphrey 10-2 Visual Field Test Mean Deviation is negatively correlated with a standard-contrast or high-contrast BCVA result; that is, a more strongly negative Mean Deviation (for example, MD of -10) is correlated with a higher logMAR value (for example, logMAR 0.9).

[0408] In other embodiments, Humphrey 10-2 Visual Field Test result or score is reported as Pattern Standard Deviation (PSD). A “Pattern Standard Deviation (PSD)” on a Humphrey 10-2 Visual Field Test refers to a measure of how irregular the visual field sensitivity is across the central vision area. In some embodiments, a Humphrey 10-2 Visual Field Test Pattern Standard Deviation score of 0.8 to 2.0 dB corresponds to normal vision. In some embodiments, a significantly elevated Pattern Standard Deviation score corresponds to greater variation in sensitivity between adjacent test points and worse vision. In some embodiments, a Humphrey 10-2 Visual Field Test Pattern Standard Deviation score greater than 2.0 dB corresponds to irregular or worse vision. In some embodiments, a Humphrey 10-2 Visual Field Test Pattern Standard Deviation is correlated with a standard-contrast or high-contrast BCVA result. In some embodiments, a Humphrey 10-2 Visual Field Test Pattern Standard Deviation is positively correlated with a standard-contrast or high-contrast BCVA result; that is, a more strongly positively Pattern Standard Deviation (for example, PSD of 10 dB) is correlated with a higher logMAR value (for example, logMAR 0.9).

[0409] A vision test score described herein can be determined based at least on results of a Visual Acuity Score (VAS). A Visual Acuity Score comprises a scale that estimates visual abilities and is calculated by having a participant read letters or symbols on a chart from top to bottom, line by line, one eye at a time. In some instances, the chart is a Snellen chart. The score is written as a fraction, with the top number representing the distance to the chart and the bottom number representing the line read. On Visual Acuity Score scale, 20/20 is rated as 100 and indicates a participant with normal vision at 20 feet. On charts with a logarithmic progression, each line is worth 5 points and each letter read is 1 point. A score of 20/40 means the person can read at 20 feet what someone with normal vision can read at 40 feet. 50 VAS points stand for 20/200. At this level (20/200), the average person has lost 50% of visual ability. Zero VAS points represents 20/2000. In some embodiments, a VAS score comprising a larger the bottom number indicates worse vision.

[0410] A vision test score described herein can be determined based at least on the results of a BCVA, Humphrey 10-2 Visual Field Test Mean Deviation (MD), Humphrey 10-2 Visual Field Test Pattern Standard Deviation (PSD), Visual Acuity Score (VAS), or a combination thereof. A vision test score described herein can be determined based at least on the results of a low-contrast BCVA Humphrey 10-2 Visual Field Test Mean Deviation (MD), Humphrey 10-2 Visual Field Test Pattern Standard Deviation (PSD), Visual Acuity Score (VAS), or a combination thereof. A low-contrast BCVA can comprise a contrast level of 2.5%, 5%, or 25% relative to a standard, or high-contrast level of 100%.

NUMBERED EMBODIMENTS

[0411] Embodiment Al. A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NO: 6-275 or 280-299, and wherein the method comprises administering to one eye of the subject the pharmaceutical composition at a dose of about 0.005 to about 20 mg of the antisense oligomer.

[0412] Embodiment A2. A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NO: 6-275 or 280-299, wherein the disease or condition comprises autosomal dominant optic atrophy (ADOA), and wherein the subject is characterized by having:

(i) a heterozygous OP Al gene variant;

(ii) clear ocular media to allow for adequate visualization of the vitreous and fundus and to achieve appropriate quality of all ophthalmic assessments;

(iii) an AREDS Clinical Lens Standard of <1 for posterior subcapsular (PSC) opacity;

(iv) a BCVA EDTRS letter score of >35 and <70 with each eye individually that is administered with the pharmaceutical composition; or

(v) any combination of (i)-(iv) .

[0413] Embodiment A3. A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NO: 6-275 or 280-299, wherein the disease or condition comprises autosomal dominant optic atrophy (ADOA), and wherein the subject is characterized by:

(1) not having a gain-of-function variant, or compound heterozygous or homozygous pathogenic or likely pathogenic variant in OP Al gene;

(2) not having only benign or likely benign variants in the OP Al gene;

(3) not having extraocular phenotypic manifestations of (syndromic) ADOA (ADOA-plus);

(4) not having been diagnosed with Behr syndrome;

(5) not having a known pathogenic mutation in another gene implicated in optic atrophy or retinal diseases;

(6) not having diabetic retinopathy with potential for development of proliferative diabetic retinopathy, diabetic macular edema, or optic neuropathy;

(7) not having or having a history of any ocular condition in either eye;

(8) not having a history of intraocular surgery or comeal surgery including refractive surgery in either eye within 12 weeks prior to the administering;

(9) not having a history of retinal photocoagulation;

(10) not having a history or presence of retinal vein occlusion;

(11) not being considered to be at risk for uveitis or ocular infection because of having any of the following: an active flare of non -infectious uveitis or an episode of infectious uveitis or other ocular infection in either eye within 12 months prior to the administering;

(12) not having dry age-related macular degeneration in either eye;

(13) not having high myopia (>6 diopters);

(14) not having a history of cancer (except a diagnosis of basal cell, squamous cell skin cancer, or carcinoma in situ of the cervix that has been successfully treated);

(15) not taking, or having taken at any time, any medication or treatment that can or might cause an optic neuropathy;

(16) not having any history of nutritional deficiency (including B12 and/or folate deficiencies), or not having a known deficiency in serum B12 or folate, not having had bariatric surgery; or

(17) any combination of ( l)-( 16) .

[0414] Embodiment A4. A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NO: 6-275 or 280-299, and wherein the antisense oligomer has any one of the following chemical structures:

[0415] Embodiment A5. The method of any one of embodiments A2-A4, wherein the method comprises administering to one eye of the subject the pharmaceutical composition at a dose of about 0.005 to about 20 mg of the antisense oligomer.

[0416] Embodiment A6. The method of any one of embodiments A1-A5, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0.005 mg to about 15 mg, about 0.005 mg to about 10 mg, about 0.005 mg to about 5 mg, about 0.005 mg to about 1 mg, about 0.01 mg to about 15 mg, about 0.01 mg to about 10 mg, about 0.01 mg to about 5 mg, about 0.01 mg to about 2.5 mg, about 0.01 mg to about 1.0 mg, about 0.01 mg to about 0.5 mg, about 0.01 mg to about 0.25 mg, about 0.01 mg to about 0. 1 mg, about 0.01 mg to about 0.05 mg, about 0.05 mg to about 10 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 2.5 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.25 mg, about 0.05 mg to about 0.1 mg, about 0. 1 mg to about 5 mg, about 0.1 mg to about 2.5 mg, about 0. 1 mg to about 1.5 mg, about 0. 1 mg to about 1.0 mg, about 0.1 mg to about 0.5 mg, or about 0.1 mg to about 0.25 mg of the antisense oligomer. [0417] Embodiment A7. The method of any one of embodiments A1-A5, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0.005 mg, about 0.01 mg, about 0.05 mg, about 0. 1 mg, about 0.2 mg, about 0.3 mg, 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.75 mg, about 2.0 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, about 5.0 mg, about 5.5 mg, about 6.0 mg, about 7.0 mg, about 8.0 mg, about 9.0 mg, about 10 mg, about 12.5 mg, about 15 mg, about 17.5 mg, or about 20 mg of the antisense oligomer.

[0418] Embodiment A8. The method of any one of embodiments A1-A5, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0.1 mg to about 1.5 mg, about 0.1 mg to about 1.4 mg, about 0.1 mg to about 1.2 mg, about 0.1 mg to about 1.0 mg, about 0. 1 mg to about 0.8 mg, about 0.1 mg to about 0.7 mg, about 0.1 mg to about 0.5 mg, about 0. 1 mg to about 0.3 mg, about 0.2 mg to about 1.5 mg, about 0.2 mg to about 1.4 mg, about 0.2 mg to about 1.2 mg, about 0.2 mg to about 1.0 mg, about 0.2 mg to about 0.8 mg, about 0.2 mg to about 0.7 mg, about 0.2 mg to about 0.5 mg, about 0.3 mg to about 1.5 mg, about 0.3 mg to about 1.4 mg, about 0.3 mg to about 1.2 mg, about 0.3 mg to about 1.0 mg, about 0.3 mg to about 0.8 mg, about 0.3 mg to about 0.7 mg, about 0.3 mg to about 0.5 mg, about 0.5 mg to about 1.5 mg, about 0.5 mg to about 1.4 mg, about 0.5 mg to about 1.2 mg, about 0.5 mg to about 1.0 mg, about 0.5 mg to about 0.8 mg, about 0.5 mg to about 0.7 mg, about 0.7 mg to about 1.5 mg, about 0.7 mg to about 1.4 mg, about 0.7 mg to about 1.2 mg, about 0.7 mg to about 1.0 mg, about 0.8 mg to about 1.5 mg, about 0.8 mg to about 1.4 mg, about 0.8 mg to about 1.2 mg, about 0.8 mg to about 1.0 mg, about 1.0 mg to about 1.5 mg, about 1.0 mg to about 1.4 mg, about 1.0 mg to about 1.2 mg, about 1.2 mg to about 1.5 mg, or about 1.2 mg to about 1.4 mg of the antisense oligomer.

[0419] Embodiment A9. The method of any one of embodiments A1-A5, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0.1 mg, about 0.2 mg, about 0.3 mg, 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, or about 1.5 mg of the antisense oligomer.

[0420] Embodiment A 10. The method of any one of embodiments A1-A9, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition in a volume of about 5 pl to about 250 pl, about 10 pl to about 250 pl, about 20 pl to about 250 pl, about 30 pl to about 250 pl, about 40 pl to about 250 pl, about 50 pl to about 250 pl, about 60 pl to about 250 pl, about 70 pl to about 250 pl, about 80 pl to about 250 pl, about 100 pl to about 250 pl, about 120 pl to about 250 pl, about 150 pl to about 250 pl, about 160 pl to about 250 pl, about 180 pl to about 500 pl, about 200 pl to about 250 pl, about 220 pl to about 250 pl, about 5 pl to about 220 pl, about 10 pl to about 220 pl, about 20 pl to about 220 pl, about 30 pl to about 220 pl, about 40 pl to about 220 pl, about 50 pl to about 220 pl, about 60 pl to about 220 pl, about 70 pl to about 220 pl, about 80 pl to about 220 pl, about 100 pl to about 220 pl, about 120 pl to about 220 pl, about 150 pl to about 220 pl, about 160 pl to about 220 pl, about 180 pl to about 220 pl, about 5 pl to about 200 pl, about 10 pl to about 200 pl, about 20 pl to about 200 pl, about 30 pl to about 200 pl, about 40 pl to about 200 pl, about 50 pl to about 200 pl, about 60 pl to about 200 pl, about 70 pl to about 200 pl, about 80 pl to about 200 pl, about 100 pl to about 200 pl, about 120 pl to about 200 pl, about 150 pl to about 200 pl, about 160 pl to about 200 pl, about 180 pl to about 200 pl, about 5 pl to about 180 pl, about 10 pl to about 180 pl, about 20 pl to about 180 pl, about 30 pl to about 180 pl, about 40 pl to about 180 pl, about 50 pl to about 180 pl, about 60 pl to about 180 pl, about 70 pl to about 180 pl, about 80 pl to about 180 pl, about 100 pl to about 180 pl, about 120 pl to about 180 pl, about 150 pl to about 180 pl, about 5 pl to about 150 pl, about 10 pl to about 150 pl, about 20 pl to about 150 pl, about 30 pl to about 150 pl, about 40 pl to about 150 pl, about 50 pl to about 150 pl, about 60 pl to about 150 pl, about 70 pl to about 150 pl, about 80 pl to about 150 pl, about 100 pl to about 150 pl, about 120 pl to about 150 pl, about 5 pl to about 150 pl, about 10 pl to about 120 pl, about 20 pl to about 120 pl, about 30 pl to about 120 pl, about 40 pl to about 120 pl, about 50 pl to about 120 pl, about 60 pl to about 120 pl, about 70 pl to about 120 pl, about 80 pl to about 120 pl, about 100 pl to about 120 pl, about 5 pl to about 100 pl, about 10 pl to about 100 pl, about 20 pl to about 100 pl, about 30 pl to about 100 pl, about 40 pl to about 100 pl, about 50 pl to about 100 pl, about 60 pl to about 100 pl, about 70 pl to about 100 pl, about 80 pl to about 100 pl, about 5 pl to about 80 pl, about 10 pl to about 80 pl, about 20 pl to about 80 pl, about 30 pl to about 80 pl, about 40 pl to about 80 pl, about 50 pl to about 80 pl, about 60 pl to about 80 pl, about 5 pl to about 60 pl, about 10 pl to about 60 pl, about 20 pl to about 60 pl, about 30 pl to about 60 pl, about 40 pl to about 60 pl, or about 50 pl to about 60 pl.

[0421] Embodiment All. The method of any one of embodiments A1-A9, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition in a volume of about 5 pl, about 8 pl, about 10 pl, about 12 pl, about 15 pl, about 18 pl, about 20 pl, about 25 pl, about 28 pl, about 30 pl, about 35 pl, about 40 pl, about 45 pl, about 48 pl, about 50 pl, about 55 pl, about 60 pl, about 65 pl, about 70 pl, about 75 pl, about 80 pl, about 90 pl, about 100 pl, about 120 pl, about 150 pl, about 160 pl, about 180 pl, about 200 pl, about 220 pl, or about 250 pl. [0422] Embodiment A12. The method of any one of embodiments Al-Al 1, wherein the method comprises administering the pharmaceutical composition to both left eye and right eye of the subject. [0423] Embodiment A 13. The method of embodiment A 12, wherein the method comprises administering the pharmaceutical composition at the same dose to both the left eye and the right eye of the subject.

[0424] Embodiment A14. The method of embodiment A12, wherein the method comprises administering the pharmaceutical composition at different doses to the left eye and the right eye of the subject.

[0425] Embodiment A 15. The method of any one of embodiments Al -A3 or A5-A14, wherein the antisense oligomer comprises a nucleotide sequence having at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 6-275 or 280-299.

[0426] Embodiment A 16. The method of any one of embodiments Al -A3 or A5-A14, wherein the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 280-283, 288, and 290-292.

[0427] Embodiment A 17. The method of any one of embodiments Al -A3 or A5-A14, wherein the antisense oligomer modulates splicing of a nonsense-mediated RNA decay-inducing exon (NMD exon) from a pre-mRNA in a cell of the subject, wherein the pre-mRNA encodes the OPA1 protein and comprises the NMD exon, thereby modulating a level of processed mRNA that is processed from the pre- mRNA, and modulating expression of the OPA1 protein in the cell.

[0428] Embodiment A18. The method of embodiment A 17, wherein the antisense oligomer:

(a) binds to a targeted portion of the pre-mRNA;

(b) modulates binding of a factor involved in splicing of the NMD exon; or

(c) a combination of (a) and (b).

[0429] Embodiment A19. The method of embodiment A18, wherein the targeted portion of the pre- mRNA is proximal to the NMD exon.

[0430] Embodiment A20. The method of embodiment A18, wherein the targeted portion of the pre- mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of 5’ end of the NMD exon.

[0431] Embodiment A21. The method of embodiment A 18, wherein the targeted portion of the pre- mRNA is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotides upstream of 5’ end of the NMD exon.

[0432] Embodiment A22. The method of embodiment A18, wherein the targeted portion of the pre- mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of 3’ end of the NMD exon.

[0433] Embodiment A23. The method of embodiment A 18, wherein the targeted portion of the pre- mRNA is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotides downstream of 3’ end of the NMD exon.

[0434] Embodiment A24. The method of embodiment A18, wherein the targeted portion of the pre- mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of genomic site GRCh38/ hg38: chr3 193628509.

[0435] Embodiment A25. The method of embodiment A 18, wherein the targeted portion of the pre- mRNA is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of genomic site GRCh38/ hg38: chr3 193628509.

[0436] Embodiment A26. The method of embodiment A18, wherein the targeted portion of the pre- mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of genomic site GRCh38/ hg38: chr3 193628616.

[0437] Embodiment A27. The method of embodiment A18, wherein the targeted portion of the pre- mRNA is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of genomic site GRCh38/ hg38: chr3 193628616.

[0438] Embodiment A28. The method of embodiment A 18, wherein the targeted portion of the pre- mRNA is located in an intronic region between two canonical exonic regions of the pre-mRNA, and wherein the intronic region contains the NMD exon.

[0439] Embodiment A29. The method of embodiment A18, wherein the targeted portion of the pre- mRNA at least partially overlaps with the NMD exon.

[0440] Embodiment A30. The method of embodiment A18, wherein the targeted portion of the pre- mRNA at least partially overlaps with an intron upstream or downstream of the NMD exon.

[0441] Embodiment A31. The method of embodiment A 18, wherein the targeted portion of the pre- mRNA comprises 5’ NMD exon-intron junction or 3’ NMD exon-intron junction.

[0442] Embodiment A32. The method of embodiment A18, wherein the targeted portion of the pre- mRNA is within the NMD exon.

[0443] Embodiment A33. The method of embodiment A 18, wherein the targeted portion of the pre- mRNA comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides of the NMD exon.

[0444] Embodiment A34. The method of any one of embodiments A17-A33, wherein the NMD exon comprises a sequence with at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO: 279.

[0445] Embodiment A35. The method of any one of embodiments A17-A33, wherein the NMD exon comprises a sequence of SEQ ID NO: 279.

[0446] Embodiment A36. The method of embodiment A18, wherein the targeted portion of the pre- mRNA is within the nonsense -mediated RNA decay-inducing exon GRCh38/ hg38: chr3 193628509 to 193628616.

[0447] Embodiment A37. The method of embodiment A18, wherein the targeted portion of the pre- mRNA is upstream or downstream of the nonsense-mediated RNA decay-inducing exon GRCh38/ hg38: chr3 193628509 to 193628616.

[0448] Embodiment A38. The method of embodiment A18, wherein the targeted portion of the pre- mRNA comprises an exon-intron junction of exon GRCh38/ hg38: chr3 193628509 to 193628616.

[0449] Embodiment A39. The method of any one of embodiments A17-A38, wherein the OPA1 protein expressed from the processed mRNA is a full-length OPA1 protein or a wild-type OPA1 protein.

[0450] Embodiment A40. The method of any one of embodiments A17-A38, wherein the OPA1 protein expressed from the processed mRNA is a functional OPA1 protein.

[0451] Embodiment A41. The method of any one of embodiments A17-A38, wherein the OPA1 protein expressed from the processed mRNA is at least partially functional as compared to a wild-type OPA1 protein.

[0452] Embodiment A42. The method of any one of embodiments A17-A38, wherein the OPA1 protein expressed from the processed mRNA is at least partially functional as compared to a full-length wild-type OP Al protein.

[0453] Embodiment A43. The method of any one of embodiments A17-A42, wherein the antisense oligomer promotes exclusion of the NMD exon from the pre -mRNA.

[0454] Embodiment A44. The method of embodiment A43, wherein the exclusion of the NMD exon from the pre-mRNA in the cell contacted with the antisense oligomer is increased by about 1. 1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about

8-fold, about 1. 1 to about 9-fold, about 2 to about 5 -fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about

9-fold, at least about 1.1 -fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold, compared to in the absence of the antisense oligomer.

[0455] Embodiment A45. The method of any one of embodiments A17-A44, wherein the method results in an increase in the level of the processed mRNA in the cell.

[0456] Embodiment A46. The method of embodiment A45, wherein the level of the processed mRNA in the cell contacted with the antisense oligomer is increased by about 1. 1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1. 1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8- fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5 -fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold, compared to in the absence of the antisense oligomer.

[0457] Embodiment A47. The method of any one of embodiments A17-A46, wherein the method results in an increase in the expression of the OPA1 protein in the cell.

[0458] Embodiment A48. The method of embodiment A47, wherein a level of the OPA1 protein expressed from the processed mRNA in the cell contacted with the antisense oligomer is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5 -fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8- fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold, compared to in the absence of the antisense oligomer.

[0459] Embodiment A49. The method of any one of embodiments A1-A3 or A5-A48, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

[0460] Embodiment A50. The method of any one of embodiments A1-A3 or A5-A48, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2’-O-methyl moiety, a 2’-Fluoro moiety, a 2’ -O-N-methyl -acetamide (2’-NMA), or a 2’-O- methoxy ethyl moiety.

[0461] Embodiment A51. The method of any one of embodiments A1-A3 or A5-A48, wherein the antisense oligomer comprises at least one modified sugar moiety.

[0462] Embodiment A52. The method of embodiment A51, wherein each sugar moiety is a modified sugar moiety.

[0463] Embodiment A53. The method of any one of embodiments A1-A3 or A5-A52, wherein the antisense oligomer comprises a 5 ’-methylcytosine (5’-MeC).

[0464] Embodiment A54. The method of any one of embodiments Al -A3 or A5-A53, wherein each cytosine of the antisense oligomer is a 5 ’-methylcytosine (5’-MeC).

[0465] Embodiment A55. The method of any one of embodiments A1-A3 or A5-A54, wherein the antisense oligomer comprises a 5 ’-methyluracil (5’-MeU).

[0466] Embodiment A56. The method of any one of embodiments A1-A3 or A5-A52, wherein each cytosine orthymidine of the antisense oligomer is a 5 ’-methyluracil (5’-MeU).

[0467] Embodiment A57. The method of any one of embodiments Al 1-A3 or A5-A55, wherein the antisense oligomer comprises a phosphorothioate linkage.

[0468] Embodiment A58. The method of any one of embodiments Al -A3 or A5-A57, wherein each intemucleoside linkage of the ASO is a phosphorothioate linkage.

[0469] Embodiment A59. The method of any one of embodiments A1-A3 or A5-A58, wherein the antisense oligomer comprises a locked nucleic acid (LNA).

[0470] Embodiment A60. The method of any one of embodiments Al -A3 or A5-A59, wherein the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

[0471] Embodiment A61. The method of any one of embodiments A1-A3 or A5-A60, wherein the antisense oligomer has any one of the following chemical structures:

[0472] Embodiment A62. The method of any one of embodiments A1-A60, wherein the pharmaceutical composition is a liquid composition.

[0473] Embodiment A63. The method of any one of embodiments A1-A62, wherein the method comprises administering the pharmaceutical composition as a bolus injection over 1 to 60 minutes, 1 to 50 minutes, 1 to 40 minutes, 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes, 1 to 5 minutes, or 1 to 3 minutes.

[0474] Embodiment A64. The method of any one of embodiments A1-A63, wherein the method comprises administering the pharmaceutical composition as a bolus injection.

[0475] Embodiment A65. The method of any one of embodiments A1-A64, wherein the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, or water for injection.

[0476] Embodiment A66. The method of any one of embodiments A1-A64, wherein the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, and water for injection.

[0477] Embodiment A67. The method of any one of embodiments A1-A66, wherein the antisense oligomer is solubilized or diluted in an isotonic solution.

[0478] Embodiment A68. The method of any one of embodiments A1-A67, wherein the antisense oligomer is solubilized or diluted in a phosphate-buffered solution with at least pH 5.8.

[0479] Embodiment A69. The method of any one of embodiments A1-A68, wherein the antisense oligomer is solubilized or diluted in a phosphate-buffered (pH 6.6 - 7.6) solution.

[0480] Embodiment A70. The method of any one of embodiments A1-A69, wherein the pharmaceutical formulation does not comprise a preservative.

[0481] Embodiment A71. The method of any one of embodiments A1-A70, wherein the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml to about 200 mg/ml.

[0482] Embodiment A72. The method of any one of embodiments A1-A70, wherein the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml to about 200 mg/ml, about 5 mg/ml to about 200 mg/ml, about 10 mg/ml to about 200 mg/ml, about 15 mg/ml to about 200 mg/ml, about 20 mg/ml to about 200 mg/ml, about 25 mg/ml to about 200 mg/ml, about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, 2 mg/ml to about 150 mg/ml, about 5 mg/ml to about 150 mg/ml, about 10 mg/ml to about 150 mg/ml, about 15 mg/ml to about 150 mg/ml, about 20 mg/ml to about 150 mg/ml, about 25 mg/ml to about 150 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, 2 mg/ml to about 100 mg/ml, about 5 mg/ml to about 100 mg/ml, about 10 mg/ml to about 100 mg/ml, about 15 mg/ml to about 100

\T1 mg/ml, about 20 mg/ml to about 100 mg/ml, about 25 mg/ml to about 100 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, 2 mg/ml to about 80 mg/ml, about 5 mg/ml to about 80 mg/ml, about 10 mg/ml to about 80 mg/ml, about 15 mg/ml to about 80 mg/ml, about 20 mg/ml to about 80 mg/ml, about 25 mg/ml to about 80 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, 2 mg/ml to about 60 mg/ml, about 5 mg/ml to about 60 mg/ml, about 10 mg/ml to about 60 mg/ml, about 15 mg/ml to about 60 mg/ml, about 20 mg/ml to about 60 mg/ml, about 25 mg/ml to about 60 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, about 40 mg/ml to about 60 mg/ml, 2 mg/ml to about 40 mg/ml, about 5 mg/ml to about 40 mg/ml, about 10 mg/ml to about 40 mg/ml, about 15 mg/ml to about 40 mg/ml, about 20 mg/ml to about 40 mg/ml, or about 25 mg/ml to about 40 mg/ml.

[0483] Embodiment A73. The method of any one of embodiments A1-A70, wherein the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 12 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 18 mg/ml, about 20 mg/ml, about 22 mg/ml, about 24 mg/ml, about 26 mg/ml, about 28 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 80 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

[0484] Embodiment A74. The method of any one of embodiments A1-A73, wherein the pharmaceutical composition is prepared by diluting a concentrate comprising the antisense oligomer.

[0485] Embodiment A75. The method of embodiment A74, wherein the antisense oligomer is present in the concentrate at a concentration of about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, or about 40 mg/ml to about 60 mg/ml.

[0486] Embodiment A76. The method of embodiment A74, wherein the antisense oligomer is present in the concentrate at a concentration of about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

[0487] Embodiment A77. The method of any one of embodiments A74-A76, wherein the concentrate is a phosphate-buffered solution.

[0488] Embodiment A78. The method of any one of embodiments A1-A77, wherein the subject is a human subject.

[0489] Embodiment A79. The method of any one of embodiments Al or A4-A78, wherein the disease or condition is associated with a deficient amount or activity of the OPA1 protein.

[0490] Embodiment A80. The method of any one of embodiments Al or A4-A79, wherein the disease or condition comprises an eye disease or condition.

[0491] Embodiment A81. The method of any one of embodiments Al or A4-A79, wherein the disease or condition comprises a cardiovascular disease or condition.

[0492] Embodiment A82. The method of any one of embodiments Al or A4-A79, wherein the disease or condition comprises a neurological disease or condition.

[0493] Embodiment A83. The method of any one of embodiments Al or A4-A79, wherein the disease or condition comprises ADOA-plus syndrome; a mitochondrial disorder; glaucoma; normal tension glaucoma; Charcot-Marie-Tooth disease; mitochondria dysfunction; diabetic retinopathy; age-related macular degeneration; retinal ganglion cell death; mitochondrial fission-mediated mitochondrial dysfunction; progressive external ophthalmoplegia; deafness; ataxia; motor neuropathy; sensory neuropathy; myopathy; Behr syndrome; brain dysfunction; encephalopathy; peripheral neuropathy; fatal infantile mitochondrial encephalomyopathy; hypertrophic cardiomyopathy; spastic ataxic syndrome; sensory motor peripheral neuropathy; hypotonia; gastrointestinal dysmotility and dysphagia; optic atrophy; optic atrophy plus syndrome; Mitochondrial DNA depletion syndrome 14; late-onset cardiomyopathy; diabetic cardiomyopathy; Alzheimer’s Disease; focal segmental glomerulosclerosis; kidney disease; Huntington’s Disease; cognitive function decline in healthy aging; Prion diseases; late onset dementia and parkinsonism; mitochondrial myopathy; Leigh syndrome; Friedreich’s ataxia; Parkinson’s disease; MELAS (Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes); pyruvate dehydrogenase complex deficiency; chronic kidney disease; Leber’s hereditary optic neuropathy; obesity; age-related systemic neurodegeneration; skeletal muscle atrophy; heart and brain ischemic damage; or massive liver apoptosis.

[0494] Embodiment A84. The method of any one of embodiments Al or A4-A79, wherein the disease or condition comprises Optic atrophy type 1.

[0495] Embodiment A85. The method of any one of embodiments Al or A4-A79, wherein the disease or condition comprises autosomal dominant optic atrophy (ADOA).

[0496] Embodiment A86. The method of embodiment A85, wherein the subject is characterized by having:

(i) a heterozygous OP Al gene variant;

(v) any combination of (i)-(iv) .

[0497] Embodiment A87. The method of embodiment A86, wherein the subject is additionally characterized by:

(1) not having a gain-of-fiinction variant, or compound heterozygous or homozygous pathogenic or likely pathogenic variant in OP Al gene;

(2) not having only benign or likely benign variants in the OP Al gene;

(4) not having been diagnosed with Behr syndrome;

(7) not having or having a history of any ocular condition in either eye;

(9) not having a history of retinal photocoagulation;

(10) not having a history or presence of retinal vein occlusion;

(12) not having dry age-related macular degeneration in either eye;

(13) not having high myopia (>6 diopters);

(16) not having any history of nutritional deficiency (including B12 and/or folate deficiencies), or not having a known deficiency in serum B12 or folate, not having had bariatric surgery;

(17) any combination of ( l)-( 16) .

[0498] Embodiment A88. The method of any one of embodiments A1-A87, wherein administering comprises administering multiple doses of the pharmaceutical composition to the human subject.

[0499] Embodiment A89. The method of embodiment A88, wherein administering comprises administering a first dose of the pharmaceutical composition to the human subject and a subsequent dose of the pharmaceutical composition to the human subject.

[0500] Embodiment A90. The method of embodiment A89, wherein the subsequent dose is lower than the previous dose following an indication that administration of the previous dose is not tolerated.

[0501] Embodiment A91. The method of embodiment A89, wherein the subsequent dose is the same as the previous dose following an indication that administration of the previous dose is tolerated.

[0502] Embodiment A92. The method of embodiment A89, wherein the subsequent dose is higher than the previous dose following an indication that administration of the previous dose is tolerated.

[0503] Embodiment A93. The method of embodiment A89, wherein the subsequent dose is the same as the previous dose following an indication that administration of the previous dose is effective.

[0504] Embodiment A94. The method of embodiment A89, wherein the subsequent dose is lower than the previous dose following an indication that administration of the previous dose is effective.

[0505] Embodiment A95. The method of embodiment A89, wherein the subsequent dose is higher than the previous dose following an indication that administration of the previous dose is not effective.

[0506] Embodiment A96. The method of any one of embodiments A1-A95, wherein the pharmaceutical composition is administered via intracerebroventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal injection, subretinal injection, topical application, implantation, or intravenous injection.

[0507] Embodiment A97. The method of embodiment A96, wherein the pharmaceutical composition is administered via intravitreal injection.

[0508] Embodiment A98. The method of any one of embodiments A1-A97, wherein the method further comprises administering an additional therapeutic agent.

[0509] Embodiment A99. The method of embodiment A98, wherein the additional therapeutic agent comprises a small molecule.

[0510] Embodiment A100. The method of embodiment A98, wherein the additional therapeutic agent comprises an antisense oligomer.

[0511] Embodiment A101. The method of embodiment A98, wherein the additional therapeutic agent comprises an ophthalmologic drug.

[0512] Embodiment A102. An antisense oligomer having any one of the following structures:

[0513] Embodiment A103. A pharmaceutical formulation comprising:

(a) an antisense oligomer, wherein the antisense oligomer comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOS: 6-275 or 280-299; and

(b) a pharmaceutically acceptable diluent; wherein the antisense oligomer is dissolved or suspended in a solution at a concentration of about

1 mg/ml to about 200 mg/ml.

[0514] Embodiment A104. A pharmaceutical formulation comprising:

(a) an antisense oligomer, and

(b) a pharmaceutically acceptable diluent; wherein the antisense oligomer is dissolved or suspended in a solution, and wherein the antisense oligomer has any one of the following chemical structures: [0515] Embodiment A 105. The pharmaceutical formulation of embodiment A 104, wherein the antisense oligomer is present in the solution at a concentration of about 1 mg/ml to about 200 mg/ml.

[0516] Embodiment A106. The pharmaceutical formulation of any one of embodiments A103-A105, wherein the antisense oligomer is present in the solution at a concentration of about 5 mg/ml to about 200 mg/ml, about 10 mg/ml to about 200 mg/ml, about 15 mg/ml to about 200 mg/ml, about 20 mg/ml to about 200 mg/ml, about 25 mg/ml to about 200 mg/ml, about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, 2 mg/ml to about 150 mg/ml, about 5 mg/ml to about 150 mg/ml, about 10 mg/ml to about 150 mg/ml, about 15 mg/ml to about 150 mg/ml, about 20 mg/ml to about 150 mg/ml, about 25 mg/ml to about 150 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, 2 mg/ml to about 100 mg/ml, about 5 mg/ml to about 100 mg/ml, about 10 mg/ml to about 100 mg/ml, about 15 mg/ml to about 100 mg/ml, about 20 mg/ml to about 100 mg/ml, about 25 mg/ml to about 100 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, 2 mg/ml to about 80 mg/ml, about 5 mg/ml to about 80 mg/ml, about 10 mg/ml to about 80 mg/ml, about 15 mg/ml to about 80 mg/ml, about 20 mg/ml to about 80 mg/ml, about 25 mg/ml to about 80 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, 2 mg/ml to about 60 mg/ml, about 5 mg/ml to about 60 mg/ml, about 10 mg/ml to about 60 mg/ml, about 15 mg/ml to about 60 mg/ml, about 20 mg/ml to about 60 mg/ml, about 25 mg/ml to about 60 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, about 40 mg/ml to about 60 mg/ml, 2 mg/ml to about 40 mg/ml, about 5 mg/ml to about 40 mg/ml, about 10 mg/ml to about 40 mg/ml, about 15 mg/ml to about 40 mg/ml, about 20 mg/ml to about 40 mg/ml, or about 25 mg/ml to about 40 mg/ml.

[0517] Embodiment A 107. The pharmaceutical formulation of embodiment A 106, wherein the antisense oligomer is present in the solution at a concentration of about 1 mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 12 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 18 mg/ml, about 20 mg/ml, about 22 mg/ml, about 24 mg/ml, about 26 mg/ml, about 28 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 80 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

[0518] Embodiment A108. The pharmaceutical formulation of any one of embodiments A103-A107, wherein the pharmaceutical composition is prepared by diluting a concentrate comprising the antisense oligomer.

[0519] Embodiment A109. The pharmaceutical formulation of embodiment A103-A108, wherein the antisense oligomer is present in the concentrate at a concentration of about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, or about 40 mg/ml to about 60 mg/ml.

[0520] Embodiment Al 10. The pharmaceutical composition of embodiment A 103 -A 109, wherein the antisense oligomer is present in the concentrate at a concentration of about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

[0521] Embodiment Al l i. The pharmaceutical formulation of any one of embodiments A103-A110, wherein the concentrate is phosphate buffered.

[0522] Embodiment Al 12. The pharmaceutical formulation of any one of embodiments A103-A111, wherein the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, or water for injection.

[0523] Embodiment Al 13. The pharmaceutical formulation of any one of embodiments A103-A112, wherein the antisense oligomer is solubilized or diluted in a solution comprising sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, and water for injection.

[0524] Embodiment Al 14. The pharmaceutical formulation of any one of embodiments A103-A113, wherein the antisense oligomer is solubilized or diluted in an isotonic solution.

[0525] Embodiment Al 15. The pharmaceutical formulation of any one of embodiments A103-A114, wherein the antisense oligomer is solubilized or diluted in a phosphate-buffered solution with at least pH 5.8.

[0526] Embodiment Al 16. The pharmaceutical formulation of any one of embodiments A103-A115, wherein the antisense oligomer is solubilized or diluted in a phosphate-buffered (pH 6.6 - 7.6) solution. [0527] Embodiment Al 17. The pharmaceutical formulation of any one of embodiments A103-A116, wherein the pharmaceutical formulation does not comprise a preservative.

[0528] Embodiment Al 18. The pharmaceutical formulation of any one of embodiments A103-A117, wherein the pharmaceutical formulation is suitable for an intravitreal injection.

[0529] Embodiment Al 19. The pharmaceutical formulation of any one of embodiments A103-A118, wherein the pharmaceutical formulation is packaged in a single-use vial. [0530] Embodiment A120. A kit comprising:

(i) a concentrate comprising an antisense oligomer (ASO), wherein the ASO comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOS: 6-275 or 280-299; and

(ii) a diluent, wherein the concentrate is miscible with the diluent; and

(iii) instructions for diluting the concentrate with the diluent.

[0531] Embodiment A121. The kit of embodiment A120, wherein the antisense oligomer is present in the concentrate at a concentration of about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, or about 40 mg/ml to about 60 mg/ml.

[0532] Embodiment A122. The kit of embodiment A121, wherein the antisense oligomer is present in the concentrate at a concentration of about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

[0533] Embodiment A123. The kit of any one of embodiments A120-A122, wherein the concentrate is phosphate buffered.

[0534] Embodiment A124. The kit of any one of embodiments A120-A123, wherein the diluent is a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, or water for injection.

[0535] Embodiment A125. The kit of any one of embodiments A120-A124, wherein the diluent is a solution comprising sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, and water for injection.

[0536] Embodiment A126. The kit of any one of embodiments A120-A125, wherein the diluent comprises an isotonic solution.

[0537] Embodiment A127. The kit of any one of embodiments A120-A126, wherein the diluent comprises a phosphate -buffered solution with at least pH 5.8.

[0538] Embodiment A128. The kit of any one of embodiments A120-A127, wherein the diluent comprises a phosphate -buffered (pH 6.6 - 7.6) solution.

[0539] Embodiment A129. The kit of any one of embodiments A120-A128, wherein the concentrate or the diluent does not comprise a preservative.

[0540] Embodiment A 130. The kit of any one of embodiments A 120-Al 29, wherein the instructions for diluting the concentrate with the diluent comprise instructions for diluting or solubilizing the ASO to a concentration of about 2 mg/ml to 200 mg/ml in the diluent.

[0541] Embodiment A131. The kit of any one of embodiments A 120-Al 30, wherein the instructions for diluting the concentrate with the diluent comprise instructions for diluting or solubilizing the ASO to a concentration of about 5 mg/ml to about 200 mg/ml, about 10 mg/ml to about 200 mg/ml, about 15 mg/ml to about 200 mg/ml, about 20 mg/ml to about 200 mg/ml, about 25 mg/ml to about 200 mg/ml, about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, 2 mg/ml to about 150 mg/ml, about 5 mg/ml to about 150 mg/ml, about 10 mg/ml to about 150 mg/ml, about 15 mg/ml to about 150 mg/ml, about 20 mg/ml to about 150 mg/ml, about 25 mg/ml to about 150 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, 2 mg/ml to about 100 mg/ml, about 5 mg/ml to about 100 mg/ml, about 10 mg/ml to about 100 mg/ml, about 15 mg/ml to about 100 mg/ml, about 20 mg/ml to about 100 mg/ml, about 25 mg/ml to about 100 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, 2 mg/ml to about 80 mg/ml, about 5 mg/ml to about 80 mg/ml, about 10 mg/ml to about 80 mg/ml, about 15 mg/ml to about 80 mg/ml, about 20 mg/ml to about 80 mg/ml, about 25 mg/ml to about 80 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, 2 mg/ml to about 60 mg/ml, about 5 mg/ml to about 60 mg/ml, about 10 mg/ml to about 60 mg/ml, about 15 mg/ml to about 60 mg/ml, about 20 mg/ml to about 60 mg/ml, about 25 mg/ml to about 60 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, about 40 mg/ml to about 60 mg/ml, 2 mg/ml to about 40 mg/ml, about 5 mg/ml to about 40 mg/ml, about 10 mg/ml to about 40 mg/ml, about 15 mg/ml to about 40 mg/ml, about 20 mg/ml to about 40 mg/ml, or about 25 mg/ml to about 40 mg/ml in the diluent.

[0542] Embodiment A132. The kit of any one of embodiments A120-A131, wherein the instructions for diluting the concentrate with the diluent comprise instructions for diluting or solubilizing the antisense oligomer to a concentration of about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 12 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 18 mg/ml, about 20 mg/ml, about 22 mg/ml, about 24 mg/ml, about 26 mg/ml, about 28 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 80 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml in the diluent.

[0543] Embodiment A 133. The pharmaceutical composition or kit of any one of embodiments A 103 or A105-A132, wherein the antisense oligomer comprises a nucleotide sequence having at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 6-275 or 280- 299.

[0544] Embodiment A134. The pharmaceutical composition or kit of any one of embodiments A103 or A105-A132, wherein the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 280-283, 288, and 290-292.

[0545] Embodiment A 135. The pharmaceutical composition or kit of any one of embodiments A 103 or A105-A134, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

[0546] Embodiment A 136. The pharmaceutical composition or kit of any one of embodiments A 103 or A105-A135, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2’-O-methyl moiety, a 2’-Fluoro moiety, a 2’-O-N-methyl- acetamide (2’-NMA), or a 2’-O-methoxyethyl moiety.

[0547] Embodiment A 137. The pharmaceutical composition or kit of any one of embodiments A 103 or A105-A136, wherein the antisense oligomer comprises at least one modified sugar moiety.

[0548] Embodiment A 138. The pharmaceutical composition or kit of embodiment A 137, wherein each sugar moiety is a modified sugar moiety.

[0549] Embodiment A 139. The pharmaceutical composition or kit of any one of embodiments A 103 or A105-A138, wherein the antisense oligomer comprises a 5 ’-methyl cytosine (5’-MeC).

[0550] Embodiment AMO. The pharmaceutical composition or kit of any one of embodiments A103 or A105-A139, wherein each cytosine of the antisense oligomer is a 5 ’-methylcytosine (5’-MeC).

[0551] Embodiment A 141. The pharmaceutical composition or kit of any one of embodiments A 103 or A105-A140, wherein the antisense oligomer comprises a 5 ’-methyluracil (5’-MeU).

[0552] Embodiment A142. The pharmaceutical composition or kit of any one of embodiments A103 or A105-A141, wherein each cytosine or thymidine of the antisense oligomer is a 5 ’-methyluracil (5’-MeU). [0553] Embodiment A 143. The pharmaceutical composition or kit of any one of embodiments A 103 or A105-A142, wherein the antisense oligomer comprises a phosphorothioate linkage.

[0554] Embodiment A144. The pharmaceutical composition or kit of any one of embodiments A103 or A105-A143, wherein each intemucleoside linkage of the ASO is a phosphorothioate linkage.

[0555] Embodiment A 145. The pharmaceutical composition or kit of any one of embodiments A 103 or A105-A144, wherein the antisense oligomer comprises a locked nucleic acid (LNA).

[0556] Embodiment A146. The pharmaceutical composition or kit of any one of embodiments A145, wherein the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

[0557] Embodiment A147. The pharmaceutical composition or kit of any one of embodiments A103 or

A 105 -A 146, wherein the antisense oligomer has any one of the following chemical structures:

[0558] Embodiment A 148. Use of an antisense oligomer for the manufacture of a medicament for treating or preventing a disease or condition characterized by a reduced expression or function of OPA1 protein in a human subject in need thereof, wherein the medicament is administered to one eye of the subject at a dose of about 0.005 mg to about 20 mg, and wherein the antisense oligomer comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOS: 6-275 or 280-299.

[0559] Embodiment A149. The use of embodiment A148, wherein the medicament is administered to the one eye of the subject at a dose of about 0.005 mg to about 15 mg, about 0.005 mg to about 10 mg, about 0.005 mg to about 5 mg, about 0.005 mg to about 1 mg, about 0.01 mg to about 15 mg, about 0.01 mg to about 10 mg, about 0.01 mg to about 5 mg, about 0.01 mg to about 2.5 mg, about 0.01 mg to about 1.0 mg, about 0.01 mg to about 0.5 mg, about 0.01 mg to about 0.25 mg, about 0.01 mg to about 0.1 mg, about 0.01 mg to about 0.05 mg, about 0.05 mg to about 10 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 2.5 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.25 mg, about 0.05 mg to about 0.1 mg, about 0. 1 mg to about 5 mg, about 0.1 mg to about 2.5 mg, about 0.1 mg to about 1.5 mg, about 0.1 mg to about 1.0 mg, about 0. 1 mg to about 0.5 mg, or about 0. 1 mg to about 0.25 mg of the antisense oligomer.

[0560] Embodiment A150. The use of embodiment A148, wherein the medicament is administered to the one eye of the subject at a dose of about 0.005 mg, about 0.01 mg, about 0.05 mg, about 0. 1 mg, about 0.2 mg, about 0.3 mg, 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.75 mg, about 2.0 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, about 5.0 mg, about 5.5 mg, about 6.0 mg, about 7.0 mg, about 8.0 mg, about 9.0 mg, about 10 mg, about 12.5 mg, about 15 mg, about 17.5 mg, or about 20 mg of the antisense oligomer.

[0561] Embodiment A151. The use of embodiment A148, wherein the medicament is administered to the one eye of the subject at a dose of about 0. 1 mg to about 1.5 mg, about 0.1 mg to about 1.4 mg, about 0.1 mg to about 1.2 mg, about 0.1 mg to about 1.0 mg, about 0.1 mg to about 0.8 mg, about 0. 1 mg to about 0.7 mg, about 0.1 mg to about 0.5 mg, about 0.1 mg to about 0.3 mg, about 0.2 mg to about 1.5 mg, about 0.2 mg to about 1.4 mg, about 0.2 mg to about 1.2 mg, about 0.2 mg to about 1.0 mg, about 0.2 mg to about 0.8 mg, about 0.2 mg to about 0.7 mg, about 0.2 mg to about 0.5 mg, about 0.3 mg to about 1.5 mg, about 0.3 mg to about 1.4 mg, about 0.3 mg to about 1.2 mg, about 0.3 mg to about 1.0 mg, about 0.3 mg to about 0.8 mg, about 0.3 mg to about 0.7 mg, about 0.3 mg to about 0.5 mg, about 0.5 mg to about 1.5 mg, about 0.5 mg to about 1.4 mg, about 0.5 mg to about 1.2 mg, about 0.5 mg to about 1.0 mg, about 0.5 mg to about 0.8 mg, about 0.5 mg to about 0.7 mg, about 0.7 mg to about 1.5 mg, about 0.7 mg to about 1.4 mg, about 0.7 mg to about 1.2 mg, about 0.7 mg to about 1.0 mg, about 0.8 mg to about 1.5 mg, about 0.8 mg to about 1.4 mg, about 0.8 mg to about 1.2 mg, about 0.8 mg to about 1.0 mg, about 1.0 mg to about 1.5 mg, about 1.0 mg to about 1.4 mg, about 1.0 mg to about 1.2 mg, about 1.2 mg to about 1.5 mg, or about 1.2 mg to about 1.4 mg of the antisense oligomer.

[0562] Embodiment A152. The use of embodiment A148, wherein the medicament is administered to the one eye of the subject at a dose of about 0. 1 mg, about 0.2 mg, about 0.3 mg, 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, or about 1.5 mg of the antisense oligomer.

[0563] Embodiment A 153. The method of any one of embodiments Al -A3 or A5-A14, wherein the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 92-96, and 166-168.

[0564] Embodiment A154. The pharmaceutical composition or kit of any one of embodiments A103 or A105-A132, wherein the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 92-96, and 166-168.

[0565] Embodiment El . A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutic agent, wherein the subject has a flavoprotein fluorescence (FPF) intensity score within a reference value range and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer.

[0566] Embodiment E2. A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising:

(1) determining a flavoprotein fluorescence (FPF) intensity score of the subject;

(2) identifying the subject as an eligible subject for treatment when the FPF intensity score determined in (1) is within a reference value range; and

(3) administering to the eligible subject a pharmaceutical composition comprising a therapeutic agent, wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer.

[0567] Embodiment E3. A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising administering to the subject a pharmaceutical composition according to a dosing regimen selected based at least in part on a flavoprotein fluorescence (FPF) intensity score that the subject has, wherein the pharmaceutical composition comprises a therapeutic agent, and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer.

[0568] Embodiment E4. The method of Embodiment E3, wherein the FPF intensity score is measured before the subject receives administration of the pharmaceutical composition.

[0569] Embodiment E5. The method of Embodiment E3, wherein the FPF intensity score is measured after the subject receives administration of one or more prior doses of the pharmaceutical composition. [0570] Embodiment E6. A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising:

(2) selecting a dosing regimen for a pharmaceutical composition for the subject based at least in part on the FPF intensity score determined in (1), wherein the pharmaceutical composition comprises a therapeutic agent, and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer; and

(3) administering the pharmaceutical composition to the subject according to the selected dosing regimen.

[0571] Embodiment E7. A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising:

(1) administering to the subject a pharmaceutical composition according to a dosing regimen, wherein the pharmaceutical composition comprises a therapeutic agent, and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer;

(2) after (1), determining a flavoprotein fluorescence (FPF) intensity score of the subject;

(3) adjusting the dosing regimen for the pharmaceutical composition based at least in part on the FPF intensity score determined in (2); and

(4) administering the pharmaceutical composition to the subject according to the dosing regimen adjusted in (3).

[0572] Embodiment E8. The method of Embodiment E7, wherein the dosing regimen for the pharmaceutical composition is selected based at least in part on a FPF intensity score measured prior to the administering in (1).

[0573] Embodiment E9. The method of any one of Embodiments E3-E8, wherein the dosing regimen comprises frequency of administration of the pharmaceutical composition, dose of the pharmaceutical composition per a single administration, time interval between administrations of the pharmaceutical composition, duration of treatment with the pharmaceutical composition, or administration route for the pharmaceutical composition.

[0574] Embodiment E10. The method of any one of Embodiments E1-E9, wherein the FPF intensity score is within a reference value range.

[0575] Embodiment El l. The method of any one of Embodiments E1-E10, wherein the FPF intensity score is determined based at least in part on detection of FPF from one or both eyes of the subject.

[0576] Embodiment E12. The method of Embodiment El l, wherein the FPF intensity score is determined based at least further in part on a parameter selected from the group consisting of Best Corrected Visual Acuity (BCVA) letter score; Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score; high-contrast Best Corrected Visual Acuity (HC BCVA) letter score; low-contrast Best Corrected Visual Acuity (LC BCVA) letter score; Humphrey 10-2 Visual Field test Mean Deviation (MD); Humphrey 10-2 Visual Field test Pattern Standard Deviation (PSD); Visual Acuity Score (VAS); Quality of life questionnaires (NEI-VFQ-25, IVI-C, EQ-5D, EQ-5D- Y); Refraction test result; Minnesota Reading (MNRead) acuity chart score; Pelli-Robson chart score; slit lamp examination result; intraocular pressure using a Tonopen; perimetry; dilated fundoscopy result; retinal nerve fiber layer (RNFL) measurement; optical coherence tomography (OCT) result; and macular ganglion cell layer/inner plexiform layer (GCL/IPL) thickness measurements; Curve Width (CW) measurement; Electroretinogram (ERG) result; fundus photography result; and any combinations thereof. [0577] Embodiment El 3. The method of Embodiment El l or El 2, wherein the detection of FPF is performed when exposing an eye of the subject for an amount of exposure time to an excitation flash. [0578] Embodiment E14. The method of Embodiment El 3, wherein the detection of FPF yields an average pixel intensity over a region of interest (RO I) of a detectable signal emitted in response to exposure to at least one excitation flash for an amount of exposure time.

[0579] Embodiment El 5. The method of Embodiment El 4, wherein the detectable signal is a fluorescent signal.

[0580] Embodiment E16. The method of Embodiment E15, wherein the fluorescent signal comprises a green light having a wavelength, or a combination of wavelengths, between about 520 nm and about 540 nm.

[0581] Embodiment El 7. The method of any one of Embodiments E14-E16, wherein the excitation flash comprises a blue light having a wavelength, or combination of wavelengths, between about 430 nm and about 470 nm.

[0582] Embodiment El 8. The method of Embodiment El 7, wherein the wavelength of blue light is about 465 nm.

[0583] Embodiment E19. The method of any one of Embodiments E13-E18, wherein the amount of the exposure time is from about 1 to about 100 ms.

[0584] Embodiment E20. The method of any one of Embodiments E13-E18, wherein the amount of exposure time is about 60 ms.

[0585] Embodiment E21. The method of any one of Embodiments E14-E20, wherein the region of interest comprises at least one region selected from the group consisting of a macular-papillary (Mac) retinal nerve fiber layer (RNFL); an ocular global macula; an ocular Superior Nasal sector; an ocular Inferior Nasal sector; an ocular Nasal sector; an ocular Superior Temporal sector; an ocular Inferior Temporal sector; an ocular Temporal sector; and an ocular peripapillary retinal nerve fiber layer (pRNFL).

[0586] Embodiment E22. The method of Embodiment E19, wherein the region of interest comprises the global macula, the ocular Temporal Inferior sector, or the ocular Temporal sector, or any combination thereof.

[0587] Embodiment E23. The method of any one of Embodiments E1-E22, wherein the FPF intensity score is indicative of a level of mitochondrial dysfunction in the eye of the subject.

[0588] Embodiment E24. The method of any one of Embodiments El, E2, or E10-E23, wherein the reference value range is a range lower than a FPF intensity score of a healthy control subject.

[0589] Embodiment E25. The method of any one of Embodiments El, E2, or E10-E23, wherein the reference value range is a range lower than an average FPF intensity score measured from a population of healthy control subjects.

[0590] Embodiment E26. The method of any one of Embodiments El, E2, or E10-E25, wherein when the pharmaceutical composition is tested on a population of test subjects suffering the disease or condition, a FPF intensity score measured from the test subjects in the population is determined to have a correlation with therapeutic efficacy of the pharmaceutical composition in the test subjects, and wherein the reference value range is a range associated with the therapeutic efficacy of the pharmaceutical composition at a reference level according to the correlation.

[0591] Embodiment E27. The method of any one of Embodiments E1-E26, wherein the genotype of the subject is unknown prior to the administration.

[0592] Embodiment E28. The method of any one of Embodiments E2 or E6-E27, wherein the genotype of the subject is unknown prior to the determining.

[0593] Embodiment E29. The method of any one of Embodiments E3-E28, wherein the dosing regimen is not selected based on the genotype of the subject.

[0594] Embodiment E30. The method of any one of Embodiments E1-E29, wherein about 0.005 to about 20 mg of the antisense oligomer is administered to one eye of the subject.

[0595] Embodiment E31. The method of Embodiment E30, wherein about 0.005 mg to about 15 mg, about 0.005 mg to about 10 mg, about 0.005 mg to about 5 mg, about 0.005 mg to about 1 mg, about 0.01 mg to about 15 mg, about 0.01 mg to about 10 mg, about 0.01 mg to about 5 mg, about 0.01 mg to about 2.5 mg, about 0.01 mg to about 1.0 mg, about 0.01 mg to about 0.5 mg, about 0.01 mg to about 0.25 mg, about 0.01 mg to about 0.1 mg, about 0.01 mg to about 0.05 mg, about 0.05 mg to about 10 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 2.5 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.25 mg, about 0.05 mg to about 0. 1 mg, about 0.1 mg to about 5 mg, about 0.1 mg to about 2.5 mg, about 0.1 mg to about 1.0 mg, about 0. 1 mg to about 0.5 mg, or about 0. 1 mg to about 0.25 mg of the antisense oligomer is administered to one eye of the subject.

[0596] Embodiment E32. The method of Embodiment E30, wherein about 0.005 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.2 mg, about 0.5 mg, about 0.75 mg, about 1.0 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2.0 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, about 5.0 mg, about 5.5 mg, about 6.0 mg, about 7.0 mg, about 8.0 mg, about 9.0 mg, about 10 mg, about 12.5 mg, about 15 mg, about 17.5 mg, or about 20 mg of the antisense oligomer is administered to one eye of the subject.

[0597] Embodiment E33. The method of Embodiment E30, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0.1 mg to about 1.5 mg, about 0. 1 mg to about 1.4 mg, about 0.1 mg to about 1.2 mg, about 0.1 mg to about 1.0 mg, about 0. 1 mg to about 0.8 mg, about 0.1 mg to about 0.7 mg, about 0.1 mg to about 0.5 mg, about 0.1 mg to about 0.3 mg, about 0.2 mg to about 1.5 mg, about 0.2 mg to about 1.4 mg, about 0.2 mg to about 1.2 mg, about 0.2 mg to about 1.0 mg, about 0.2 mg to about 0.8 mg, about 0.2 mg to about 0.7 mg, about 0.2 mg to about 0.5 mg, about 0.3 mg to about 1.5 mg, about 0.3 mg to about 1.4 mg, about 0.3 mg to about 1.2 mg, about 0.3 mg to about 1.0 mg, about 0.3 mg to about 0.8 mg, about 0.3 mg to about 0.7 mg, about 0.3 mg to about 0.5 mg, about 0.5 mg to about 1.5 mg, about 0.5 mg to about 1.4 mg, about 0.5 mg to about 1.2 mg, about 0.5 mg to about 1.0 mg, about 0.5 mg to about 0.8 mg, about 0.5 mg to about 0.7 mg, about 0.7 mg to about 1.5 mg, about 0.7 mg to about 1.4 mg, about 0.7 mg to about 1.2 mg, about 0.7 mg to about 1.0 mg, about 0.8 mg to about 1.5 mg, about 0.8 mg to about 1.4 mg, about 0.8 mg to about 1.2 mg, about 0.8 mg to about 1.0 mg, about 1.0 mg to about 1.5 mg, about 1.0 mg to about 1.4 mg, about 1.0 mg to about 1.2 mg, about 1.2 mg to about 1.5 mg, or about 1.2 mg to about 1.4 mg of the antisense oligomer.

[0598] Embodiment E34. The method of Embodiment E30, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0.1 mg, about 0.2 mg, about 0.3 mg, 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, or about 1.5 mg of the antisense oligomer. [0599] Embodiment E35. The method of Embodiment E30, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition in a volume of about 5 pl to about 250 pl, about 10 pl to about 250 pl, about 20 pl to about 250 pl, about 30 pl to about 250 pl, about 40 pl to about 250 pl, about 50 pl to about 250 pl, about 60 pl to about 250 pl, about 70 pl to about 250 pl, about 80 pl to about 250 pl, about 100 pl to about 250 pl, about 120 pl to about 250 pl, about 150 pl to about 250 pl, about 160 pl to about 250 pl, about 180 pl to about 500 pl, about 200 pl to about 250 pl, about 220 pl to about 250 pl, about 5 pl to about 220 pl, about 10 pl to about 220 pl, about 20 pl to about 220 pl, about 30 pl to about 220 pl, about 40 pl to about 220 pl, about 50 pl to about 220 pl, about 60 pl to about 220 pl, about 70 pl to about 220 pl, about 80 pl to about 220 pl, about 100 pl to about 220 pl, about 120 pl to about 220 pl, about 150 pl to about 220 pl, about 160 pl to about 220 pl, about 180 pl to about 220 pl, about 5 pl to about 200 pl, about 10 pl to about 200 pl, about 20 pl to about 200 pl, about 30 pl to about 200 pl, about 40 pl to about 200 pl, about 50 pl to about 200 pl, about 60 pl to about 200 pl, about 70 pl to about 200 pl, about 80 pl to about 200 pl, about 100 pl to about 200 pl, about 120 pl to about 200 pl, about 150 pl to about 200 pl, about 160 pl to about 200 pl, about 180 pl to about 200 pl, about 5 pl to about 180 pl, about 10 pl to about 180 pl, about 20 pl to about 180 pl, about 30 pl to about 180 pl, about 40 pl to about 180 pl, about 50 pl to about 180 pl, about 60 pl to about 180 pl, about 70 pl to about 180 pl, about 80 pl to about 180 pl, about 100 pl to about 180 pl, about 120 pl to about 180 pl, about 150 pl to about 180 pl, about 5 pl to about 150 pl, about 10 pl to about 150 pl, about 20 pl to about 150 pl, about 30 pl to about 150 pl, about 40 pl to about 150 pl, about 50 pl to about 150 pl, about 60 pl to about 150 pl, about 70 pl to about 150 pl, about 80 pl to about 150 pl, about 100 pl to about 150 pl, about 120 pl to about 150 pl, about 5 pl to about 150 pl, about 10 pl to about 120 pl, about 20 pl to about 120 pl, about 30 pl to about 120 pl, about 40 pl to about 120 pl, about 50 pl to about 120 pl, about 60 pl to about 120 pl, about 70 pl to about 120 pl, about 80 pl to about 120 pl, about 100 pl to about 120 pl, about 5 pl to about 100 pl, about 10 pl to about 100 pl, about 20 pl to about 100 pl, about 30 pl to about 100 pl, about 40 pl to about 100 pl, about 50 pl to about 100 pl, about 60 pl to about 100 pl, about 70 pl to about 100 pl, about 80 pl to about 100 pl, about 5 pl to about 80 pl, about 10 pl to about 80 pl, about 20 pl to about 80 pl, about 30 pl to about 80 pl, about 40 pl to about 80 pl, about 50 pl to about 80 pl, about 60 pl to about 80 pl, about 5 pl to about 60 pl, about 10 pl to about 60 pl, about 20 pl to about 60 pl, about 30 pl to about 60 pl, about 40 pl to about 60 pl, or about 50 pl to about 60 pl.

[0600] Embodiment E36. The method of any one of Embodiments E30-E35, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition in a volume of about 5 pl, about 8 pl, about 10 pl, about 12 pl, about 15 pl, about 18 pl, about 20 pl, about 25 pl, about 28 pl, about 30 pl, about 35 pl, about 40 pl, about 45 pl, about 48 pl, about 50 pl, about 55 pl, about 60 pl, about 65 pl, about 70 pl, about 75 pl, about 80 pl, about 90 pl, about 100 pl, about 120 pl, about 150 pl, about 160 pl, about 180 pl, about 200 pl, about 220 pl, or about 250 pl.

[0601] Embodiment E37. The method of any one of Embodiments E1-E36, wherein the method comprises administering the pharmaceutical composition to both left eye and right eye of the subject.

[0602] Embodiment E38. The method of Embodiment E37, wherein the method comprises administering the pharmaceutical composition at the same dose to both the left eye and the right eye of the subject.

[0603] Embodiment E39. The method of Embodiment E37, wherein the method comprises administering the pharmaceutical composition at different doses to the left eye and the right eye of the subject.

[0604] Embodiment E40. The method of any one of Embodiments E1-E39, wherein the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NO: 6-275 or 280-299.

[0605] Embodiment E41. The method of any one of Embodiments E1-E39, wherein the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 280-283, 288, and 290-292.

[0606] Embodiment E42. The method of any one of Embodiments E1-E41, wherein the therapeutic agent further comprises a gene editing molecule.

[0607] Embodiment E43. The method of Embodiment E42, wherein the gene editing molecule comprises CRISPR-Cas9.

[0608] Embodiment E44. The method of any one of Embodiments E1-E41, wherein the therapeutic agent comprises the antisense oligomer, and wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

[0609] Embodiment E45. The method of any one of Embodiments E1-E41, wherein the therapeutic agent comprises the antisense oligomer, and wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2’-O-methyl moiety, a 2’-Fluoro moiety, or a 2’-O-methoxyethyl moiety.

[0610] Embodiment E46. The method of any one of Embodiments E1-E41, wherein the therapeutic agent comprises the antisense oligomer, and wherein the antisense oligomer comprises at least one modified sugar moiety.

[0611] Embodiment E47. The method of Embodiment E46, wherein each sugar moiety is a modified sugar moiety.

[0612] Embodiment E48. The method of any one of Embodiments E1-E47, wherein the antisense oligomer comprises a 5 ’-methylcytosine (5’-MeC).

[0613] Embodiment E49. The method of any one of Embodiments E1-E48, wherein each cytosine of the antisense oligomer is a 5 ’-methylcytosine (5’-MeC).

[0614] Embodiment E50. The method of any one of Embodiments E1-E49, wherein the antisense oligomer comprises a 5 ’-methyluracil (5’-MeU). [0615] Embodiment E51. The method of any one of Embodiments E1-E50, wherein each cytosine or thymidine of the antisense oligomer is a 5 ’-methyluracil (5’-MeU).

[0616] Embodiment E52. The method of any one of Embodiments E1-E51, wherein the antisense oligomer comprises a phosphorothioate linkage.

[0617] Embodiment E53. The method of any one of Embodiments E1-E52, wherein each intemucleoside linkage of the ASO is a phosphorothioate linkage.

[0618] Embodiment E54. The method of any one of Embodiments E1-E53, wherein the antisense oligomer comprises a locked nucleic acid (LNA).

[0619] Embodiment E55. The method of any one of Embodiments E1-E54, wherein the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

[0620] Embodiment E56. The method of any one of Embodiments E1-E55, wherein the therapeutic agent comprises the antisense oligomer, and the antisense oligomer has any one of the following chemical structures:

or a pharmaceutically acceptable salt thereof.

[0621] Embodiment E57. The method of Embodiment E56, wherein the antisense oligomer has any of the following structures:

[0622] Embodiment E58. The method of any one of Embodiments E1-E41, wherein the therapeutic agent comprises the vector, and wherein the vector comprises a viral vector encoding the antisense oligomer.

[0623] Embodiment E59. The method of Embodiment E58, wherein the viral vector comprises an adenoviral vector, adeno-associated viral (AAV) vector, lentiviral vector, Herpes Simplex Virus (HSV) viral vector, or retroviral vector.

[0624] Embodiment E60. The method of any one of Embodiments E1-E59, wherein the pharmaceutical composition is a liquid composition.

[0625] Embodiment E61. The method of any one of Embodiments E1-E60, wherein the method comprises administering the pharmaceutical composition as a bolus injection over 1 to 60 minutes, 1 to 50 minutes, 1 to 40 minutes, 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes, 1 to 5 minutes, or 1 to 3 minutes.

[0626] Embodiment E62. The method of any one of Embodiments E1-E61, wherein the method comprises administering the pharmaceutical composition as a bolus injection.

[0627] Embodiment E63. The method of any one of Embodiments E1-E62, wherein the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, or water for injection.

[0628] Embodiment E64. The method of any one of Embodiments E1-E62, wherein the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, and water for injection.

[0629] Embodiment E65. The method of any one of Embodiments E1-E64, wherein the antisense oligomer is solubilized or diluted in an isotonic solution.

[0630] Embodiment E66. The method of any one of Embodiments E1-E65, wherein the antisense oligomer is solubilized or diluted in a phosphate-buffered solution with at least pH 5.8.

[0631] Embodiment E67. The method of any one of Embodiments E1-E65, wherein the antisense oligomer is solubilized or diluted in a phosphate-buffered (pH 6.6 - 7.6) solution.

[0632] Embodiment E68. The method of any one of Embodiments E1-E67, wherein the pharmaceutical formulation does not comprise a preservative.

[0633] Embodiment E69. The method of any one of Embodiments E1-E68, wherein the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml to about 200 mg/ml.

[0634] Embodiment E70. The method of any one of Embodiments E1-E69, wherein the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml to about 200 mg/ml, about 5 mg/ml to about 200 mg/ml, about 10 mg/ml to about 200 mg/ml, about 15 mg/ml to about 200 mg/ml, about 20 mg/ml to about 200 mg/ml, about 25 mg/ml to about 200 mg/ml, about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, 2 mg/ml to about 150 mg/ml, about 5 mg/ml to about 150 mg/ml, about 10 mg/ml to about 150 mg/ml, about 15 mg/ml to about 150 mg/ml, about 20 mg/ml to about 150 mg/ml, about 25 mg/ml to about 150 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, 2 mg/ml to about 100 mg/ml, about 5 mg/ml to about 100 mg/ml, about 10 mg/ml to about 100 mg/ml, about 15 mg/ml to about 100 mg/ml, about 20 mg/ml to about 100 mg/ml, about 25 mg/ml to about 100 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, 2 mg/ml to about 80 mg/ml, about 5 mg/ml to about 80 mg/ml, about 10 mg/ml to about 80 mg/ml, about 15 mg/ml to about 80 mg/ml, about 20 mg/ml to about 80 mg/ml, about 25 mg/ml to about 80 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, 2 mg/ml to about 60 mg/ml, about 5 mg/ml to about 60 mg/ml, about 10 mg/ml to about 60 mg/ml, about 15 mg/ml to about 60 mg/ml, about 20 mg/ml to about 60 mg/ml, about 25 mg/ml to about 60 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, about 40 mg/ml to about 60 mg/ml, 2 mg/ml to about 40 mg/ml, about 5 mg/ml to about 40 mg/ml, about 10 mg/ml to about 40 mg/ml, about 15 mg/ml to about 40 mg/ml, about 20 mg/ml to about 40 mg/ml, or about 25 mg/ml to about 40 mg/ml.

[0635] Embodiment E71. The method of any one of Embodiments E1-E70, wherein the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 12 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 18 mg/ml, about 20 mg/ml, about 22 mg/ml, about 24 mg/ml, about 26 mg/ml, about 28 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 80 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

[0636] Embodiment E72. The method of any one of Embodiments E1-E71, wherein the pharmaceutical composition is prepared by diluting a concentrate comprising the antisense oligomer.

[0637] Embodiment E73. The method of Embodiment E72, wherein the antisense oligomer is present in the concentrate at a concentration of about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, or about 40 mg/ml to about 60 mg/ml.

[0638] Embodiment E74. The method of Embodiment E72, wherein the antisense oligomer is present in the concentrate at a concentration of about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

[0639] Embodiment E75. The method of any one of Embodiments E1-E74, wherein the concentrate is a phosphate-buffered solution.

[0640] Embodiment E76. The method of any one of Embodiments E1-E75, wherein the disease or condition is associated with a deficient amount or activity of the OPA protein.

[0641] Embodiment E77. The method of any one of Embodiments E1-E76, wherein the disease or condition comprises an eye disease or condition.

[0642] Embodiment E78. The method of any one of Embodiments E1-E76, wherein the disease or condition comprises a cardiovascular disease or condition.

[0643] Embodiment E79. The method of any one of Embodiments E1-E76, wherein the disease or condition comprises a neurological disease or condition.

[0644] Embodiment E80. The method of any one of Embodiments E1-E76, wherein the disease or condition comprises ADOA-plus; a mitochondrial disorder; glaucoma; normal tension glaucoma; Charcot-Marie-Tooth disease; mitochondria dysfunction; diabetic retinopathy; age-related macular degeneration; retinal ganglion cell death; mitochondrial fission-mediated mitochondrial dysfunction; progressive external ophthalmoplegia; deafness; ataxia; motor neuropathy; sensory neuropathy; myopathy; Behr syndrome; brain dysfunction; encephalopathy; peripheral neuropathy; fatal infantile mitochondrial encephalomyopathy; hypertrophic cardiomyopathy; spastic ataxic syndrome; sensory motor peripheral neuropathy; hypotonia; gastrointestinal dysmotility and dysphagia; optic atrophy; optic atrophy plus syndrome; Mitochondrial DNA depletion syndrome 14; late-onset cardiomyopathy; diabetic cardiomyopathy; Alzheimer’s Disease; focal segmental glomerulosclerosis; kidney disease; Huntington’s Disease; cognitive function decline in healthy aging; Prion diseases; late onset dementia and parkinsonism; mitochondrial myopathy; Leigh syndrome; Friedreich’s ataxia; Parkinson’s disease;

[0645] Embodiment E81. The method of any one of Embodiments E1-E76, wherein the disease or condition comprises Optic atrophy type 1.

[0646] Embodiment E82. The method of any one of Embodiments E1-E76, wherein the disease or condition comprises autosomal dominant optic atrophy (ADOA).

[0647] Embodiment E83. The method of any one of Embodiments E1-E82, wherein the pharmaceutical composition is administered via intracerebroventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, implantation, or intravenous injection.

[0648] Embodiment E84. The method of any one of Embodiments E1-E82, wherein the pharmaceutical composition is administered via intravitreal injection.

[0649] Embodiment E85. The method of any one of Embodiments E1-E84, wherein the method further comprises administering an additional therapeutic agent.

[0650] Embodiment E86. The method of Embodiment E85, wherein the additional therapeutic agent comprises a small molecule.

[0651] Embodiment E87. The method of Embodiment E85, wherein the additional therapeutic agent comprises an antisense oligomer.

[0652] Embodiment E88. The method of Embodiment E85, wherein the additional therapeutic agent comprises an ophthalmologic drug.

[0653] Embodiment E89. The method of any one of Embodiments E1-E88, wherein the subject is a human subject.

[0654] Embodiment E90. The method of any one of Embodiments E1-E39, wherein the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 92-96, and 166-168.

EXAMPLES

[0655] The present disclosure will be more specifically illustrated by the following Examples. However, it should be understood that the present disclosure is not limited by these examples in any manner.

Example 1: Identification of NMD-inducing Exon Inclusion Events in Transcripts by RNAseq using Next-Generation Sequencing.

[0656] Exemplary genes and intron sequences are summarized in Table 1 and Table 2 (SEQ ID NOS indicate the corresponding nucleotide sequences represented by the Gene ID Nos). The sequence for each intron is summarized in Table 3 and Table 4. Table 5 lists sequences of OPA1 antisense oligomers of this disclosure.

Table 1. List of exemplary target gene sequences. Table 2. List of exemplary target gene sequences.

Table 3. Sequences of exemplary target introns in pre-mRNA transcripts.

Table 4. Exemplary target gene intron sequences aatgttcattttaagcctactttagtagcctttatttgggctagagatgtattcctctttctgatatttatgggttatctgtttaacccttttatatctccctttcc cgatttgtaaatagagactggcaagactttttaccctgagtagagcaccaaacatggcttgtttctgcccacactgtagtacctgaggggaagtaaa tgggactttaaaagcaatttatgctcttttatagtgaaatatccctctactatcccgaaagactgtacctacaatatcctccactccttccccctgtagt tactatagagatgacttttcggttcttcactgccataatgatcaaaatcctaatcatgagatttttatcatccaggcatgtgaggtttactgatgcataaa accgcaagtactttttgtgttttttaatgttttttctctcttatctctgaaagtctaagtagatcatcatttttgatgtctatagtagcaactaataaattttcc ctgtatcttctcagcaaaagaactcaagcagagacagaagatagaactaccatggtagttttgcttcctatggatatgtcacatacatagaaattttta caatgacctttttatatatgtatttcagaatttcagaatggcctcaatgcctaataggaagaaatacttgaaatttttaaattagggcttggttttgtgagga gctagtaaaggttttctctttcagctttagctgtttctgcggaggattccgctctttctccatcagtttcatagccctggaatgtagaaaagctctggtttc aagaccatgatatccatttctgtcagggtgagttttaaatttatttcatgatgcaaacaatatattgaacaacaggacatgaactgttctgttgtaagtgg ctgaattttatcagtaaagcacatcaaaataaaatataccccaatgctagtaagacctagagtgacagatgaaaatagctgtgtattctctaagaa aatatataaaaatatcatctcatcaatctttaatgtttgttttataaatctaaatgtttttatatgtttcctaggaaatataggtctaattttttactttaccacca gctgtcttttattttactctttttttgagacggagtttcgctctgtgctaggctagagtgcagtggcactatctcagctcactgcgacctctgcctcccgg gttcaagcgattctcctgcctcagtctcccgagtagctgggatacaggcacatgccactacaccaggctaattttgtatttttagtagagacggggttt cttcatgtggtcaggctggtctcgaactcccgacctcaggtgatccgcctgcctcggcctcccagagtgctgggatacaggcatgagccaccgca cctggccagctgtcttttaatataacatatgataatgtgatgttccattaaactaagcggagaggaaacatgctggtaaaccatgtgtgagtattcatt gtaccagaaaggcaaatgatacattttatcctaaaattcaaatttataaacatcttaacactgtgatcataaatactactaatctagcatataaatatattt gtaggcggggcacggtggctcacgcctgtaatcccagcactttgggaggctgaggtgggcagatcacgaggtcaggagatcgagaccatcctgg ctaacatggtgaaaccccatctctactaaaaatacaaaaaaaattagctgggtgtgctggcgggcacctgtagtcccagctacttgggaggctgagg caggagaatggcgtgaccccaggaggcagagctccagcctgggcgactccgtctcaaaaaaaaagaaaaaagaaatatatttgtaatattctact aacctatatcattaactttttatataacttttttattttaccaaataagtaaccttttatagccctggctatactaaacatcctaacttttttgtttaatgtat tagtttttaagtatgccccagatgtcaagtaatgtggattttctataataatttaggatatatgcatgaagtcagtagtatttacatttaaaactaaaaca atttatactaatacagtttatacatttcatactaatttagctacagtggataaatatttaatggaacaaagtaaatcaaagtaccttttcaaatgaatggaaa taaatccacataacaattttttatgaccacactatacagtgtgatggcatgccaaatgatcataatgtggaatatgtatttcttcatggctttcaagattc tgttctttagtttgtgggctcctctccaactgctgtctcctcacagtttaggcgactgtttataattctgtccatcctgcataaacacacacagtcaaaat gaaaaaaagctctatcagcagatctgtgctgctgtacagaaatgggaaaacaatgaagtttgcattatcttttttctaattaccagatcgtttttggagc tatttaggcatacgcttttaaggaaaaaagaaaaaaagagtgtaccttttgtttctaacaaaggtgtatctatatatgaaataaaaaatggggatagt tatgacaaagtatttagaaataggaataaaatctaaaataacttttcatagcatggacaagactataatgtctacctcaataagcaaatcatttaaaaa tttttcatgtatatttgctgccatgatgtgtgtgattgctaaataaccaatgaatgaagatcaacaaggatttaaatgaagaagaatatggatttaactat ttctcctgtgaaataagttcatatttacaagttttgattttcagaaattagacaatatttttaaaggctgggatgacaacttctgcctctaccaagaagtca aagcacagttatgtgaatcatcataaatcacatcatttttatatattttgtatttataatgtatgtgactactttaaaacctgtataaaataaaatgtttttta atattttattttagaatatagcattaataacaatttgaagtagtttacacaatacctgtgagttttatttttgttttatatgaaataattttagtgctttactgg cttcatgctatggatgcatctctgtgtacgagtagcagatctttcctggaactgaatttaaaagcaagcatttggctccactaaatctctgaaaatg caactgtctttgcatttatacataattcgctactatggtacagaaatggatacaatacaaaaatatttccttataagatacactgtgaccaatgagctttt taaatagctgtaatcagtaacatgtatttgacttttcaaaacacatttctggagggatatcagtgctttatttccccaaatatctgaatccctatgctttagtac aaaacaactctgaagaatttagtaaccatatgtgtgatctctgtttttctaactagtctttcataagaaatgactagaatagcaacagggaaatgatgc cttttaaggtttttgtttctcaatataaaattttggtgaaccatttttatgataaatacaggtatttttactttctaaatcactgatttaaaatactttgataaat atgcatataaagtcagtgtttttaactctcaatactatcaaaaaaatttaactgctgtacatctgtataaacctaattctattcaactaaaatattttaaac atttag SEQ ID NO: 5

Gene: OPA1

Intron: GRCh38/ hg38:chr3 193593374 to 193614710

Intron Sequence: gtaagtgcaggctctaatctggccccgttaattctggggcctcttgagagtggggctgtcttatctctatctccaaaaatgtgcaggtgactctcaggcc aggccgacggcagttggagaattcccagatgttcttgaggacccagaatgacaggagccctggctgggcttacgttcggagccggcttcaatactg gcccttttctctggccctacccaacccgaaaattctggacgcctctcaatcttggcccgtctctattgtccttttgtctctgccctttacacccttgtgtcttc agtgttctgtctgtctctggttgcctcttttgccttttttctgtcctctccctgccaggtttggctctgtccatgagtcacctctctccacatttctcctaactctc ggtgtcttctttttcttccatttccacgccatgtgtacattgcatcttcaggtacctgggctcttctatcggggaaaggggcgtccgtctctttccctagccc gctgatagaagtcagaactagagcaatgacgcacacggtgtcagagacggtgattcgagatgccctttcaatagcagcttttttctgtgtttcgggag ggagacttactttttgatgcaaggtcgtgaacgtggcaccacctttctaatctcaatcattgttgccctggggtggtttaattctaaatagaaaatcataga aatcttttcatttctgtgcgttactatatgcattgtaatgagattaaattggattttataggaaattttgttctagtatcattagataccttcaagcttagctcattg ttgcaggcatttgataggaagtaagatgcatcaagcaaaattggaaaaacgtggttttcctgaattaacttctaagcagttgttttgaattttttccagacct ttttaagtggtatagataatttatcgtgtttataaggaatggaatgcattcgttagtttgtttttgttttgttttgagacggagtcttgctctgtcgtccaggctg gagtgcagtagcgctatctcggctcactgcaacctccgcctcccaggttcaagcaattctcctgtctccgcctccggagtagctggaattacaggcac gcgccagcacgcctagctaatttttgtatttttagtagagagggggtttcaccattttggccaggctggtctcgaactcctgacctcatgtgatccaccct cctcgacttcccaaagtgctgggattacaaccgtgagccaccgcgcccggcccaatttgttttatataggttaactggagtccaaaatacagaactag atgagataacaatagttaacagtgttagtcagttagaattattgcataggtatttttaatctcatggaattttagtctttgagtaagttcacagcccttggtatt aaagtaagttatttacaacccttgcatttctacttctcaatatttagtgaggaaacatatctgattttctttaaataaaaagagaaaagactgcagaagatag cattctctgttggagcaattaagatgtataagaagaactacaaagacggagttttaaaacaaactgatttataagtggtatttatttaattggctgtcattgg gctaaattatttctaaagttaccatggatgccattgagtcatggcttaaaaatgtctcctggtgatggcacagtttagctacctaaagaagtagagatgtg ggaagccagaagccccaagctctgcagtttttcttttgctatagttcctttgcatgttgtgaaagaatacagttaaattcctgctccctaacagatgagag cataagcatttctttgggcatacatatgtaaatacatgctcatggacatgtgaaaagatcaatactaacatttgggtgcaataaataattgtgtaaaattatt tttaaaagaattacatattaggaaatgatatattgattaaaagtgatagtcaatgaacaagagagtagatttctgggggaaacctattttgcatcatacttg atttttagttttgactgaatattgaagtctatattcaaaattcttttcctttagaactgtaaaggcattgctgcattttcttctaatgtaattgtttattgctgctgag aattcttatgacaatctgattttttcatcttcatgattatcttgtttttcccttcatggaatctgttagggtcttgactttatcctttatcctaaatttctcaaggcttg gaccaggtgtgggtttggttttgttttcttttgctactcatttgacttggcacactcagtgggcctttccctttatctttcttcatttctgagacgtttttctctctta ttttttattatcttcctttcatttttcctgtcctttttctttctagacatctcttaggaggatagtggtcctcttagattgatatgttatgtccgtgatttccaaagtaa gatttgtactcgtcgtctgttaaaaggaaaagcatacatataccctatgtatatatgcacacttttttatttttaaattatatatgtatctgtactaattatttacat tgtaagtcaaccctaacataatcttaaaggataagatacaaaacatactgcatctagaagcttcagtactttcttcctgaatcccagtagatccttttgttca tcccacgggatgcattccgcccccatcctcccactccctttggataccacattaccacagctctgcatcacttaactttcctcttatgtttttcaccttttttttt tttttttttttgcattttatgtcctggggaatttccttaattcatttcatggttttactgttgatttttttaatattggccatcgcaacttttcttttcttttcctttcctttcc L LLccL LLccL LLccLL L LcL LL LcL L LLcLL L LcL LL LcttaaL L LLcLL L LcL LL LcL L LLcL L L LctgttcL L L LcLL L LcL LL LcL L LLcL L L LcLL Lcacacaggatcttggcg tg t tgtccaggctggcctcgaactcctgggctcaggtaatcctctcaccttggcctcccaaaatgccaggattacaggcgtgcgccactgcatttggcgg caacttaatttttttatttttatttttccttttagaggacacctagcactgagcattgcaacttttcatttccatgaacttttaagaaaactcttaaagacatgttta attctgtacactttctattgttctttgattgctgtttttgaataacaacaaggagtacgccttagcattttgatggtatcctcttaatagtcgcaataatagtccc cttggcgctctgtatactctcaagtcttaaatgttttgtatgcagctgtacgttgacagttgaatggtctcgctccaagtggatcagcaagaacataaaga atcatttaactggtacaggctgcggcttgtgaattccctattaacaccaaagaagacgtgtgagactccgtactgaaactaaagacgacttgtgagttc cacactgagatcaaataagtctttatgatggtgacagagagtggtgtcaacgcctaaagttttggtaatctctctaaatgaggggctgaccaaaagg gggaacttaactgtattagacataattttgagaaacatgggtatgtggatggtaatggaggaaatgggtgtagatgagattgcctagggagagtgaga agtaggttaggtctaagccttgatgagttcccaacatttccaagggtagttgaggatactgaaaatgagtggccagtgagatagaggtaaagctaga gactgcccaggggagaggaattttcaacaatgaggaggtgtcaacattgtcaggtatgctgagaggtcagataaaaccagaattgagcaaaatgg ccatggaagcctatggtgccctccgtaagagctgtttcgctgaagtgatagaaacggaaatcaggctgggcacagtggctcactcctgtaatccca gcactttgggaggccgaggtgggcggatcacctgaggttaggagttcgagaccagcctggccaacatggtgaaaccctgtctctactaaaaataca aaaagtagccaggtgtggtggcaggtccctgtaatcccagctactcaggaggctgaggcaggagaatcgcttgagccccagaggcggaggttgc agtgagcagagatcgagccactgcactccaacctgggtgacagagcaagactccgtttcaaaaaaaaaaaaaaaaaaagaaatggaaatcagga tggtttggcttttattttaataaaatagctagagcagggaaatggggtactttttttcccccttttaagatgagacatagccaggtgcagtggcttacacct gtaatcccaacactttgaaagggagggtcgcttgagctcaggagtttgagaccagcctaggcaacatagcaagaccttgtctctactaaaattcaaaa aaaattaactgggcatgctggcacacacctctagtcccagctatttatgaagctgaggcaggaggatcacacttgagcccagatacgtggggctgc agtgagccctgataatgccattgcactccacgttgggcaacagagcaagacttcgtctcaaaaataaataaataccctgtctcaaaaataaaaaataa atatgggaggagagatttgacttagattcctcaaagggcaggaggaaagagaatccaaacagtgattcacctttaatgggagaaagatcgctaatt tacatgaggaagaagaggatggtggagatacagtaggtgaacagtttttgtatgaggaagtgaacatgtgtcattctaatagctccattctctgtga agtagagggcaaggtcatctactgagagtggggaggtcaagagagataaggggagattagaagagctcttctagcagagagtggaagaatgaat tgctaagagagatgaagtaggatgtaagtagttttgagggccctgtgagatgtgcttccagtgggtgtgattttctccagtagtgctttatttccctgg gtacaggcagagagaaaaacaataaggctcatgtagggtttgtattttgttggacaagtcaaacagaaaagtcagaggacgagggagttagaatgt ttgcaaaagagtatgaaacgatgaaccgcataatctaaggtggtaagtgggtgaatagataaggaggatgtgaataggtaaggagaagaaagaa atatcagatatgatatgatggcgactctctaatacagctatatgccattttaaccgattaagaaactaaggctttagaaaatcataatttgccctaac tgcacagctagtaagcagtggaaatgtgattggaaccagagtctctgactcaatagactaaatggatgtaaggatgtagttgaaagaagggtgagc taaacgtgtggaaccatgagctctttctctggttgatatccctctctgtaagtgataacatgggtcacgctggataaaacctgtggtgatggtgacttt cctttgtccttcctcctgtgcctagtctggcgagtatctgcctttccctttcctttctcatgctgccacctaactttaggctcttcccctacatctgggtaact gaaataagatcacctttttgttccccttctgatttactttgacctaacatatctttactattttctttaaataatgtttcatagtctattctactcaggaactctg tagttccccatgcctacgaaaaaaagttaagcctcagcctatattcagtgactcttcaatggatattcagtccagttttactcctcctatgagcctctat gccagctcctgggtctctgccctttcatgtctcagctctgcacccttctttctcttttttattcttttttttttttgtacttttttggttttctttttggtttctttttttgt tatttataaacctccatcacacttcatcctatggagttttgaaccacagcaaggtgcagtatcatcctggggctctggaggaagtggcagggagtcc aaaatgtcacctagcttctatctggggccacatgtatttctgcatctgctgcttcccacactctgcccacaagtgtcgctgtggaaataatttgagatt tactgtctggctgaccctagtttcaatctcttttccaccatttgctaatcatctaccttgggcaaaacatagaattaaaagaaaacttcagacaagttaaat ttgatggagtttaattgagcaaagaaaaaaaatgatccacaaattgggcagtctccagaatcaccgcagattcagagagactccaggggtgcctcgt ggtcagaacaaatttatagacagaaaaggtaaagtgacctacaggaatcagaattgagacatagaaacagtgagatggttacagctcggcgtttgc ctatttgaacgcagtttgaacactcagcagtctatgagtggttgaagtatggccgctgggattggccaacactcagctgttatacagatgcatactac taagtaggttttcgattttgtctgcctatttgagctaggtacagttcgtccacaaggactcaaatataaaagtacggagtcctcttcgggccatatttagt tcgcttaacaattcccccttttggtcagcccctcaatttagagagatgaccaaaactttaggcgtgacaccactctctgtcaccatcataaagactat tggtctcagtgtggaactcacaagtcgtctttagtttcagtatggagtctcacacatctctttggtgtaatagggaattcacaagtgcaactttgtacc agctaaatgattctttatgttctgctgatccagtggagtaagaccattcaactgtcaatgtacagctgcatacaaaacatttaagactgagagtataca gtgcaccaaggggactatattatgactgtaagaggacaccgtcaaaatgctaaggtgtactcctaataaaagttctatgaaatgaactgaaccaa atcagccaagtaaggttcagacaatataagcagttcagcagtatggggtctgatggtcagagtctcagtggagtatgatagtgataaggatcat agtcgctgtaaagtagctgactaaagaggtgctcgttttcatgttaccttgtaatacaagtcataataactgaaaacctgctagaagagatataaa gatagaaaccctggaaaacccaagctgccatcaccactaggatgcctgcaaaccaactgtagtgctcctataaacatatcgtgggttcctttct ctgagagatttctttatgtacttggtggcagtgtctaaggaaacagcagtatcagccaccttttaaattaagctttttgtagtaacagaatcaggggag ggatagtacaaaatcagttttgtttaacaccaaacataggcctccagcttgagcaaaaagaagatctaagactgcatgatctccattaagtgttttcgt tgaatatgtatgtgtcatgtgcctttctgagagtagcttctacccatctgaaaccctgggaggtctgatggctaccaaatccaagaattttcccaatata caaatagttttaaattccgtacaaatggtacttcactaccaccaagagtgagcccccaggaaccccagtggaatctttccccggtagaaactagcta tcctcgtctatttcgaggctagtgctaatttcagttatgatcattttggcctccaagtataagggctatcatgagaattttcaggggaagcaattcgaaag gcaggagcaggccaggccagataacaagaaccaaaccaaccaaggaggcagaacagaatatgcagatctccacagacccaatagagaccctc aggggtggaaaagggggccacctagtgtatttgagcagggatcattcaggtttgttcgaccatgaatctgtagctcctgaataacatccagtggga aatttacttttctatggcccctttgtagtgtgtgtaagggtgtataaccacatctagtaaaaagagaccctactggatatacaagcaatcactgtactaa cataagtaattcccaaatctgagtatgtgatgcctgcaagcacaatatacgttttgtaggcatcatttggatttgttttttatatttggtgtgatcgactttatc agtgaaaaagagtgtgtttttagtgagtgtaggaaagcaagtactagtgatgtttagagtatcaagaatagctttccattcttccctggggtttcaggg tgactcatgggaaacgtggaggggcactggcacccttggaatcatttcctgattttttggcattagcccacaaacccaacagttaccctggttttgtgc tagagcataagctgagctgaagccatccactgattatggtcccatggattttcatgtaaggaaaaggaaaggattagggaaaaaaataaggaaaac agaaaaacacataaggctttcatggtggtagagaagtctgatctgtgatctagggaaagctgtctgtaaccaggatgctgtctgctctgggaagag atttccctggtcagctttacctaaagtctccaacgggtatatagtaccaggagtctgagggggcccttttgaatgtgagatgtggacccatggttcaa agccctgaagcttctctgcactgtgggtggtaagaaggactggtatggtcccatccaacgaggtcaagagtgatcttcttctgatgtcatttccggaa ggcccagtctccaaatccagaccatggagggtttgattgtcctcagtggtggatctgaaatgctccttacctggtggaagtatactttggcgtaat acataaagcctgcagtatttagtcatatcagagtttaagagagcaggagaagcatgagatgctattattagggacatgggcctcccagtgactatttc ataaggggtcaatttatgttttccaacaggatgaatctgatgccataaaaccaaaaggtagtacctttggccaaggcaactcaatgattcagtaac tggacagtttcagttttcaaatgccatttgttctttcaagctttcctgaagactgagggtaataaggacaatggtaatgcaactgtgtcagtaacacctat taactgctttataactgctcagtaaaatgagtcctctatcactggagacttttagagggatcccccataaaggaaaaacattttctaataatttcttagct atggtcacagcatcagctttcctacatgggaaggcctttatccaaccagaaaacatgcaaactattacaagaacatactgataccccattgagggtgg taactgaatgaagtccatctgtaaatgttcaaatggtccatcaggtggtggaaatatactgcctctagtttttctgggatatgagtttgacaagtcaaaca tgatataagccattttagtaatgtcagaatagtcaccccaccagtattttttcataatttggatcactttgtctgttccatgatgagctgtggagagctttca ataatggaagcttcaaagattcaggaaggaccaggcggccgtccgggccctttgtgagtctttgcttcacgtaaatttacatccttttagataccagttt tgtttttgcaaatcagatgcgtgcactgttataaataggtcatcgtaaggaaatggctggataatctatggagttcattcagatgcgtatctgatg gttccagcactagctgatgagcataaaaatctgctaaagcatttcactgatatttgggttcatttctacaagtatgagcttcagtctaataacagcaatct gcatttgtaacaggatagcagaaaggagctcatctgtttggagtccatttttgatggggatcccactagaggtgagaaaccttcgtagtttccatatcat gccaaaatcacgtactactccaaaagcatgtctactatccgtaaatatttactgactgtcctagctgtgtgacatgtcaggtaagggcagaaagtct gcaggtgggctgactgactgaagagtcgcttctctattaactcattttgggtggtaacagcatatcctgactgatatttttttttctgagtttttggcata ggacccatcaacaaaaagtgtaattcaggatatccagtggagtatctgtatagcaacacgaggggccactatttctgatactacactcacaccgt gtggtctcaccatcatcaggcagagataacagagtagcagcattaagtagattacagccttttagatgaagataagaaggagataggagaagtaat cataagatgtagtctactcactgaaaaatgctgggtttgattggaatttaatagactttccacagcgtgtgggacttgcaaataagtcatttcctaaaa ccagatctgatgaagcttctaccagctggctgctgctactgcttttaaacaataggatatgcctagagactgggcctaatgcaggctatagtatgca gtggtcctatgtttagcaccgtgttcctgatatacatcatgaacaaacaaagtgaaaggtttagtgtaatttggaagtcctaaagctgggggctgtgt aaggccaacttcatttggctaaaagcctgctcatgactgtcttcccaaggtaaaggctctggtacagcatttttagtgagctcatacagtggtgaagcta taaggaaaaatttggaacccaggatctgcaatatcctgcaagcctaagaaagccttttgtcttttggttgcaggtcgaggaaaactttaaataggtttta tcctctcaggtaagagggaaatcccttcagcagccaagtcatgtcccaaatagtggactttttcttttgaaaatgaagttttggccaggcatggtggct aacgcctgtaatcccagcactttgggaggctgaggcaggcggatcacctgaggtcgggagttcaaggacagcctgaccaacatggagaaaccct gtctctactaaaaatacaaaatagccaggcgtggtggtgcatgcctgtaatcccagctactcgggaggctgaggcaggagaatcgctgaaccca ggaggcagaggttgtggtgagccagtatcacaccattgcactccagcctgggcaacaagagtgaaactccatctcaaaaaaaaaaaaaaagaaaa aagaaaagaaaaaatgaagtttttccatgaagccctgtgacctttatatgcaagttgctgtaaaaggtaaactgagtcaatttccgggcactccttaat aggagagcataacaataagtatctacatactgaatgagagtagaattttgaggaaactgtagtgtcattaactcctgatgcagtgcctggggaaaata tgaaggggctcagtaaaccctgtggcatacactccaggtgtattgctgatttttccaagtaaaggcaaacaagtattgactttctttatggaatgctag agaaggctgagccaagatctattactgtggacaacttggaatcagtgggtacattaggttataaagtattaggatttgggactacaggaaatcttggtat tacaattttataatgcctgtaaatctggaacaaatctccagtctcatccattttgttttttaactggtaggatggagtgtacaggggctggtgcatggaa tatgagtcctgtttaataaatctctacaattggtgagagccctaaattgcttcaggttttagtggatatgtggtaattaggcaaaggtttagaatgatc tgtagtacttttataggttctacacttttaatcttcctatatcagtgggaagaggcccataaacataggtgttttcgaaagatcaggggtatacaggct tgagtttcgatcttatcaattctgcctgtagacagcataacaattctagttcaggagaatcaggaaaactctaagatatttctgtttctgaggaaaatttt aggtgcccttttagctttgaaagtaaatcttgccctaccaagtttactggaacagtatcacgtagtaaaaaactgtgtttttctgaaagggggctcagagt taatggatgggtcagatatgggaacctctggaactgatttgaaacccctgtcacagaaatgacctttttactctaagggatttgttggcttattaaggt ggggtttatggtagatagagtagccctggtatccataaggactatacacaactccctatttattttaacctctgtttccccatgttcctttaaaggtatacg gggagcaatccactggagaatcccttagagcctcctttaagtgaatattgtcaggaggactaaggtctcttgggctccctctagtggtgaaacagttt ggcctagagggaggtttatcagccgacaatcccttttccagtgccctggttgtttgcaatacaggcagacatctggggtaaagaaatctgttctggg acctctgatttgatttttttaatatataattttaaaaatattttccaaagtgtgactaaaaaaatttttttttattatactttaagttttagggtacatgtgcacaac gtgcaggtttgtacatatgtatacatgtgccatgtggtgtgctgcacccataactcatcatttacataggtatatctcctaatgctatccctcccccctc ccccaaccccacaacaggccccagtgtgtgatgttcccctcctgtgtccaagtgttctcactgttcagttcccacctacgagtgagaacatgcggtgt tggttttttgtcctgtgatagtttgctgagaatgatggtttccagctcatccatgtccctacaaaggacataactcatcattttttatggctccatagtatt ccatggtgtatatatgccacattttctaatccagtctatcatgtggacattgtgtggttccaagtctttgctatgtgaatagtgctgcaataaacatac gtgtgcatgtgtctttatagcagcatgatttataatcctttgggtatatacccagtaatgggatggctgggtcaaacggtatttctagttctagatccctga ggaatgccacactgacttccacaatggtgaactagtttacagtcccaccaacagtgtaaaagtgttcctatttctccacatcctctccagcacctgtg tttcctgactttttaatgatgccattctaactggtgtgagtggtatctcatgtggttttgatttgcatttctctgatggccagtgatgatgagcattttttcatg tgtcttttggctgcataaatgtcttcttttgagaagtgtctgttcatatccttcacccactgtgatggggtgtttgtttttctctgtaagtttgtttgagttcttt gtagattctggatatagccctttgtcagatgagaagtttcagaaattttctcccattctgtaggtgcctgttcactctgatggtagtttcttttgctgtgcag aagctctttactttaatgagatcccatttgtcaattttggcttttgtgccatgcttttggtgttttagacatgaagtcctggccatgcctatgtcctgaatgg tatgcctaggttttcttctaggatttttatggttttaggtctaaataagtctttaatctatctgaattaatttttgtataaggtgtaaggaagggatccagtttc agctttctacatatggctagccagttttcccagcaccatttataaatagggaatcgtttccccgtttctgtttttgtcaggtttgtcaaagatcagatagtg tagatatgcggcgtatttctgagggctctgtctgttccatggcctatatctctgttttggtaccagtaccatgctgttttggtgactgtagcctgtatagt tgaagtcaggtagcgtgatgcctccagctttgttctttggctaggattgactggcaatgcaggctcttttttggttccatatgaactttaaagtagtttttt ccaatctgtgaagaaagtctttggtagctgatggggatggcatgaatctataaattaccctgggcagtatggccattttcacgatattgattctccta cccatgagcatggaatgttcttccatttgttgtatcctcttttatttcctgagcagtggtttgtagttctcctgaagaggtctttcacatccctgtatgtgg attcctaggtattttattctctttgaagcaatgtgaatgagagtcactcatgatttggctctctgtttgtctgtatggtatataagaatgctctcttttgttct tgtagtctgctagcggtctatcaattttgtgatcttttcgaaaaaccagtactggattcatgattttttgaagggttttttgtgtctctatctccttcagttct gctctggtctatttatttctgccttctgctggcttttgaatgtgtttgctctgcttctctagttcttttaatgtgacgtagggtgtcaattttagatctttccta ctttctctgtgggcatttagtgctataaatttccctctacacactgctttgaatgtgtcccagagatctggtatgtgtgtctttgttctcatggtttcaaag aacatctttacttctgccttcatttcgtatgtacccagtagtcatcaggagcaggtgttcagtttccatgtagtgagcagttttgagtgagtttctaatc ctgagttctagtttgatccactgtggtctgagagacagtttgtataattgtatcttttacattttctgaggagagcttatttccaactatgtggtcaatttt ggaataagtgcagtgtggtgctaagaagaacgtatgttctgtgatttggggtggagagttctgtagatgtgtataggtccgctggtgcagagctga gtgaattcctggatatcctgtaactttctgtctcgtggtctgtctaatgtgacagtggggtgtaaagtctcccatatgtgtgtgggagtctgagtc tctttgtaggtcactcagggctgctttatgaatctgggtgctcctgtatggtgcatatatatttaggatagtagctcttctgtgaatgatccctttacc atatgtaatggccttctttgtctcttttgatctttgtggtttaaagtctgttttaccagagactaggatgaaacccctgcctttttttgttttccatttgctggt agatctcctccatccctttattttgagcctatgtgtgactctgcacgtgagatgggtttcctgaatacagcacactgatgggtctgactctttatccaattt gccagtccgtgtcttttaatggagcatttagcccatttacatttaaggttaatatgtatgtgtgaatttgatcctgtcattctctcaacatttgctgtctgta aaggattttatttctccttcactatgaagctagtttggctggatatgaaattctgggtgaaaattcttttctttaagaatgtgaatatggcctccactctct tctggcgtgtagagtttctgccgagagatcagctgtggtctgatgggcttcccttgtgggtaacctgacctttctctctagctgccataacattttttcct tcatttcaactttggtgaatctgacaattatgtgtctggagtgctcttttcgaggagtatctttgtggcattctctgtgtttcctgaatttgaatgtggcctg cctgctagatggggaagttctcctggataatatcctgcagagtgttttccaacttggttccatcttcccgtcactttcaggtacaccaatcagacgtag atttggtcttttcacatagtcccatatttcttggaggctttgttcgtttctttttattcttttttctctaaacttctcttcccgcttcatttcattgatttgatcttccatc actgataccctttcttccagtgatcgaatcggctactgaggctgtgcatccgtcacgtagttctcgtgcctggttttcagctccatcaggtcctttaag gactctctgcattagtattctagtagccgtcgtcgaatttttttcaaggtttttaacttctttgccatgggttcgaacttcctccttagctggatagtttga tgtctgaagtcttctctctcagctcgtcaaagtcatctctgtccagctttgttccgtgctggtgaggagctgcattcctttggaggaggagaggtgct ctgatttttagaattttcagtatttttgctctgtttcttccccatctttgtggttttgtctacctttggtctttgatgatggtgatgtacagatgggtttttggtgtgg atgtcctttctgtttgtagttttccttctaacagtcaggaccctcagctgcaggtctatggagtttgctggaggtccactccagaccatgtttgcctgggt atcagcagcggaggctgcagaacaacgaatattggtgaacagcagatgttgctgcctgatcgttcctctggaagttttgtctcagaggggtacccgg ccatgtgaggtgtcagtctgcccctactggggggtgcctcccagtaggctatcgggggtcagggacccactgaggaggcagtctgtctgtctc agatctcaagctgtgtgctgggagaaccactgctctcttccaagctgtcagacagggacatttaagtctgcagaggtttctgctgccttttgttcggctat gccctgcctgcagaggtggagtctacagaggaaggcaggcctccttgagctgcagtgggctccacccagttcgagcttcccagctgctttttttacct gctcaagcctccgcaatggcgggcacccctcccccagcctcgctgccacctgcagtttgatctcagactgctgtgctagcaatgagcgaggctcc atgggcataggacccgctgagccaggcgcgggatatagtctcctggtgtgctgttgctaagaccatcggaaaagcgcagtatagggtgggagtg acccaattttccaggtgctgtctgtcacccctttcctggctaggaaagggaattccctgacccctgtgctcctgggtgaggcgatgcctcgccctgc tttggctcatgctcggtgcgctgcacccactgtcctgcacccactgtctgacaatccccagtgagatgaacccagtacctcagttggaaatgcagaaa tcacccgttttctgcgtcgctcaagctgggagctgtagactggagctgtcctattggccatctggaaccgcccgatgtgatttaaaatgagaacga gatggtccctttggtcctggtccctgtaactgtgcaatgaaggggcataagctatagccttttgaggttttttttgctctagagtctctcaaaatgctt agctaggttgggcacgatggctcacgcctgtaatcccagcactttggaaggccaaggtgggaggatcacgaggtcaggagatcaagaccatcctg gctaagatggtgaaatcccatctctactaaaaatacacagattagctgggcatggtggcacacgcctgtagtcgcagctactcgggaggctgaggc aagagaatgcttgaacctgggaggcagaggttgcagtgagccgagatgcgccactacactctagcctgggtgacagagcaagactccacctca aaaaaaaaaaaaaaaaaaaaaaaagttcagctaaggccaccaattcagtcacatctctaacttcccatgcaactatgttttttagtaaactgctaagt tcaggatggagtccatttataagtaaagcagttaatgctgtttcagcccctgcagggaatactccttgctgtactttgagcccaggatgtttcacaaatat ttctaagcgacttctgtaatctgaaactggttcatcttttcttttcttttttttttgctacaagatgtatgatggaccaattttttgtggaaaaattttaggaactg aatgtaaaaggttttcagcgatttttctagctatttttggtccttctgtgaggagctcttagagggccctttaaaatgtcctcctcaggtttgtcccattctg ctgctgccatccatttctgagcttcaccagcccccagtatcatatgaataaatggtaaattcatgaagtcctggatcgtaagctcctataggattctaaa ttcctcagtaaatttttgagacttttccctggaccagggaagtcctcacaatggggctaagctcagttttagaccatggagtgaaagtagtacagca ggcaggcctggctgatataaggtctcactttgtaagacatctgtctaactccttttttttttttttttttttttaaatcatcttcagggtgaaagtgtaatttaaca aaaagtttagtggactcagagtatgtaggtagagatggacaaagaaggaacagtccgagttagatcagtcaaagtacagtcctctttcttcatgtcctt gaaatagcctaatagtgtggcttattttgagtgaaggccttagcccttaaggcaattaaatttactgtggagagaagagctaatctaatggggagaag gagcctttgtacaggtgtggtagtgtggttctttgagtgacaagatttctgtttgccagatggtaggagaagtctgtgtgtctgctttctctctatggcc taggatcactgtggtgaatgaaaaacctgtctcagggcctgactcagataattcccttaaaacccggctaaggtcatagatgaataatcagtaattgaa cagaagctctgcaatagaaaagaagccagataatatttttggaaatttaattatatttacagattttattttatacagtagacatggaattaaatttattacat tatgtctaatttactctttgctgttttgatttgctgtttgacaatacatgtcctgtaaactatttccttttaactttttctcaatttatggtgcttattttccccata aagactaccaattttttttttaactatttgttacacatactgaatctagagtgtaattaagctactttcatactggttaagtcaaatatagcaaatgctacta taaaaatttactatccaaaaatgtgtctcaagccccaactgatggtttcaaattctgtataataatatgcagcatgtgtttgcaaagctggctgtact gtgatgctgagaatgatgagtcactcagctaaactgagtgattttgagacttgtgtacaaattgatggtgaatgtaagcatgcaaagagagaccttag ctagcagtaccctttttgaaatcactctgacatcaagtttgaaaatgtgggcaataatcagaggtggtaaggtggccaggctttagctgaatactttttta actggttcagtctgagggctgaaagccccagatttaaacagtatttagaatttgaagcagtcaagtatagtttaatggtgtcaggtttgtaacaaagttt ctggctagacttctactagaaatgtaaaagtgcatgtgaatcagctttttaaaaaagtaataataatgaaaaacatttctacaactagaactaaagaaaa gatttgtcctttctaataggaaaacacatctggagaagtgctggcaactagcagaacagtaggaccatcagaatcaactgaagtgaaagtgacgg ggagctgaggggaacacagatagtttgacttcagtcagacagaataaacatgatgaaccgataacctgtgattcccagcctggggttactactggag ttttaggtgtcctggaaagtataataccggtcttcaaaaagtctacagaaagcatagatttccacataatgctgcacaggctaacgaataatcaagttt ctttggtttggcctggatttatatccattcagtttgtggacactactgaatatttatgtcatgtgatcaaaagtctgatatgatttgataatgaaacatga aaaaaatagtaaaaccaaccatttttaacctacactactatctgaggtatgatgacatacattaaaaccacctcttaataaatgctcttgttaatcaaaa atttgaaaacgtatgtccactggaggaaaaaagacatagccctggatgtgaactgaatatactgagactcggagaccttcagaactacctgaagatg aatcgaagtgctgcctactttagagaatggactaatttaatttgggagtcagcagatgctgtatatcagtcatcatatataccggtgacaagaccactt agttcattcccttttttagattctgtaagatatgtgttccagtgaaatgatttgcaaaatgagacattttattttctgtgcttttgttctatcatgtttctgatgg tcataagcatctcacagaagtaagaaatatggcgattcagaaggcaacaagcacatttataatttatagaaaatatttgaaggactttttcatggcccaa atcatgaaaagtagtagtatgttttaagtataatataaatataatacataatgttctttctgcaacatatactctcatctttttttttttttttttttttgagac ggagtctcactctgtcacccggctggagtacagtggtacgatctggcccactgcaacctctgcctcccgggttcaagcgattctcctgcctcagcct cccaagtagctgggatacaggctcctgccaccacgcctagctaatttttgtatttttagtagagacagggtttcaccaggttggccaggatggtctga tctctgacctcatggtccgtccacctctgcctcccaaagtgtgggatacaggcgtgagccacccagcagtctgatctaattttatagtttatgtgta cctccccagctgaagtatctcttttcttttttcccgcgtgtttagtgtcactcatctttatagcatagctcaatgtcacttcatgaagccttccataacctttgt agctccataatatattcttctgagtgtttaaaacactgccatatgaaacactatttactttggcttacattctactatctaatcggccatttctgtactaaa tctttttctcagagcacctgggatagtctgtgtctagtaaaatcagtgatgatttaactcggtagagtagaggctgattaaagtaaataaatctggtg atgccaacaaaattttggtcccctcaattttttgctctcatacctgcaaatctccctggccttcatatttggcaaccattgaggagaacaaggctgtaaa agtagttcatgtactgatattctgaatggaataagcagagtgctaagtaggactgcttttctgggatttctatgcaacaaataatgtagtaactgga aatccaagtcaagacactggcagatcgatgtcttttgaggacccttggctcatagatgatgcctctccctatatccttacatagcaaaaggggcca ggcagctctggcctttttttgtaaggccaataactccagaaacctcatgacctcatcacctcccaaaggccccacctctcaatactatcacattgtgagg ctaggtttcaacatatgaatgtgggagacaaaaattcagaccatagtataatatttcaagatactaaactctctctaccaaactcattaacttttaggtt agcacagtattttcatgatattttggtttctggagtatactaattttctgatctgatgttataataaaaaaaaacaggactttgtacgtgaaatgagactg agataaggaagctgattcagagatggagatttaaaaaaagagagatgagagatgagatctgcagtgtcaaactgacaatagccaggagtcagga gatataagagactatatcatctgtgatgtaatgattatttatgtatttataaatactactgtattttatatattatatacatgttttaaaaatatttttgtacc atttctgaaagaaaaatgtctaagctgggaaaatatttatgaaaaatgtggtttgtacatctgaggagtgtatctgcacagtaggtgcatagatttctt cctctcctgtccacatggcctagctagaggctgtgtggccatcacttggtatttagggtaagactggtgcacaaaatcaaagacaggtaaccttgg tataagtgtagtatcatgtaaatagcttttctatgtctaattctgttttcttcctactttttcaggaggtcaatttcagtcatttcaactatctttacataatagtg tgaaatcagctaattatgtacatcttatagctcagaaaagttttgaagtatatacaaatgctagtcaggaaaaaagattcagtcatgtaatctgtacat tctactatttaaatcaaccaatatatagatatgatttagtgcagtaattctgctggctaacctatctcatttggtggtggttagtacttcagagtactcacc atagtttcatttatgttttcagcatcacttcctggtttttctcaattccatggctgtggaatcaattcatatgtatatttagcttcggtgagcaaaaacatagcta gaaaaagaaaagaagtgagtttcctacctggtaaataaagtcgatgtgtaagccaaggaggacttcttttgaatggtactttaacaatccctgttctg tatactgtgaatatatcatttaaatagcctaataaattggatgctaggctgagccacctatactttagttttgttatggaaagaagggagaggagcaagt atgttcttatatgtactagaaataagaatgtagctgtagtacacatgttctaagtttttttcgtaagacaactgaaatgagtcccataggcctgctattt aacattctaagatatgactaaggtaatgatgagcttttgaatctgacaattcaagagatatccataatgaatactgattcattttctacatgctgaaagct aatgttcattttaagcctactttagtagcctttatttgggctagagatgtattcctctttctgatatttatgggttatctgtttaacccttttatatctccctttcc cgatttgtaaattagagactggcaagactttttaccctgagtagagcaccaaacatggcttgtttctgcccacactgtagtacctgaggggaagtaaa tgggactttaaaagcaatttatgctcttttatagtgaaatatccctctactatcccgaaagactgtacctacaatatcctccactccttccccctgtagt tactatagagatgacttttcggttcttcactgccataatgatcaaaatcctaatcatgagatttttatcatccaggcatgtgaggtttactgatgcataaa accgcaagtactttttgtgttttttaatgttttttctctcttatctctgaaagtctaagtagatcatcatttttgatgtctatagtagcaactaataaattttcc ctgtatcttctcagcaaaagaactcaagcagagacagaagatagaactaccatggtagttttgcttcctatggatatgtcacatacatagaaattttta caatgacctttttatatatgtatttcagaatttcagaatggcctcaatgcctaataggaagaaatacttgaaatttttaaattagggcttggttttgtgagga gctagtaaaggttttctctttcagctttagctgtttctgcggaggattccgctctttctccatcagtttcatagccctggaatgtagaaaagctctggtttc aagaccatgatatccatttctgtcagggtgagttttaaatttatttcatgatgcaaacaatatattgaacaacaggacatgaactgttctgttgtaagtgg ctgaattttatcagtaaagcacatcaaaataaaatataccccaatgctagtaagacctagagtgacagatgaaaatagctgtgtattctctaagaa aatatataaaaatatcatctcatcaatctttaatgtttgttttataaatctaaatgtttttatatgtttcctaggaaatataggtctaattttttactttaccacca gctgtcttttattttactctttttttgagacggagtttcgctctgtgctaggctagagtgcagtggcactatctcagctcactgcgacctctgcctcccgg gttcaagcgattctcctgcctcagtctcccgagtagctgggatacaggcacatgccactacaccaggctaattttgtatttttagtagagacggggttt cttcatgtggtcaggctggtctcgaactcccgacctcaggtgatccgcctgcctcggcctcccagagtgctgggatacaggcatgagccaccgca cctggccagctgtcttttaatataacatatgataatgtgatgttccattaaactaagcggagaggaaacatgctggtaaaccatgtgtgagtattcatt gtaccagaaaggcaaatgatacattttatcctaaaattcaaatttataaacatcttaacactgtgatcataaatactactaatctagcatataaatatattt gtaggcggggcacggtggctcacgcctgtaatcccagcactttgggaggctgaggtgggcagatcacgaggtcaggagatcgagaccatcctgg ctaacatggtgaaaccccatctctactaaaaatacaaaaaaaattagctgggtgtgctggcgggcacctgtagtcccagctacttgggaggctgagg caggagaatggcgtgaccccaggaggcagagctccagcctgggcgactccgtctcaaaaaaaaagaaaaaagaaatatatttgtaatattctact aacctatatcattaactttttatataacttttttattttaccaaataagtaaccttttatagccctggctatactaaacatcctaacttttttgtttaatgtat tagtttttaagtatgccccagatgtcaagtaatgtggattttctataataatttaggatatatgcatgaagtcagtagtatttacatttaaaactaaaaca atttatactaatacagtttatacatttcatactaatttagctacagtggataaatatttaatggaacaaagtaaatcaaagtaccttttcaaatgaatggaaa taaatccacataacaattttttatgaccacactatacagtgtgatggcatgccaaatgatcataatgtggaatatgtatttcttcatggctttcaagattc tgttctttagtttgtgggctcctctccaactgctgtctcctcacagtttaggcgactgtttataattctgtccatcctgcataaacacacacagtcaaaat gaaaaaaagctctatcagcagatctgtgctgctgtacagaaatgggaaaacaatgaagtttgcattatcttttttctaattaccagatcgtttttggagc tatttaggcatacgcttttaaggaaaaaagaaaaaaagagtgtaccttttgtttctaacaaaggtgtatctatatatgaaataaaaaatggggatagt tatgacaaagtatttagaaataggaataaaatctaaaataacttttcatagcatggacaagactataatgtctacctcaataagcaaatcatttaaaaa tttttcatgtatatttgctgccatgatgtgtgtgattgctaaataaccaatgaatgaagatcaacaaggatttaaatgaagaagaatatggatttaactat ttctcctgtgaaataagttcatatttacaagttttgattttcagaaattagacaatatttttaaaggctgggatgacaacttctgcctctaccaagaagtca aagcacagttatgtgaatcatcataaatcacatcatttttatatattttgtatttataatgtatgtgactactttaaaacctgtataaaataaaatgtttttta atattttattttagaatatagcattaataacaatttgaagtagtttacacaatacctgtgagttttatttttgttttatatgaaataattttagtgctttactgg

Example 2: OPA1 Non-productive Splicing Event Identification and Validation.

[0657] A novel nonsense-mediated decay (NMD) exon inclusion event (Exon X) was identified in the OP Al gene which leads to the introduction of a premature termination codon (PTC) resulting in a nonproductive mRNA transcript degraded by NMD (FIG. 1A). As NMD is a translation-dependent process, the protein synthesis inhibitor cycloheximide (CHX) was used to evaluate the true abundance of the event. FIG. IB shows an increase in OPA1 transcripts containing the NMD exon in HEK293 cells with increasing cycloheximide dose. Treatment of HEK293 cells with cycloheximide for 3 hr increases nonproductive mRNA transcripts. Other ocular cell lines also validated for the presence of the NMD exon (ARPE-19, Y79).

Example 3: OPA1 NMD Event is Conserved in Primate Eyes.

[0658] FIG. 1C shows reverse transcription PCR data from the macular and peripheral regions of both eyes of a human donor. OD: oculus dexter (right eye), OS: oculus sinister (left eye). The non-productive transcript was also detected in human retinal tissue. The abundance of the transcript in the tissue is potentially higher than what is detected by RT-PCR given that NMD is presumed active and therefore the transcript is being continuously degraded in vivo.

Example 4: ASO-1 Decreases Non-Productive OPA1 mRNA and Increases OPA1 Expression in a Dose-Dependent Manner In Vitro.

[0659] HEK293 cells were transfected with different doses of ASO-1, an antisense oligomer according to some embodiments of the present disclosure, which has the sequence of SEQ ID NO: 36, or nontargeting (NT) ASO. RNA was isolated 24 hours after transfection and analyzed for impact on nonproductive OPA1 mRNA (FIG. 2A) and OPA1 mRNA expression (FIG. 2B) similarly to in Example 11. For protein analysis, cells were lysed with RIPA buffer 48 hours after transfection and western blots were probed with antibodies targeting OPA1 and -actin, as shown in FIG. 2C. Multiple bands correspond to different isoforms of OPA1. Data represent the average of three independent experiments (* P<0.05 by one-way ANOVA compared to “NO ASO” group). The Non-targeting ASO targets an unrelated gene. [0660] Transfection of HEK293 cells with ASO-1 showed dose-dependent reduction in non-productive transcripts by RT-PCR (FIG. 2A). ASO-1 treatment results in a dose-dependent increase in both total OPA1 mRNA (FIG. 2B) and OPA1 protein (FIG. 2C) in HEK293 cells. Western blot analyses showed an increase in multiple OPA1 protein isoforms (FIG. 2C). A nontargeting (NT) ASO control directed against an unrelated gene did not have any effect on any of these three parameters. These results indicate that ASO-1 specifically reduces this non-productive splicing event and increases total OPA1 mRNA and protein expression in HEK293 cells in a dose-dependent manner.

[0661] To calculate the half maximal effective concentration (EC50) in HEK293 cells, ASO-1 was delivered by free-uptake and levels of the non-productive transcript were detected by a primer-probe qPCR assay and normalized to house-keeping gene RPL32. A dose-dependent reduction in nonproductive transcripts was seen by free-uptake and the EC50 for ASO-1 was determined as 5.63 pM (FIG. 2D)

Example 5: ASO Microwalk Evaluated by RT-qPCR.

[0662] In one experiment, microwalk was conducted to test ASOs that have sequences listed in Table 7. Briefly, about 30,000 HEK293 cells per well were treated gymnotically with 20 pM one of the 20 exemplary ASOs (free uptake) listed in Table 7 for 72 hours. After the treatment, the cells were harvested for analysis. RT-PCR reactions were conducted for products corresponding to Exon 7 or Exon 7x inclusion and full-length.

Table 5. Exemplary OPA1 ASO sequences

Table 6. Exemplary OPA1 ASO sequences

Table 7. Exemplary OPA1 ASO sequences

Example 6: ASO-1 Increases Total Cellular ATP Levels in OPAl^+/_ HEK293 Cells

[0663] Since most patients with ADOA show haploinsufficiency of OPA1, a cellular model of OPA1 haploinsufficiency was engineered in HEK293 cells by using CRISPR-Cas9 gene editing to insert one nucleotide in one OPA1 allele. Western blot analyses show reduced OPA1 protein levels in OPA1+/- HEK293 cells compared to isogenic OPA1 ^/+ HEK293 cells (FIG. 3A). The process yielded an OPAl^+/~ HEK293 cell line that showed reduced OPA1 expression compared to an isogenic control cell line (OP A l HEK293) that underwent the gene editing process but did not develop any mutations (FIG.

3A).

[0664] OPA1^+/+ or OPAl^+/~ HEK293 cells were treated with Vehicle or 10 pM ASO-1 by gymnotic delivery for 72 hours and assessed for effect on OP Al mRNA expression (FIGS. 4A-4B) and OPA1 protein (FIGS. 4C-4D) 72 hours post treatment. Data are plotted relative to the Vehicle group separately for both OPA1^+/+ and OPAl^+/~ cells. Representative western blots for data in (FIGS. 4C-4D) can be found in FIG. 4E. *P<0.05, **P<0.01 by unpaired t-test.

[0665] ASO-1 increased OP Al mRNA and OPA1 protein in both OPA1^+/+ and O7 7^+/_HEK293 cell lines by free-uptake (FIGS. 4A-4D), indicating that the presence of one WT allele is sufficient for ASO-1 to increase protein productionASO-1.

[0666] Mitochondria are primary centers of ATP production in the cell through oxidative phosphorylation. Given the critical role of OPA1 in the maintenance of mitochondria bioenergetics, 6VN / ~ HEK293 cells were examined to determine whether total cellular ATP levels were reduced. [0667] OPA1^+/+ or O7 7^+/_HEK293 cells were treated with Vehicle or 10 pM ASO-1 by free-uptake for 96 hours and total cellular ATP levels were measured and normalized to total protein levels (FIG. 3B). All values were plotted relative to the mean ATP level of WT OPA1^+/+ HEK293 cells. The mean ± standard deviation of at least three independent experiments (each with 3 replicates) conducted on separate days are shown. *P<0.05, ****P<0.0001 by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test.

[0668] Total cellular ATP levels were found to be significantly lower in OP A l^+/~ HEK293 cells compared to isogenic WT control cells (FIG. 3B). ASO-1 treatment increased ATP levels in OPAl^+/~ HEK293 cells but did not modulate ATP levels in OPA1^+/+ cells (FIG. 3B). These results support the concept that ASO-1 can positively modulate the reduced ATP phenotype found in OPAl^+/~ cells. Example 7: ASO-1 Increases OP Al Expression in ADOA Patient Fibroblast Cells in a Variantindependent Manner

[0669] Previous studies have used primary dermal fibroblasts derived from patients with ADOA OP Al mutations as an in vitro model of disease pathophysiology. To test whether ASO-1 would increase OPA1 expression and potentially modify disease progression in ADOA, dermal fibroblasts from three unrelated ADOA patients (designated as F34, F35 and F36) with different OPA1 pathogenic variants were evaluated (FIG. 5A).

[0670] WT and all three patient fibroblast cells were treated with 40 nM ASO-1 by transfection and harvested after 24 hours for analysis of non-productive splicing (FIG. 5B) and OP Al mRNA levels (FIG. 5C). OPA1 protein levels (FIG. 5D) were determined by western blot 72 hours after transfection and normalized to P-actin. All data are plotted relative to the WT Vehicle group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by unpaired t-test.

[0671] Patient fibroblast cells had OP Al mRNA levels ranging from 58-68% and OPA1 protein levels ranging from 36-47% of levels measured in WT fibroblasts (FIGS. 5C and 5D, compare Vehicle bars across the different genotypes). Treatment with ASO-1 reduced non-productive splicing and increased OP Al mRNA and protein in all three patient cells (FIGS. 5B-5D), supporting the mutation-independent nature of the TANGO approach.

Example 8: ASO-1 Enhances the Ability of ADOA Patient Fibroblast Cells to Respond to Mitochondrial Stress

[0672] The respiratory function of the three patient cells was compared to WT fibroblast cells by measuring the oxygen consumption rate (OCR) of live cells under basal conditions and upon treatment with different electron transport chain (ETC) inhibitors using the Seahorse extracellular flux analyzer. Specifically, basal respiration, ATP-linked respiration, maximal respiration, and spare (reserve) mitochondrial respiratory capacity were measured (FIGS. 6A-6D).

[0673] FIGS. 6A-6D show histograms demonstrating the reduction of mitochondrial function in ADOA patient-derived fibroblast cells oxygen consumption rate (OCR; pmol/min/cell) under both basal and stress conditions was measured by a Seahorse XFe96 extracellular flux analyzer. Data are normalized to mean WT values for each parameter. Biological replicates were pooled from separate experiments conducted on two different days. **P<0.01, ****P<0.0001 by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test.

[0674] All three ADOA patient cells showed a significant reduction in these four parameters indicating a deficit in mitochondrial respiratory function under basal and stress-mediated conditions (FIGS. 6A-6D). [0675] FIGS. 7A-7D show histograms of F34, F35 and F36 patient fibroblast cells that were treated with 20, 40 or 60 nM ASO-1 or Vehicle by transfection; their oxygen consumption rate (OCR; pmol/min/cell) was measured on a XFe96 extracellular flux analyzer under basal and stress conditions. Basal respiration (FIG. 7A), ATP-linked respiration (FIG. 7B), maximal respiration (FIG. 7C), and spare respiratory capacity (FIG. 7D) were reported. Data are normalized relative to the Vehicle group for each cell type. Statistical analysis was by ordinary ANOVA followed by Dunnett’s multiple comparison test against Vehicle; *P<0.05; **P<0.01; ***P<0.001; and ****P<0.0001.

[0676] Treatment with ASO-1 significantly increased the four OCR parameters in F35 and F36 fibroblast cells in a dose-dependent manner. In F34 fibroblast cells, ASO-1 treatment resulted in a dose-dependent increase in maximal respiration and spare-respiratory capacity, while increases in basal or ATP-linked respiration were not observed (FIGS. 7A-D). Maximal respiration is measured upon the addition of the uncoupler FCCP, which mimics a physiological energy demand by stimulating the respiratory chain to operate at maximum capacity. The difference between maximal and basal respiration is the spare respiratory capacity, a measure of mitochondrial reserve. The increase in maximal respiration and spare respiratory capacity in all three patient cells with different pathogenic variants shows that ASO-1 enhances the ability of OP Al haploinsufficient cells to respond to conditions that induce mitochondrial stress (FIG 7D)

Example 9: Surrogate ASO ST-1102 Produces Lasting Reduction in Non-productive Splicing and Increases OPA1 Protein in Rabbit Retinas

[0677] Sequence alignments of the non-productive splicing event suggested that it is well conserved in rabbit, pig, dog, and several nonhuman primate species (chimpanzee, rhesus monkey, green monkey). Interesting, both mouse and rat lacked conservation of the 3 ’ AG acceptor splice site of the event and therefore, rodents could not be used as models for in vivo proof-of-mechanism studies. While the event is conserved in rabbits, the ASO-1 target region is not perfectly conserved between humans and rabbits. Therefore, a rabbit surrogate ASO, ST- 1102, with identical sequence homology to the rabbit OPA1 target region was created for proof-of-mechanism studies. A single, bilateral intravitreal (IVT) injection of PBS or ST-1102 at 0.04, 0. 12 or 0.23 mg/eye was administered to female adult New Zealand White (NZW) rabbits and retinas were collected 7-, 14-, and 28-days post-injection for pharmacological assessments of non-productive splicing, OPA1 protein and ASO exposure (FIG. 8A).

[0678] Non-productive splicing was measured by RT-PCR using rabbit retinal RNA by quantifying the relative abundance of non-productive OPA1 mRNA transcripts to total OPA1 transcripts (FIG. 8B). Data are plotted relative to the PBS injected group. Statistical significance (P<0.0001 vs. PBS injected group) was observed at all time-points for all dose groups. RT-PCR gel images are included in FIG. 9. OPA1 protein was quantitated in retinal lysates by western blot and normalized to actin (***P<0.001, ***p<0.0001 vs. PBS treated group) (FIG. 8C). Data are plotted relative to the PBS injected group. Western blot images are included in FIG. 10. ST-1102 was detected in retinal tissue on Day 29 by HELISA at all three dose levels (FIG. 8D) Statistical analysis by one-way ordinary ANOVA followed by Dunnett’s multiple comparisons test.

[0679] A significant dose-related decrease in the non-productive transcript was observed in rabbit retinal tissue as early as 7 days post-injection by RT-PCR. The levels of non-productive transcripts further dropped at both 14- and 28-days post-injection (FIG. 8B and FIG. 9). No changes in retinal OPA1 protein levels were observed 7 days post-injection however significant dose-related upregulation of OPA1 protein levels were seen 14 days post-injection that remained significantly elevated at 28 days postinjection (FIG. 8C and FIG. 10). Retinal exposure of ST-1102 was measured only 28 days post-injection. ST-1102 retinal levels increased with dose but were not strictly dose-proportionate (FIG. 8D). Taken together, these data indicate that a single IVT administration of ST-1102 significantly reduced the levels of non-productive transcripts and increased OPA1 protein in the rabbit retina for at least four weeks.

Example 10: ASO-1 Produces Lasting Reduction in NMD Exon Inclusion, Increase in OPA1 Protein, and Exposure in Cynomolgus Monkey Retinal Tissue

[0680] The non-productive exon splicing event and the ASO target sequence is perfectly conserved between cynomolgus monkeys and humans, therefore this species was used to evaluate pharmacology, exposure, and tolerability of ASO-1. A single, bilateral IVT injection of PBS or ASO-1 was administered to cynomolgus monkeys.

[0681] Pharmacology was assessed in the Vehicle (PBS-injected), and 0.1, 0.3, and 1.0 mg/eye ASO-1 treatment groups.

[0682] Retinas were collected from indicated treatment groups at 4 or 8 weeks (FIG. 11 A) Presence of non-productive exon-containing RNA transcripts in retinal tissue was assessed by qPCR and expressed relative to total OPA1 transcripts. At 4 weeks after a single injection of ASO-1, detectable non-productive RNA transcripts in the retinal samples were reduced by approximately 56% (0.1 mg/eye), 76% (0.3 mg/eye), and 94% (1.0 mg/eye) when compared to PBS-injected eyes (FIG. HA).

[0683] FIG. 11B shows histograms OPA1 protein quantitated in retinal lysates by ELISA and normalized to total protein in the sample. The 0.3 and 1.0 mg/eye treatment groups were also evaluated at 8 weeks and found to maintain similar reductions. A corresponding increase in retinal OPA1 protein quantified by ELISA was observed. At week 4, the level of increase was 31%, 47%, and 44% in the retinal tissue at ASO-1 doses of 0.1, 0.3, and 1.0 mg/eye, respectively (FIG. 11B). The 0.3 and 1.0 mg dose levels were also assessed in an additional cohort at week 8, and elevated OPA1 protein levels were maintained. Percent increase could not be determined since there were no control animals at week 8; however, the OPA1 level measured by ELISA was comparable and slightly higher than the respective week 4 dose group levels (FIG. 11B). These data indicate that a single IVT administration of ASO-1 was effective for at least 8 weeks in significantly reducing the levels of non-productive mRNA transcripts and increasing OPA 1 protein in the retina.

[0684] Retinal tissue levels of ASO-1 were quantitated by HELISA at weeks 4 and 8. Samples were normalized by tissue weight. The exposure was dose -dependent but not strictly dose proportional in retinal tissue (FIG. 11C). At week 8 compared to week 4, mean ASO-1 concentration in the retina decreased by 34% in the 0.3 mg/eye group and remained at a similar level for 1 mg/eye dose (increased by 6%) (FIG. 11C). This sustained retention through week 8 suggests slow clearance and long tissue half-life of ASO-1 in retina.

[0685] Data in FIGS. 11A-11C are presented as mean ± standard deviation. Each data point represents one retina. Statistics were not performed for week 8 where N=2. *P<0.05; **P<0.01; ***P<0.001 vs. Vehicle (PBS-treated) group. Example 11: ASO-1 and OPA1 Protein Are Detected in Cynomolgus Monkey RGCs After IVT Administration

[0686] The loss of visual function in ADOA is due to the dysfunction and loss of RGCs, thus therapeutic targeting of this cell type can lead to successful treatment of ADOA. Sections of Modified Davidson’s- fixed paraffin-embedded retina from the Vehicle and 0.1, 0.3, and 1.0 mg/eye ASO-1 treatment groups were evaluated by RNAscope™ in situ hybridization to detect ASO-1.

[0687] FIGS. 12A-12B are exemplary images labeled with treatment type and ASO-1 dose per eye. PBS-injected eyes did not demonstrate any detectable signal, indicating that the probe was highly specific (FIGS. 12A-12B)

[0688] FIG. 12A is an exemplary image of RNAscope™ staining with a specific probe to ASO-1 performed on MDF -fixed, paraffin-embedded cynomolgus monkey eyes to demonstrate cellular location of ASO-1 (red) in RGCs. Increased ASO-1 signal is correlated with increased dose. Nuclei were counterstained with hematoxylin (blue).

[0689] FIGS. 13A-13D show histograms of the quantitation of mRNA copies (dots) per cell and percent positive RGCs in foveal (FIGS. 13A-13B) and peripheral retina (FIGS. 13C-13D) showed a dose-related increase in ASO-1 detected in retinal ganglion cells (RGCs) after intravitreal (IVT) administration to cynomolgus monkeys. Statistics by one-way ANOVA with Dunnett's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared to Vehicle (PBS-treated).

[0690] By visual inspection (FIG. 39A) as well as quantitative histological assessment (FIGS. 13A-D), a dose-related increase was observed in ASO-1 signal in the RGC layer at week 4.

[0691] FIG. 12B is an exemplary image of immunofluorescence staining with OPA1 antibody, which shows apparent dose-related protein increase (red) in the RGCs in/near the fovea. Nuclei were stained with DAPI (blue) and the magnification was 40X.

[0692] By visual inspection after antibody staining, OPA1 protein immunofluorescence was observed in the RGC layer of the retina. The intensity of immunofluorescence appeared to increase with ASO-1 dose in the RGC cell layer, including RGCs in the foveal region (FIG. 12B) at 4 weeks after ASO-1 treatment. However, results of attempted quantitation of the immunofluorescence intensity showed an increasing trend but were not statistically significantly different from Vehicle (FIGS. 14A-14B). A substantial signal for OPA1 protein was apparent in the RGCs at baseline (FIG. 12B, PBS).

[0693] The foregoing preclinical data support the TANGO disease-modifying approach in ADOA. As demonstrated by the data, the exemplary antisense oligomer, ASO-1, reduced non-productive exon inclusion, increased total OPA1 mRNA and protein expression in all three patient fibroblast cell lines; increased ASO-1 dose increased mitochondrial respiration in two fibroblast cell lines. The data further suggest that the ASO mediated increase in OPA1 protein expression is disease-modifying in ADOA in a mutation-independent manner.

Example 12: Material and Methods

[0694] The materials and methods described below pertain to Examples 2-11. [0695] Animal Care

[0696] In-life phase of the rabbit and monkey studies were carried out by external contract research organizations (CROs) (Absorption Systems, USA [rabbit] and Alta Biosciences, USA [monkey]). All animal experiments were conducted in accordance with the National Research Council’s Guide for the Care and Use of Uaboratory Animals under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Stoke Therapeutics and/or contracted facility.

[0697] Human Tissue

[0698] Human retinal sample was obtained from the Uions Eye Institute for Transplant & Research (Tampa FE), which complies with regulations from the FDA, the Eye Band Association of America (EBAA), as well as the State of Florida.

[0699] Bioinformatic Analysis

[0700] Bioinformatic RNA-sequencing analysis of the Sequence Read archive with the identifiers SRP026048 (12 samples), SRP107937 (31 samples), SRP174668 (36 samples) and ERP003613 (4 samples) was performed by using the previously described bioinformatic tools, transcript database, and quality control of samples to identify NMD splicing events, exon exclusion and skipping, and alternative splice junctions and intron events (Lim 2020).

[0701] Conservation of the NMD exon inclusion event on the OP Al gene across 100 different species was performed by using the human PhastCons 100-way conservation scores database, as previously described (Lim 2020).

[0702] Cells and Cell Cultures

[0703] All cells used in the study were cultured at 37°C in a humidified incubator with 5% CO2.

HEK293 cells were obtained from ATCC and cultured in DMEM high glucose with 10% FBS. HEK293 OPA1+/- cells were generated as a cellular model of haploinsufficiency with CRISPR technology at GenScript. Briefly, the OPA1 gene was targeted and mutated by transient co-transfection of plasmids carrying gRNA and Cas9. Confirmation of the knockout was performed with Sanger sequencing followed by full length sequencing of the OP Al gene. HEK293 cells that underwent the CRISPR process but maintained the OPA1 WT sequence in both alleles were called OPA1^+/+ HEK293 cells.

[0704] Primary human dermal fibroblasts: WT primary dermal fibroblasts were purchased from ATCC (PCS-201-012). The three human primary dermal fibroblasts from ADOA patients were obtained from the National Eye Institute. Fibroblast cells were grown in an enriched medium (1: 1 basic fibroblast media and AmnioMaxTM-II Complete medium) at 37°C in 5% CO2. The composition of the basic fibroblast media was as follows: DMEM high glucose with 10% heat inactivated FBS, glutamine (2 mM final), sodium pyruvate (1 mM final), and antibiotics.

[0705] Treatment with Cycloheximide

[0706] For any in vitro analysis of non-productive transcript levels, cells were treated with their respective culture medium containing cycloheximide (50 pg/ml) for the 3 hours immediately before harvesting. Control cells received culture medium containing an equivalent volume of DMSO. [0707] RNA extraction

[0708] For cultured cells, total RNA was extracted using the Qiagen RNeasy Kit (Qiagen, 74106) or RNeasy 96 Kit (Qiagen 74181) following manufacturer’s instructions. RNA isolation from tissue was performed using the QIAzol reagent as described previously (Lim 2020).

[0709] cDNA Synthesis

[0710] cDNA synthesis was conducted using SuperScript™ IV Reverse Transcriptase (Thermo Fisher, 18090010) following manufacturer’s instructions.

[0711] ASO Uptake: Transfections and Free Uptake

[0712] For transfection, diluted Lipofectamine RNAiMAX reagent (0.9 pl in 100 pl OptiMem) was mixed with ASO at indicated concentration in Eppendorf tubes for 30 min and placed in 24-well plate. Cells (100,000 cells, suspended in 400 pl media) were added to 24-well plates and incubated for 5-6 hours at 37°C in 5% CO2. Media was then either replaced entirely or an additional 500 pl fresh media was added to wells and plates were returned to incubator until harvesting.

[0713] For free uptake, HEK293 cells (25,000-30,000 cells suspended in 450-490 pl culture media) were added to 24-well plates containing concentrated ASO-media (10-50 pl) to obtain final desired ASO concentration. Cells were incubated for 72-96 hours.

[0714] RT-PCR and qPCR Analysis

[0715] RT-PCR was performed as previously described (Lim 2020) with the primers and cycle conditions listed in Tables 8-9. The intensities of the non-productive and productive OPA1 mRNA transcripts from the RT-PCR gel images were calculated using the Multi Gauge software (Fujifilm V2.3). Relative non-productive splicing was calculated as the intensity of the (non-productive transcript / [the non-productive + productive transcript]) and normalized to the Vehicle group.

[0716] qPCR was also performed as previously described (Lim 2020) with primer-probe assay information and cycle conditions listed in Tables 10-12. The 2-AACt method was used to calculate relative expression levels. Total OPA1 mRNA assessments in cell-based studies were normalized to house-keeping gene RPL32. Similarly, for EC50 evaluation, levels of non-productive transcript were also normalized to RPL32. For rabbit and cynomolgus monkey studies, levels of non-productive transcript were calculated relative to total OPA1 levels.

[0717] ATP Measurement

[0718] OPA1^+/+ or O7 7^+/_HEK293 cells were seeded at 3 x 10⁵ cells in a T-25 flask in cell culture media containing 10 pM ASO-1 or an equivalent volume of Vehicle and incubated for 96 hours. Cells were washed twice with cold PBS and harvested with 120 ml cold ATP assay buffer. Samples were centrifuged at 13,000 x g for 5 min at 4°C and the supernatants were collected and kept on ice. An aliquot of lysed sample was set aside to be quantified by BCA assay for total protein concentration. The remaining sample was deproteinized using the Deproteinizing TCA kit (Abeam, ab204708) and ATP levels were measured in the lysates using the ATP assay kit (Abeam, ab83355) according to the manufacturer’s instructions. [0719] In Vivo Assessment of ST-1102

[0720] Adult female NZW rabbits received a bilateral single IVT injection of 30 pl containing PBS (N=3 per time point) or ST-1102 (0.04, 0.12, or 0.23 mg/eye). Clinical ophthalmic examinations were performed on days 8, 15 and 29. For non-productive splicing and OPA1 protein analysis, each dose group included three animals for each time-point. Retinas were divided along the nasal temporal axes into two halves and collected in separate tubes, flash frozen and stored below -70°C until analysis. The nasal halves were used to isolate RNA while the temporal halves were used for protein analysis. ASO exposure was only measured on Day 29 and included three animals per dose group. Whole retinas were used for analysis.

[0721] In Vivo Assessment of ASO-1

[0722] 1.8-2.0-year-old male and female cynomolgus monkeys received a bilateral single 50 pl IVT injection of PBS (N=4 animals per time point) or ASO-1 (0.1, 0.3, and 1.0 mg/eye; N=4 per dose) and followed for 4 weeks. A separate cohort at 0.3 and 1.0 mg/eye (N=2 per dose) were followed an additional 4 weeks. Ocular tissues were harvested at weeks 4 or 8. One eye (OS) was dissected, and retinal tissue was snap frozen, and the fellow eye (OD) was fixed intact in modified Davidson’s fixative, processed and embedded in paraffin.

[0723] Protein Extraction and OPA1 Protein Assessment by Western Blot

[0724] Protein was extracted from rabbit retinal tissues in Red RING tubes containing zirconium beads by adding RIPA buffer containing protease and phosphatase inhibitors (10 pl per mg tissue) followed by homogenization with a Bullet Blender Storm 24. Samples were then passed through the QIAshredder column by centrifugation. For protein extraction from cells, cell pellets were lysed in RIPA buffer and passed through the QIAshredder columns. Protein concentration in the flow through was measured by the BCA assay.

[0725] hnmunoblotting was carried out as previously described (Lim 2020) with the following modifications. The following primary antibodies used were as follows: a-OPAl protein (Abeam 119685), primary monoclonal antibody against a-P-actin (Abeam ab8226), a- -tubulin (Bio-Rad VMA00222). Secondary antibodies used were either goat anti-mouse HRP antibody (Santa Cruz, sc-2005) or IRDye® 800CW Donkey anti-mouse IgG secondary antibody (Licor, 926-32212). For HRP detection, membranes were scanned on a Typhoon 9500 scanner and Multi Gauge software (Fujifilm V2.3) was used to quantify all OPA1 isoforms and actin (or p-tubulin as indicated) in each sample lane. For fluorescent secondary antibody detection, membranes were scanned on an Odyssey CLx Imaging System and hnageJ was used for quantification of all isoforms of OPA1 and P-actin bands. OPA1 protein levels were normalized to P- actin or p-tubulin as indicated and expressed relative to the average OPA1 expression levels of the Vehicle group.

[0726] OPA1 Protein Quantitation by ELISA

[0727] Cynomolgus monkey tissue homogenate was prepared by adding retina to pre-filled tubes with zirconium beads (SPEX SamplePrep) at a 20 mg/ml concentration using IX Lysis buffer (Ray Biotech) plus 100X protease and phosphatase Inhibitor cocktail (Thermo Fisher). Sample tubes were added to a pre-cooled cryoblock and lysed in a SPEX 1600 Mini G homogenizer at 1425 rpm for 5 min. After homogenization, samples were centrifuged at 10,000 x g for 5 min at 4°C, and supernatant was collected and transferred to a new polypropylene tube for analysis. The relative quantity of OPA1 protein in monkey retinal tissues was measured by an ELISA. Briefly, Nunc plates (Thermo Fisher) were coated with the capture mouse monoclonal antibody against OPA1 protein 500 ng/ml final concentration (BD Transduction Labs, 612007) overnight at 4°C. After the plates were washed, blocked with SuperBlock T20, (300 pl, Thermo Fisher) for 60 min at room temperature on a plate shaker and rinsed, the test samples, OPA1 calibration curve (Origene, 311417) and Vehicle (100 pl) were added to the plates and incubated for 2 hours at room temperature on a plate shaker. After plates were washed, the detection antibody HRP-rabbit monoclonal anti-OPAl antibody, 1000 ng/ml final concentration, (Novus Biologicals) was added (100 pl), and plates were incubated for 60 min at room temperature in the dark on a plate shaker. After plates were washed, the signal was developed with TMB substrate (Thermo Fisher) for 20 min and stopped in accordance with manufacturer’s instructions. The plates were read at 450 nm by SpectraMax 5Me (Molecular Devices) and a four-parameter logistic model was used to generate the standard curve and the quantity of OPA1 in each test sample was extrapolated. OPA1 protein levels in each sample were normalized to the total protein content determined by the BCA assay.

[0728] Mitochondrial Respiratory Functions Measured By Seahorse XFe96 Analyzer

[0729] For characterization of mitochondrial bioenergetics in ADOA patient fibroblast cells, 10,000 fibroblast cells were plated in Seahorse XFe96 PDL plate for 24 hr. The Seahorse XF Cell Mito Stress Test Kit (Agilent 103015-100) was used to assess mitochondrial function following manufacturer’s instructions. The final concentration of the ETC modulators was: oligomycin (1.5 pM), FCCP (1 pM) and rotenone/antimycin (0.5 pM). Cell counts for each well were captured by staining with Cyquant and imaged using an ImageXpress (Molecular Devices). The Wave software program (Agilent Technologies V2.6.1) was used to normalize the OCR to the cell count and reported as pmol/min/cell.

[0730] To study effect of ASO-1, fibroblast cells were treated with different doses of ASO-1 by transfection using Lipofectamine RNAiMAX for 72 hours. Cells were then trypsinized and re -plated on Seahorse XFe96 PDL plate for additional 24 hours and assay was performed as described above.

[0731] Quantification of ASO In Vivo by Hybridization Enzyme Linked Immunosorbent Assay (HELISA)

[0732] Prior to the measuring of ASO, retinal tissues were processed in tubes containing zirconium beads by adding homogenate buffer containing proteinase K (0.25 mg/ml) followed by homogenization with Spex 1600 MiniG homogenizer for 2x1 min at 1200 rpm. Then the homogenate was incubated at 550°C for an hour for tissue lysis. The concentrations of ST-1102 in rabbit and ASO-1 in monkey retinal tissues were measured by using an established HELISA for quantifying ST-1102 or ASO-1, respectively with the following modifications of previously described methods (Burki 2015; Straarup 2010). Briefly, the complementary capture probe with biotin at 3 ’ end was coated onto a NeutrA vidin ELISA plate (Pierce 15507). Separately, the 5’ digoxigenin-labeled detection probe was incubated with a standard curve of ST-1102 samples or test samples. After the complex was hybridized with the capture probe, an anti-digoxigenin antibody conjugated to alkaline phosphatase (Sigma, 11093274910) was added to detect the bound analyte, and ATToPhos® (Promega, SI 000) was used as a substrate for fluorescent measurements with excitation wavelength of 435 run and emission wavelength of 555 nm. The standard curves of ST-1102 and ASO-1 were calculated with a four-parameter logistic, and the quantity in each test sample was extrapolated.

[0733] In Situ Detection of ASO-1 by RNAscope™

[0734] Whole globes were harvested and fixed for 24 hours in Modified Davidson’s fixative (MDF), then subjected to routine processing and paraffin embedding. A specific probe directed against the ASO-1 sequence was generated and used for detection by custom miRNAscope™ LS RED in situ hybridization (probe dilution 1:5000). RNA in situ hybridization was performed on automation platform using the RNAscope™ Red Reagent Kit (Advanced Cell Diagnostics, Inc., Newark, CA) according to the manufacturer’s instructions. Briefly, 5 pm MDF -fixed, paraffin-embedded tissue sections were pretreated with heat and protease prior to hybridization with the target oligo probes. Preamplifier, amplifier, and AP- labeled oligos were then hybridized sequentially, followed by chromogenic precipitate development. Each sample was quality controlled for RNA integrity with RNAscope™ probe specific to PPIB RNA and for background with a probe specific to bacterial dapB RNA. Specific RNA staining signal was identified as red, punctate dots. Samples were counterstained with Gill’s Hematoxylin. Regions of interest (ROI) included RGC layer in or near the fovea and RGC layer in peripheral retina. Visual scoring was performed by a qualified scientist who was blinded to treatment groups but not controls. RNAscope™ quantification is based on number of dots per cell and not on intensity. Percentage of cells positive is scored visually based on number of cells with >1 dot/cell. Quantitative image analysis was performed using HALO® software on 1-mm lengths of each ROI.

[0735] Immunofluorescence Detection of OPA1 Protein

[0736] Eyes were fixed and embedded as described above. Immunostaining was performed on Leica automation platforms using the Bond™ Polymer Refine Detection Kit (Leica Biosystems). Briefly, 5 pm MDF fixed tissue sections were pretreated with heat and target retrieval solution prior to incubation with the target antibody (Novus Anti-OPAl antibody NB 110-55290 at 5 pg/ml), and polymer. Fluorescent whole slide images were acquired using 3DHistech Panoramic SCAN II digital slide scanner equipped with a 40x objective. Images were obtained from the regions of interest as described above. Quantitative image analysis was performed using HALO® software on 1-mm lengths of each ROI.

[0737] Biostatistical Analysis

[0738] Differences among groups were determined by ordinary one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test or by unpaired t-test, as indicated. All statistical comparisons were performed with GraphPad Prism. Levels of significance began at P=0.05.

[0739] A significant dose-related decrease in the non-productive transcript was observed in rabbit retinal tissue as early as 7 days post-injection by RT-PCR. The levels of non-productive transcripts further dropped at both 14- and 28-days post-injection (FIG. 8B and FIG. 9). No changes in retinal OPA1 protein levels were observed 7 days post-injection however significant dose-related upregulation of OPA1 protein levels were seen 14 days post-injection that remained significantly elevated at 28 days postinjection (FIG. 8C and FIG. 10). Retinal exposure of ST-1102 was measured only 28 days post-injection. ST-1102 retinal levels increased with dose but were not strictly dose-proportionate (FIG. 8D). Taken together, these data indicate that a single IVT administration of ST-1102 significantly reduced the levels of non-productive transcripts and increased OPA1 protein in the rabbit retina for at least four weeks. [0740] Treatment with ASO-1 significantly increased the four OCR parameters in F35 and F36 fibroblast cells in a dose-dependent manner. In F34 fibroblast cells, ASO-1 treatment resulted in a dose-dependent increase in maximal respiration and spare-respiratory capacity, while increases in basal or ATP-linked respiration were not observed (FIG. 7A-7D). Maximal respiration is measured upon the addition of the uncoupler FCCP, which mimics a physiological energy demand by stimulating the respiratory chain to operate at maximum capacity. The difference between maximal and basal respiration is the spare respiratory capacity, a measure of mitochondrial reserve. The increase in maximal respiration and spare respiratory capacity in all three patient cells with different pathogenic variants shows that ASO-1 enhances the ability of OP Al haploinsufficient cells to respond to conditions that induce mitochondrial stress (FIG. 7D)

Table 8. Exemplary RT-PCR primers

Table 9. Exemplary PCR Thermocycling Conditions

[0741] qPCR Assay Information

[0742] Probe-based qPCR assays were either purchased from Thermo Fisher or custom-designed by IDT. qPCR was performed with the following cycle conditions: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles (95°C for 15 sec, 60°C for 1 min)

[0743] The commercially purchased assays were as follows: human OPAL. Hs01047013_ml, human RPL32-. Hs00851655_gl. [0744] Tables 10-12 comprise exemplary sequences of the custom-designed qPCR assays.

Table 10. Primers and Probes Used in qPCR Assay for Detection of Human Non-productive OPA1 transcript.

Table 11. Primers and Probes Used in qPCR Assay for Detection of Cyno Non-productive OPA1 Transcript.

Table 12. Primers and Probes Used in qPCR Assay for Detection of Cyno Total OPA1 Transcripts.

Example 13. OcuSciences Ocumet Beacon

[0745] In this Example, OcuMet Beacon™ (OcuSciences Inc., Ann Arbor, MI) is an instrument that was used to stimulate, detect, and quantitate retinal mitochondrial flavoprotein light emission and compute the flavoprotein fluorescence (FPF) intensity, which has been found for the first time to be correlated with eye condition in ADOA. Patients with ADOA were found to have high FPF scores.

[0746] Stimulation, detection, quantitation

[0747] OcuMet Beacon™ Imaging Technique

[0748] The OcuMet Beacon™ is a scanning laser ophthalmoscope that comprises a confocal infrared scanner and a metabolic detector. Measureent of retinal FPF using OcuMet Beacon™ has been reported in Ahsanuddin et al. (2023) Front. Ophthalmol. 3: 1110501. The confocal infrared scanner captures a 60° x 21.5° infrared image in a region of interest (ROI), and the metabolic detector enhances wavelengths near the emission peak of FPF (520-540 nm) and produces a corresponding 17° x 21.5° FPF heatmap that colors areas of increased mitochondrial dysfunction (dashed oval, FIG. 30). The metabolic detector comprises an excitation band-pass filter tuned to 465 nm and an emission band-pass filter adjusted to enhance retinal FPF and minimize contaminating signals from other retinal fluorophores. The OcuMet Beacon™ has automatic focusing and interfaces with customized image-quality assessment and analysis software.

[0749] Exemplary Standard OcuMet Beacon Output

[0750] Using the data collected, the OcuMet Beacon™ generates a Flavoprotein Fluorescence (FPF) Report (FIG. 30) comprising patient information (e.g., patient identifier, birthdate, eye image date, eye image time, and the eye examined); an infrared image of the fundus in a wide-angle view; a Region of Interest (ROI) box (box with dashed lines) highlighting the area of the retina captured on a metabolic FPF image; the (metabolic) FPF image, which is a colorized image of the ROI that shows the measured FPF signal, wherein the highest signal value is represented with red and the lowest signal value is represented with black; an FPF score representing the average level of flavoprotein fluorescence in the Region of Interest (ROI) (box with dashed lines); a histogram of pixel values binned to form a frequency distribution from within the ROI; and a Curve Width (CW) value, which is the degree to which the FPF signal varies across the ROI. The CW value represents the width of the histogram at half of the maximum frequency value and is correlated with heterogeneity within the ROI. The higher the FPF score, the greater the right-shift of the histogram.

[0751] Beacon’s algorithm data maps are derived from OCT maps

[0752] Beacon’s algorithm interprets retinal image data with regions mapped in accordance with Optical Coherence Tomography (OCT) maps, which are cross-sectional images of the retina used to gauge retinal thickness. Analysis of the macular-papillary (Mac) retinal nerve fiber layer (RNFL) comprises assessing the entire region within the circle (FIG. 16A). Analysis of the peripapillary (Ppy) optic nerve (FIG. 16B) entails the assessment of five sectors: 1: Temporal (T); 2: Superior (S); 3: Nasal (N); 4: Inferior (I); and 5: Center (C).

[0753] The flavoprotein fluorescence (FPF) report (FIG. 32) also comprises retinal images of each measured eye (e.g., right eye, or oculus dexter (OD); and left eye, or oculus sinister (OS)) and false- colored maps of these images (green represents normal FPF, yellow represents a moderate increase in FPF, and red represents significant increase in FPF). From the retinal images and false-colored maps, (1) an optic nerve rim flavoprotein fluorescence profile is generated for the assessed sectors of each measured eye: Temporal (TMP); Superior (SUP); Nasal (NAS); and Inferior (INF), and (2) false-colored maps corresponding to the Optic Nerve Hypoplasia (ONH) stress index of each measured sector of each eye. [0754] A high concentration of mitochondria is found at the optic nerve head and peripapillary zone. Analysis of en face views of the optic nerve and OCT-related peripapillary optic nerve zones of exemplary ADOA patient data collected using Beacon indicate that the temporal (TMP or T) zone exhibits the greatest increase of FPF consistent with macular papillary retinal nerve fiber layer (RNFU) loss in ADOA.

Example 14. OSPREY: an open-label study to investigate safety, tolerability, and exposure of the Antisense Oligonucleotide (ASO) ASO-1 in Patients with OPA1 Autosomal Dominant Optic Atrophy (ADOA) [0755] OSPREY study objectives

[0756] ASO-1 is an investigational antisense oligonucleotide (ASO) treatment designed to upregulate OPA1 protein expression with the aim of stopping or slowing vision loss in ADOA. The OSPREY study described herein endeavors to achieve the primary objective of evaluating the safety, tolerability, and exposure (serum) of single ascending doses of ASO-1 in a human Phase 1 study, in addition to the secondary objective of evaluating changes in visual function, ocular structures, and quality of life after single doses of ASO-1. The reference to serum represents the measurement of ASO-1 in patient serum as a measure of systemic exposure and is not a measurement of ASO-1 locally within the eye itself.

[0757] OSPREY recruitment criteria

[0758] Key criteria used in the inclusion of study participants: those who (1) are clinically confirmed to have ADOA with the heterozygous OPA1 variant predicted to cause haploinsufficiency; (2) are between the ages of about 6 and 55 years old (divided into two cohorts: cohort A comprising those between at least 18 years old and less than 55 years old, and cohort B comprising those at least 6 years old and less than 18 years old); and (3) have Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity letter scores of from at least 35 to 70 letters for each eye.

[0759] Key criteria used in the exclusion of study participants: those who have (1) ADOA plus or Behr syndrome; (2) gain-of-function or compound heterozygous OPA1 variants; (3) conditions, medications, or surgical operations that could affect optic nerve function; and/or (4) high myopia (worse than -6 diopters (>6D)).

[0760] OSPREY study design - dosages and assessments

[0761] Intravitreal, 50-pl doses of ASO-1 are administered to the pars plana in one eye (the eye determined to have the more severe disease condition) of each patient following a 3+3 ascending dose design over the course of 48 weeks. The study sample size N is 60 (48 participants in cohort A and 12 participants in cohort B). Cohort A comprises four dose levels (0.1, 0.3, 0.5, and 0.7 mg of ASO-1) with an option to add two dose levels, capped at a maximum total dosage of 1.2 mg of ASO-1 per eye according to the Safety Monitoring Committee (SMC).

[0762] Ophthalmic assessments include Best Corrected Visual Acuity (BCVA) tests, low-contrast BCVA (Sloan) tests, Minnesota low vision reading (MNREAD) acuity tests, slit lamp examinations, static automated perimetry (24-2), full-field electroretinograms (ffERG), photopic negative response (PhNR) measurements, spectral-domain optical coherence tomography (SD-OCT), and OcuMet Beacon™ flavoprotein fluorescence (FPF).

[0763] Other assessments include quality-of-life (QOL) assessments, blood and/or urine for safety and exposure pharmacokinetics (POK) evaluation, biomarkers that may be present in the serum, plasma, or whole blood), and/or other adverse events.

[0764] OSPREY study design -progression of dose administration in cohort A (> 18 to <55 years old) [0765] The progression of dose administration in cohort A follows the general scheme of having an administration of a dosage of the ASO per eye followed by approval from the Safety Monitoring Committee (SMC), who determines whether the cohort should continue with the same dosage or escalate to the next dosage level, with the SMC evaluating after each administration (FIG. 33).

[0766] Cohort A begins with 0.1 mg ASO-1 per eye at a sample size of N=l, followed by approval from the Safety Monitoring Committee (SMC), which provides a recommendation on whether to continue dosing, and then whether to recommend escalating to the next dosage level of 0.3 mg ASO-1 per eye. After dosage with 0.3 mg/eye, the Safety Monitoring Committee (SMC) provides a recommendation on whether to continue administration at the same dose and then whether to recommend escalating to the next dosage level of 0.5 mg ASO-1 per eye. After dosage with 0.5 mg/eye, the Safety Monitoring Committee (SMC) provides a recommendation on whether to continue administration at the same dose and then whether to recommend escalating to the next dosage level of 0.7 mg ASO-1 per eye. Safety follow-ups are performed 48 weeks after the first dosage.

[0767] Six or fewer patients may be added to any dosage level that is tolerated with fewer than two patients experiencing dose-limiting toxicities (DLT) during corresponding evaluation steps by the SMC (four-pointed stars). Three additional patients may be added for safety assessment and the SMC may recommend de-escalation to a lower dosage during corresponding evaluation steps (five-pointed stars in FIG. 33)

[0768] OSPREY study design -progression of dose administration in cohort (>6 to <18 years old) [0769] Cohort B begins with an ASO dosage recommended by the Safety Monitoring Committee (SMC) from cohort A (sample size N = 1, >12 to 18 years old), followed by review by the SMC, who provides a recommendation on whether to continue administration at the same dose. After confirmation by the SMC from the previous step, the sample size increases to N = 2, >6 to 18 years old, followed by another round of review by the SMC (FIG. 34). Safety follow-ups are performed 48 weeks after the first dosage. Three patients may be added after the second evaluation by the SMC. The dosage may be escalated or deescalated in a 3+3 fashion if necessary to find an optimal dose in pediatric participants. After the 48-week safety follow-up, six or fewer patients may be added if the dosage is tolerated well, or the highest dose level tested is well-tolerated, or confirm safety or continue to the second endpoint.

Example 15. FALCON - A non-interventional, natural history study of mitochondrial dysfunction in OP Al-Autosomal Dominant Optic Atrophy (ADOA)

[0770] A natural history study (FALCON) was run to evaluate and compare functional and anatomic markers of ADOA disease progression; provide multimodal functional and structural assessments of an OPA1 study cohort to expand ADOA phenotypic characterization (structural and functional neuro- ophthalmic parameters); and potentially identify biomarkers for monitoring treatment effects, particularly to distinguish the effects of aging from those of disease in visual functions and structures. FPF was identified as a biomarker for monitoring treatment effects, particularly to distinguish the effects of aging from those of disease in visual functions and structures. The FALCON study aimed to identify variables that significantly change with ADOA disease progression but minimally change with normal aging.

[0771] Published, normative data for healthy subjects were used to estimate age effects on variables of interest. Statistical methods that could estimate and remove age effects from ADOA group were employed, assuming that the age effect was linear and similar in both healthy and disease populations. Publications of known validated biomarkers that change minimally with age were used. Feature selection techniques from machine-learning algorithms were used to analyze the data and identify potential biomarkers while accounting for the confounding effects of age.

[0772] FALCON - Vision Tests and Other Assessments

[0773] The FALCON study comprised vision tests and other assessments to potentially identify biomarkers for monitoring treatment effects and distinguishing the effects of aging from those of disease in visual functions and structures. The following aspects of the FALCON study were performed to identify variables that significantly change with ADOA disease progression but minimally change with normal aging.

[0774] FALCON -Initial study design and participant demographics

[0775] Initial study demographics comprised 48 patients in three age cohorts (8-17 years old, 18-40 years old, and 41-60 years old) across ten sites (five in the US, two in Italy, two in the UK, and one in Denmark). All data points are averages for both of each patient’s eyes (Table 14A).

Table 14A. Initial study demographics of pre-enrolled FALCON study participants

[0776] Participant demographics at 12 months comprised 45 patients in three age cohorts (8-17 years old, 18-40 years old, and 41-60 years old) across ten sites (five in the US, two in Italy, two in the UK, and one in Denmark). All data points are averages for both of each patient’s eyes (Table 14B).

Table 14B. Demographics of enrolled FALCON study participants meeting participation criteria at 12 months

BCVA, best corrected visual acuity; max, maximum; min, minimum; n, number; 0PA1 gene, optic atrophy; SD, standard deviation.

[0777] A total of 47 participants are currently enrolled in FALCON and completed both the 6- and 12- month assessments. Current participant demographics can be found in Table 14C.

Table 14C. Demographics of currently enrolled FALCON study participants who have completed both the 6- and 12-month assessments

[0778] FALCON - study duration

[0779] The initial and 12-month timepoints of the study was run over the course of 24 months, with assessments performed at baseline, 6 months, 12 months, 18 months, and 24 months.

[0780] FALCON - recruitment criteria

[0781] Key criteria used in the inclusion of study participants: those who have been clinically diagnosed with ADOA, confirmed to have the heterozygous OPA1 variant, and scored >5 letters in an Early Treatment of Diabetic Retinopathy Study (ETDRS) test.

[0782] Key criteria used in the exclusion of study participants: those who had gain-of-function (GOF) mutations in OPAL, compound heterozygous, homozygous pathogenic or likely pathogenic, benign or likely benign OP Al gene variants; or those who had extraocular phenotypic manifestations of (syndromic) ADOA (ADOA-plus).

[0783] FALCON - assessments and endpoints

[0784] A series of assessments were done for the FALCON study, including Quality of life questionnaires (NEI-VFQ-25, IVI-C, EQ-5D, EQ-5D-Y); Refraction*, Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chart, Best Corrected Visual Acuity (BCVA)*, MNRead acuity chart*; Pelli-Robson chart*; low-contrast BCVA*; slit lamp examination; measurement of intraocular pressure using a Tonopen only; perimetry; dilated fundoscopy; Humphrey visual field test, retinal nerve fiber layer (RNFL) measurements, optical coherence tomography (OCT), and macular ganglion cell layer/inner plexiform layer (GCL/IPL) thickness measurements; Electroretinogram (ERG), and fundus photography. * Denotes assessments that were done pre-dilation. Primary endpoints included assessing changes from baseline to 24 months using the following tests: ETDRS, BCVA, Humphrey, RNFL, OCT macular GCL/IPL thickness. Secondary endpoints included assessing changes at 6, 12, and 18 months with BCVA, Humphrey 10-2, RNFL, OCT GCL/IPL, low-contrast (LC) Visual Acuity (25%, 5%, and 2.5%), ERG PhNR, QOL (NEI-VFQ-25, IVI-C, EQ-5D/EQ-5D-Y) and reading speed (MNRead). Exploratory endpoints included validating the correlation between retinal mitochondrial dysfunction and flavoprotein fluorescence (via OcuMet Beacon™) and analysis of whole blood, plasma, and serum.

[0785] FALCON - genotyping

[0786] A saliva/buccal swab for genotyping was collected for each study participant. Historical genetic testing (i.e., genetic testing from participant record) results were used to determine eligibility. FALCON cohorts have a lower proportion of missense mutations than in other study cohorts due to exclusion of ADOA Plus (initial proportions can be found in Table 15A and proportions at 12 months can be found in Table 15B).

Table 15A. Initial proportion of types of OPA1 mutations of FALCON study participants

Table 15B. Proportion of types of OPA1 mutations of FALCON study participants at 12 months

*OPA1 genotype total includes only patients who completed at least one post-baseline visit.

[0787] FALCON- OCT GCL/IPL

[0788] Optical Coherence Tomography was performed according to standard procedures known in the art (Table 16). Ganglion Cell-Inner Plexiform Layer (GCL/IPL) thickness and retinal nerve fiber layer (RNFL) thickness of participants in the FALCON study were similar to previously reported values (Barboni et al. Am J Ophthalmol. 2011: 158: 628-636. Barboni et al. Ophthal. 2011: 118: 2076-2080. Corajevic et al. Acta Ophthalmol. 2018: 96: 251-256. Yu-Wai-Man et al. Prog Ret & Eye Res. 2011: 30:81-114).

Table 16. Comparison of FALCON OCT results with reference values

[0789] FALCON -Best Corrected Visual Acuity (BCVA) - Early Treatment Diabetic Retinopathy Study (ETDRS)

[0790] Original Sloan ETDRS charts were used to measure refractions and visual acuity (VA): Chart R (Precision Vision 2110), used for refraction; Chart 1 (Precision Vision 2111), used to test the right eye (OD); and Chart 2 (Precision Vision 2112), used to test the left eye (OS). These charts use high-contrast Sloan letters of equal difficulty having 5 letters in each of 14 lines. Each chart has a designated sequence of letters. Participants only visualize Chart 1 and Chart 2 on the light box once refraction was completed. [0791] A light box was mounted on a wall, countertop, or on a stand manufactured for the light box. The height of the light box was positioned such that the top of the third row of letters is 125.5 ± 5. 1 cm from the floor. A distance of either 1 meter or 4 meters was required between the participant’s eyes and the front of the VA chart.

[0792] All testing began at a distance of 4 meters between the VA chart to the participant’s eyes. Participants were required to be able to read letters in the English alphabet. Trial frames with best corrected manifest refraction were used to read charts. Chart 1 (Precision Vision 2111) for the right eye (OD) was tested first, followed by testing with Chart 2 (Precision Vision 2112) for the left eye (OS). Testing procedures were executed as in standard practice. Briefly, participants occluded the eye that was not being tested. Each letter attempted by the participant is scored as either right or wrong, and each letter read correctly is scored as one point. Eyes reading 19 or fewer letters correctly at 4 meters were additionally tested at 1 meter.

[0793] BCVA test results are recorded in logMAR. A logMAR value of 0 is representative of standard vision, positive values represent poor vision, and negative values represent good vision. A best-corrected visual acuity reading worse than 0.5 logMAR is correlated with low vision, and a best-corrected visual acuity worse than 1.3 logMAR is associated with blindness.

[0794] FALCON cohorts with ADOA participants between 41 and 60 years of age performed the worst in the BCVA tests (highest logMAR, lowest letter scores), while those in cohorts comprising participants 40 or younger performed better with lower logMAR and higher letter scores (Table 17). Table 17. BCVA logMAR and visual acuity letter scores

[0795] Baseline Visual Acuity appears to worsen (logMAR increases) after age 27 (FIG. 15).

[0796] FALCON -Best Corrected Visual Acuity (BCVA) - high/low contrast

[0797] Testing methodology for high- and low-contrast BCVA tests are the same as the methodology for BCVA ETDRS tests, except that study participants read charts at both 4 meters and 1 meter. The charts used include the Sloan Revised Low-Contrast Charts at 25% (Precision Vision 2154 and 2154A - scrambled), 5% (Precision Vision 2152 and 2152A - scrambled), and 2.5% (Precision Vision 2151 and 2151 A - scrambled). An illuminator cabinet designed for the low-contrast Sloan charts were used. Scoring is the sum of the correct responses at 4 meters and 1 meter.

[0798] Baseline High/Low Contrast Visual Acuity is correlated with functional and anatomic parameters (FIG. 16). Results from the high- or low-contrast contrast BCVA indicate that logMAR at 2.5% contrast (i.e., low contrast) was strongly negatively correlated with the thickness of the Global ganglion cell layer (GCL), Nasal GCL, and Temporal GCL, Global retinal nerve fiber layer (RNFL), Nasal RNFL, and Temporal RNFL, meaning that thicker ocular anatomical parts comprising the nasal sector, temporal GCL, and global RNFL were correlated with lower logMAR values (i.e., better vision) in a low-contrast (2.5%) visual acuity test. A correlation coefficient with a larger negative value corresponds to a stronger negative correlation. Results from the high- or low-contrast contrast BCVA also indicate that logMAR at high-contrast (100% contrast) was strongly negatively correlated with Humphrey 10-2 Vision Field test Median Deviation. A normal visual field has a Humphrey Median Deviation of zero. The worse the visual field Mean Deviation, the higher the negative value corresponding to Mean Deviation. A higher Humphrey score (i.e., a more negative value) is thus correlated with a higher logMAR (i.e., worse vision) in a standard-contrast BCVA.

[0799] FALCON- GCL/IPL thickness

[0800] Baseline visual acuity appears to worsen (logMAR Increases) after threshold GCL/IPL thickness [0801] Baseline visual acuity appears to worsen after the Ganglion Cell Layer-Inner Plexiform Layer (GCL/IPL) thickness is measured at about 53 pm because logMAR increases at this 53-pm threshold (FIG. 17). Slopes for Nasal GCL > 53 pm and < 53 pm are different. When Nasal GCL was greater than about 53 pm, little to no change was observed in logMAR from baseline visual acuity tests. When Nasal GCL < 53 pm, logMAR increases as Nasal GCL decreases.

[0802] FALCON - HC BCVA andLC BCVA Delta Letter Score

[0803] Baseline difference between high-contrast (HC) BCVA and low-contrast (LC) BCVA (2.5%) scores of FALCON participants is greater than seen in historical controls (Table 18).

Table 18. Baseline differences between HC BCVA and LC BCVA (2.5%)

[0804] In the FALCON study, the difference between the high-contrast BCVA score and the 2.5% low- contrast BCVA score (Delta Letter Score acquired by subtracting the latter from the former) was found to be 10-15 letters higher than normal historical controls, and scores of the participants in the FALCON study were similar to those of multiple Sclerosis (MS) patients. A higher DELTA appears to indicate the presence of ‘disease’ for both ADOA and MS. Historical control values were acquired from * Little et al. Invest Ophth Vis Sci. 2013 54(1) - logMAR values normalized to the 100 Letter Score Scale; and ** Balcer et al. Neurology 2003 61 - logMAR values normalized to 100 Letter Score Scale.

[0805] Baseline HC BCVA minus LC BCVA (2.5%) (Delta Letter Score) is weakly correlated with Global GCL/IPL (FIG. 18). Delta HC/LC BCVA plotted against Global GCL/IPL thickness shows a relatively flat slope. The poor correlation between HC/LC BCVA Delta and GCL/IPL thickness suggests excess Delta may be due to factors(s) other than Global GCL/IPL.

[0806] FALCON -Humphrey 10-2 visual field threshold test

[0807] Baseline Humphrey Visual Field (10-2) Mean Deviation (MD) appears to worsen with age after the age of 27 (FIG. 19). For patients younger than 27 (<27 y), MD changes minimally (there is a slight decrease in absolute value with age). For patients who were at least 27 years old (> 27 y), MD increases linearly in absolute value. [0808] Older Patients (41-60 y) have thinner GCL/IPL, poorer Visual Fields (corresponding to more strongly negative mean deviation values in a Humphrey 10-2 test, and larger pattern standard deviation values, indicating more focal defects), and slower reading speed (Table 19).

Table 19. Mean values of FALCON cohorts in Visual Field, Reading Speed, GCL/IPL measurements, and RNFL measurements.

GCL, ganglion cell layer; IPL, inner plexiform layer; MD, Mean Deviation; PSD, Pattern Standard

Deviation; VF, Visual Field.

[0809] Participants in the FALCON study had a low percentage of missense mutations and those with an OP Al mutation have profound deficits in visual function, the disease worsening with age.

[0810] BCVA was high to moderately highly correlated to Humphrey 10-2 Mean Deviation, RNFL, and GCL/IPL thickness. The difference between high-contrast and 2.5% low-contrast BCVA exceeded published normal control data by 10-15 letters, suggesting that the increased difference is due to disease burden. Baseline high completion rates and relatively low intra-patient variability observed in these assessments indicate reliability and potential for repeat use, as would be required in ADOA interventional clinical studies. A follow-up of patients in FALCON for two years will provide valuable information on ADOA disease progression.

[0811] FALCON -Assessments at 12 months

[0812] FALCON- 12-month HCVA, LCVA, and RNFL thickness

[0813] FALCON participants experienced minimal changes in visual acuity and Global retinal nerve fiber layer (RNFL) thickness over 12 months, and these observations are consistent with the slow- progressing nature of ADOA. Results from evaluations for High-Contrast Letter Acuity (FIG. 20A) and Low-Contrast (2.5%) Letter Acuity (FIG. 20B) show little change in EDTRS letter changes at the 6- and 12-month visit assessments. Variability is relatively low, indicating the data are of good quality. Changes in RNFL thickness were also minimal over the course of 12 months (FIG. 20C).

[0814] Patients with higher baseline High-Contrast (HC) Visual Acuity (VA) showed greater decline in Low-Contrast (LC) Visual Acuity (VA) at 12 months. Patients with a baseline HC visual acuity of <0.3 logMAR experienced approximately a 5. 1-letter decrease in LC (2.5%) VA (FIG. 21A), whereas all patients completing the 12-month study visit experienced approximately a 1-letter decrease in LC (2.5%) VA (FIG. 21B)

[0815] FALCON - 12-month Photopic Negative Response (PhNR)

[0816] The photopic negative response (PhNR) test is a visual electrophysiology test used to assess inner retinal integrity and retinal ganglion cell (RGC) function. Ganglion cell pathology affects the amplitudes of the PhNR. Reduced amplitudes are correlated with various forms of optic neuropathy.

[0817] Patients with highest baseline BCVA showed greatest decline in photopic negative response (PhNR) measurements base ratio (peak to trough) at 12 months. FALCON participants with low baseline BCVA (<0.3 log MAR, or visual acuity better than 20/40) had approximately a -40% change in phNR base ratio at 12 months (FIG. 22A). Participants with baseline BCVA >0.3 to <0.6 logMAR, or visual acuity 20/40 to <20/80, had approximately 35% change in phNR base ratio at 12 months (FIG. 22B). Participants with the baseline BCVA (>0.6 to <0.9 logMAR, or visual acuity 20/80 to 20/160) experienced approximately 18% change in phNR base ratio at 12 months (FIG. 22C).

[0818] FALCON - 12-month HC and LC ETDRS letter score change

[0819] FALCON participants did not have significant changes in their High-Contrast ETDRS Letter Scores at 12 months. Participants with low baseline BCVA (<0.3 logMAR) visual acuity (Va) experienced approximately a -1.4 HC letter score change (FIG. 23 A), participants with baseline BCVA of <0.3 to <0.6 logMAR experienced approximately a +0.9 HC letter score change (FIG. 23B), participants with baseline BCVA of 0.6 to <1 logMAR experienced approximately a +0.4 HC letter score change (FIG. 23C), and participants with baseline BCVA of >1 logMAR experienced approximately a +0.9 HC letter score change (FIG. 23D) at 12 months.

[0820] Only participants with a visual acuity (“Va”) of <0.3 logMAR experienced a Low-Contrast (2.5%) letter score change of at least 5 letters at 12 months. Participants with low baseline BCVA (<0.3 logMAR) experienced approximately a -5.1 LC letter score change (FIG. 23E), participants with baseline BCVA of <0.3 to <0.6 logMAR experienced approximately a +0.9 LC letter score change (FIG. 23F), participants with baseline BCVA of 0.6 to <1 logMAR experienced approximately a +1. 1 LC letter score change (FIG. 23G), and participants with baseline BCVA of >1 logMAR experienced approximately a - 0.5 LC letter score change (FIG. 23H) at 12 months.

[0821] A larger percentage of participants (-15%, or 7 out of 46 participants) experienced more than a 5- letter loss (“> 5 Letter Loss”) at 12 months when evaluated with for Low-Contrast (2.5%) Best Corrected Visual Acuity (FIG. 24, right column). Because a higher percentage of patients experienced > 5 letter loss (above noise level) indicates that Low-Contrast (2.5%) BCVA is likely a more sensitive indicator of visual loss over the short duration of 12 months. Several patients had a decline of more than five letters when evaluated with the Low-Contrast (2.5%) BCVA Letter Score (FIG. 26). About 4% of participants (2 out of 46 participants) experienced more than a 5 -letter loss at 12 months when evaluated for High- Contrast Best Corrected Visual Acuity (FIG. 24, left column).

[0822] Participants with the best initial Visual Acuity had the highest representation in the group of patients who experienced a greater than 5 -letter loss at 12 months when evaluated with Low-Contrast (2.5%) ETDRS BCVA. Participants with an initial Best Corrected Visual Acuity (BCVA) of <0.3 logMAR experienced about a 15-letter loss from baseline (FIG. 25A, first column), those with a BCVA of >0.3 to <0.6 logMAR experienced about a 10-letter loss from baseline (FIG. 25A, second column), and those with a BCVA of >0.6 to <1 logMAR experienced about a 12-letter loss from baseline (FIG. 25A, third column) at 12 months. No participants with a BCVA of >1 logMAR were evaluated (FIG.

25 A, fourth column). The distribution of patients with > 5 letter loss was also evaluated for patient groups with different initial visual acuity. About 32% of participants with an initial Best Corrected Visual Acuity (BCVA) of <0.3 logMAR (FIG. 25B, first column), about 10% of participants with a BCVA of >0.3 to <0.6 logMAR (FIG. 25B, second column), and about 12% of participants (FIG. 25B, third column) experienced a greater than 5-letter loss from baseline at 12 months. Those with a BCVA of >1 logMAR (FIG. 25B, fourth column) were not evaluated.

[0823] FALCON - Genotype Correlations with Visual Acuity Score (VAS)

[0824] Visual Acuity Score (VAS) is a scale that estimates visual ability. VAS is calculated on ETDRS charts as 1 point for each correctly read letter. Higher values indicate better vision. The formula for calculating VAS is 100 - 50 x logMAR, and 20/20 is rated as 100 on the VAS scale. 50 VAS points is equivalent to 20/200, and 0 VAS points is equivalent to 20/2000.

[0825] Patients who had greater than 5 letters lost (“> 5 Letter Loss”) were evaluated with regard to their OPA1 mutation type. Of these participants who had more than five letters lost when evaluated with the Low-Contrast (2.5%) BCVA Letter Score, some patients had missense mutations and others had nonsense mutations in OPA1 (FIG. 26).

[0826] Participants with missense mutations showed a greater Low-Contrast (2.5%) EDTRS letter change than the full study group. Participants with missense mutations experienced about a -1 ETDRS letter change with the High-Contrast (HC) Visual Acuity Score (VAS) (FIG. 27, first column), whereas participants from the full study group experienced close to no ETDRS letter change with HC VAS at 12 months (FIG. 27, second column). Participants with missense mutations experienced about a -4 ETDRS letter change with the Low-Contrast (LC) (2.5%) Visual Acuity Score (VAS) (FIG. 27, third column), whereas participants from the full study group experienced close to no ETDRS letter change with LC VAS (FIG. 27, fourth column).

[0827] A small group of FALCON participants (small N) were further evaluated for correlation between LC (2.5%) Letter Change and the types of OPA1 missense mutations they had. FALCON participants with a C-terminal coil-coil domain missense mutation in OPA1 showed the greatest 12-month letter loss. When these participants were evaluated for their visual acuity, they had about a -3 HC Letter Change (FIG. 28A, square points) and about a -7.5 LC (2.5%) Letter Change (FIG. 28B, square points) at 12 months. Participants with a missense mutation in the GTPase domain of OPA 1 had about a +0.75 HC Letter Change (FIG. 28A, circular points) and about a -2.5 LC (2.5%) Letter Change (FIG. 28B, circular points) at 12 months. Participants with a missense mutation in the dynamin domain of OPA1 had about a -2.5 HC Letter Change (FIG. 28 A, triangular points) and about a -0.5 LC (2.5%) Letter Change (FIG. 28B, triangular points) at 12 months. The 12-month HC and LC Letter Changes associated with various missense mutations in the OPA1 domain can be found in Table 20.

Table 20. OPA1 missense mutations in various OPA1 mutations and HC/LC letter changes at 12 months (“12M”)

[0828] FALCON - Minnesota Maximum Reading Speed (MnRead)

[0829] Minnesota Maximum Reading Speed (MnRead) charts are continuous-text reading acuity charts for measuring reading acuity, critical print size, and maximum reading speed (words per minute) in both normal and low-vision participants. Normal charts were used in the FALCON study, and each chart contained 19 different test sentences with print sizes that progressively decrease in size moving down the chart. The charts were viewed by participants at 40 cm, measured from the participant’s outer canthus to the front of the chart. Due to the close working distance, +2.50 DS were added to each participant’s 4- meter refraction result. Charts were illuminated evenly without any glare or shadows. The start time was initiated when a sentence was uncovered, and the participant began by reading the first complete sentence in its entirety. The time stopped when the participant reached the end of the sentence. This process was repeated until the participant was unable to read any words in a sentence.

[0830] FALCON participant age cohorts and the entire study group showed no differential progression pattern when examined based on initial BCVA and evaluated with MnRead. The maximum reading speeds of the 8- to 17-year-old cohort were about 170 words/min (FIG. 29 A), 18- to 40-year-old cohort were about 195 words/min (FIG. 29B), 41- to 60-year-old cohort were about 120 words/min (FIG. 29C), and all participants were about 180 words/min (FIG. 29D) at 12 months, with little change relative to their baseline reading speeds.

[0831] BEACON - OcuMet Beacon™ Sub-Study

[0832] BEACON is a sub-study of the FALCON study in which the OcuMet Beacon™ Device (OcuSciences) was used to assess the correlation between Flavoprotein Fluorescence (FPF) measurements and mitochondrial dysfunction and oxidative stress in the eyes of ADOA patients.

[0833] BEACON - Study design and participant demographics

[0834] Nineteen patients in three age cohorts (8-17 years old, 18-40 years old, and 41-60 years old) completed the BEACON test across seven sites (five in the US, two in the UK). All data points are averages for both of each patient’s eyes (Table 21).

Table 21. Demographics of participants who completed the BEACON test

[0835] BEACON- Optic disc FPF and pRNFL

[0836] All participants in the BEACON test had each eye evaluated by OcuMet Beacon™. Flavoprotein fluorescence (expressed as dB gray scale units (GSU)) was plotted against peripapillary retinal nerve fiber layer (pRNFL) thickness (pm). Significant negative correlations between FPF and pRNFL thickness were found across all Garway-Heath visual field sectors (i.e., the thicker the pRNFL, the lower the FPF readout; the thinner the pRNFL, the higher the FPF readout), for example, in the global optic disc (FIG. 35A), but these negative correlations were not found in the temporal regions, temporal (T) (FIG. 35B) and temporal inferior (TI) (FIG. 35C). The pRNFL thickness in the temporal region (T) tended to be thinner at about 50-pm thick or less, when compared to the thickness measurements of other Garway- Heath visual field sectors. The pRNFL in the temporal region was also found to have higher FPF between about 5 dB GSU and 14 dB GSU.

[0837] BEACON - Optic disc FPF and age

[0838] Study participant age in years was plotted against flavoprotein fluorescence (reported in decibel (dB) grayscale units (GSU)) as measured by OcuMet Beacon™ and data from three groups (younger than 18, between 18 and 40 years of age, and older than 40). Data was plotted without RNFL normalization for the global optic disc (FIG. 36A), Temporal Inferior sector (FIG. 36B), Temporal sector (FIG. 36C), and with RNFL normalization for the global optic disc (FIG. 36D), Temporal Inferior sector (FIG. 36E), Temporal sector (FIG. 36F). Data from the group comprising participants younger than 18 years of age had global optic disc FPF readings averaging less than about 0.1 dB GSU after data normalization, temporal inferior sector FPF readings averaging less than about 0. 1 dB GSU after data normalization, and temporal sector FPF readings averaging about 0.2 dB GSU after data normalization. Data from the group comprising participants 18 years of age or older to 40 years of age or younger had global optic disc FPF readings averaging about 0.12 dB GSU after data normalization, temporal inferior sector FPF readings averaging about 0.1 dB GSU after data normalization, and temporal sector FPF readings averaging about 0.3 dB GSU after data normalization. Data from the group comprising participants older than 40 years of age had global optic disc FPF readings averaging about 0.15 dB GSU after data normalization, temporal inferior sector FPF readings averaging about 0.15 dB GSU after data normalization, and temporal sector FPF readings averaging about 0.4 dB GSU after data normalization. Higher global, temporal inferior sector, and temporal sector optic disc FPF readings were each positively correlated with an increase in participant age. Temporal sector FPF readings exhibited the highest differences between the three age groups in this study.

[0839] Spearman’s rho correlations: optic disc FPF correlation coefficients ranged from r = 0.77 to 0.81 across Global (FIG. 37A) and Garway -Heath sectors, including the Temporal sector of the optic disc (FIG. 37B) and Temporal Inferior sector of the optic disc (FIG. 37C). A significant age-dependent increase in FPF decibel (dB) grayscale units (GSU) was observed. The macular FPF correlation with age was r = 0.39 (FIG. 37D). The Temporal Inferior sector is where the correlation to FPF and age is highest, though generally there was a less pronounced effect of aging on FPF. Each data point is the average of FPF measurements from both eyes of one patient.

[0840] BEACON - Optic disc FPF and letter score

[0841] Study participant letter scores from high-contrast (HC) and low-contrast (EC) Best Corrected Visual Acuity (BCVA) tests were plotted against flavoprotein fluorescence intensity (reported in dB GSU) as measured by OcuMet Beacon™. Data from three groups (younger than 18, between 18 and 40 years of age, and older than 40) not normalized to pRNFL were plotted for HC BCVA (FIG. 38A) and LC BCVA (FIG. 38B), and data normalized to pRNFL were also plotted for HC BCVA (FIG. 38C) and LC BCVA (FIG. 38D). Higher letter scores are associated with better performance in BCVA tests, while lower letter scores are associated with poorer performance.

[0842] In the high-contrast BCVA tests, the group comprising participants younger than 18 years of age had letter scores that averaged at about 60 letters and FPF readouts that averaged at about 0.05 dB GSU; the group comprising participants between 18 and 40 years of age had letter scores that averaged at about 50 letters and FPF readouts that averaged at about 0.1 dB GSU; and the group comprising participants older than 40 had letter scores that averaged at about 45 letters and FPF readouts that averaged at about 0. 15 dB GSU. In the low-contrast BCVA tests, the group comprising participants younger than 18 years of age had letter scores that averaged at about 38 letters and FPF readouts that averaged at about 0.05 dB GSU; the group comprising participants between 18 and 40 years of age had letter scores that averaged at about 25 letters and FPF readouts that averaged at about 0. 1 dB GSU; and the group comprising participants older than 40 had letter scores that averaged at about 13 letters and FPF readouts that averaged at about 0. 15 dB GSU.

[0843] BEACON - Optic disc FPF and Humphrey 10-2 Visual Field Mean Deviation

[0844] Flavoprotein fluorescence (FPF) readings for the global optic disc (in units of decibel (dB) gray scale units (GSU), FIG. 39A) and global macula (in units of linear gray scale units (GSU), FIG. 39B) were plotted against Humphrey 10-2 Visual Field (HVF) threshold test mean deviation (MD). There is a moderate, negative correlation between global disc FPF and HVF mean deviation based on the calculated correlation coefficient r = -0.51 and p = .031 (FIG. 39 A). No significant correlation was found between global macular FPF and HVF mean deviation (FIG. 39B).

[0845] BEACON - FPF hierarchical clustering

[0846] A hierarchical cluster analysis was performed using data compiled from all the assessments run during the BEACON study (FIG. 40), including the Humphrey Visual Field, low-contrast BCVA, high- contrast BCVA, pRNFE thickness, ganglion cell-inner plexiform layer (GCEIPE), MNRead Speed, and optic disc FPF tests for various visual sectors such as the inferior nasal (IN or NI) sector; superior nasal (SN or NS) sector; inferior temporal (TI or IT) sector; and superior temporal (ST or TS) sector. Sample size was N=10/19, where each row represents data from a single patient and only data for patients completing all assessments were included. Patients older than 40 years of age had the worst visual functions, thinner than average OCT, and higher FPF. Patients younger than 18 years of age tended to have better visual function, thicker OCT, and lower FPF.

[0847] Based on these analyses, optic disc FPF was found to worsen with age and significantly correlated with visual function and pRNFE. Given that the entire study cohort comprises participants with similar thinning of temporal RNFE that is greater than expected for normal aging and a worse FPF score with increasing age, disease progression appears unrelated to temporal RNFE thinning.

[0848] BEACON - Garway-Heath visual field sectors and optic disc flavoprotein fluorescence (FPF) [0849] The relationship between Garway-Heath visual field sectors (Global (G); Temporal (T);

Temporal Superior (TS); Nasal Superior (NS); Nasal (N); Nasal Inferior (NI); and Temporal Inferior (TI)) and optic disc flavoprotein fluorescence (FPF) was assessed using the Beacon instrument in three groups divided by age (study participants ages 8-17, ages 18-40, and ages 41-60) (FIG. 41). BEACON study participants were found to have higher average optic disc FPF levels globally and in the temporal sectors, particularly the temporal inferior (TI) sector for those in the cohort comprising participants between the ages of 41 and 60. Participants were observed to have low to moderate optic disc FPF levels in the nasal sectors.

[0850] BEACON -Low Contrast at 25%, 5% and 2.5% Letter Acuity & logMAR showed strong correlations to FPF Temporal Inferior and Nasal Inferior

[0851] Parameters comprising LogMAR, Visual Acuity Score (VAS), LogMAR Low-Contrast Letter Acuity (2.5%, 5%, and 25%), Visual Acuity Score (VAS) Low-Contrast (2.5%, 5%, and 25%), Humphrey Automated Perimetry (AP) Mean Deviation (MD), and Humphrey Automated Perimetry (AP) and Pattern Standard Deviation (PSD) were evaluated to determine their level of correlation with non- normalized flavoprotein fluorescence (FPF) data (FIG. 42). Visual acuity based on standard contrast, Best Corrected Visual Acuity Charts (LogMAR), and visual acuity based on Low-Contrast Best Corrected Visual Acuity Charts (LogMAR LC 2.5%, 5%, and 25%) showed the strongest correlation to FPF Temporal Inferior (TI) and Nasal Inferior (NI) data (e.g., the higher a participant’s LogMAR, the greater the FPF score in the TI and/or NI visual field sectors; the higher a participant’s LogMAR with a Low-Contrast (2.5%) BCVA, the greater the FPF score in the TI and/or NI visual field sectors, etc.). Visual Acuity Score (VAS) Low-Contrast (2.5%, 5%, and 25%) appeared to be negatively correlated with FPF scores in the Nasal Inferior (NI), Nasal Superior (NS), Temporal (T), and Temporal Inferior (TI) visual field sectors.

[0852] The oldest age groups had the highest FPF scores across the time period studied, consistent with the notion that the older patients had a more advanced stage of the disease and thus, higher mitochondrial stress, which correlates with higher FPF scores. The oldest age group (41- to 60-year-olds) showed the highest temporal (FIG. 43B, triangular points) and global (FIG. 43A, triangular points) non-normalized FPF scores. The middle age group (18- to 40-year-olds) had the second-highest temporal (FIG. 43B, square points) and global (FIG. 43A, square points) non-normalized FPF scores. The youngest (8- to 17- year-olds) age group had the lowest temporal (FIG. 43B, circular points) and global (FIG. 43A, circular points) non-normalized FPF scores.

[0853] Participants who were 8 to 40 years old showed greatest increase in FPF scores at both 6 and 12 months. The percent change from baseline in normalized FPF score in the Temporal visual field sector for the youngest (8- to 17-year-olds) age group (FIG. 44, circular points) was about a 9% increase. The percent change from baseline in normalized FPF score in the Temporal visual field sector for the middle age group (18- to 40-year-olds) (FIG. 44, square points) was about a 10% increase. The percent change from baseline in normalized FPF score in the Temporal visual field sector for the oldest age group (41- to 60-year-olds) (FIG. 44, triangular points) was about a 1% increase. The combined scores from all age groups showed an increase in the percentage change from baseline in the FPF score in the Temporal visual field sector at both 6 and 12 months of the study.

[0854] Temporal Inferior (TI) FPF scores for all age groups except the oldest age group showed changes from baseline (FIG. 45A and FIG. 45B). TI (normalized to RNFL thickness) FPF scores (FIG. 45B) show increases of 10% or more in participants ages 8 to 40 years old. Combined scores also show increased FPF scores at 12 months.

[0855] Based on current medical understanding of ADOA from a clinical and anatomical perspective, the disease is related to changes on the temporal side of the optic disc, rather than on the nasal side. This correlation seen in the FPF assessments, supported by the observations that nothing of note was found on the nasal side during the 12 months observed. Normalized nasal FPF scores showed minimal changes from baseline for all age groups (FIG. 46A) and by comparison with the initial Best Corrected Visual Acuity (FIG. 46B). These data are consistent with the anatomy and phenotype of ADOA.

[0856] Macular FPF scores of participants showed little change over 12 months (FIG. 47). Peripapillary FPF appears to be more sensitive in showing changes over 12 months. [0857] FPF global disc scores of participants with the best initial vision (as assessed by baseline Best Corrected Visual Acuity, represented as Baseline LogMAR <0.3) showed the greatest percentage of change from baseline when scores were normalized for RNFL thickness (FIG. 47, circular points), followed by participants with Baseline LogMar >0.3 to <0.6 (FIG. 47, square points).

[0858] Evaluation of participants using functional assessments including, for example, Humphrey Visual Field (10-2) Mean Deviation, Humphrey Visual Field (10-2) Pattern Standard Deviation, MnRead Maximum Reading Speed, QoL Measures; structural assessments including, for example, GCL/IPL, and/or RNFL; and BEACON FPF scores including, for example, Nasal Peripapillary scores and/or Macular scores did not show overall consistent change at 12 months.

[0859] High Contrast (HC) and Low-Contrast (LC 2.5%) BCVA declined the most in patients with initially good BCVA, and several patients had a decline of more than 5 Letters (above ‘noise’ level). The observed decline was 2 letters in HC BCVA and the observed decline was 5 letters in 2.5% LC BCVA. Patients with a greater than 5 letter loss at 12 months were the most prevalent in vision group that had initially good vision, though the group size was small (small N).

[0860] Together with findings from Baseline analysis, Low-Contrast BCVA may represent the most sensitive visual function test in ADOA, supported by the findings that LC (2.5%) showed 7 out of 46 (15.22%) patients had > 5 Letter Loss at 12 months. Patients with missense mutations in ADOA may show greater decline at 12 months than the study group as a whole, though the group size was small (small N). Participants with the C-terminal Coil-Coil domain (CC) missense mutations experienced the greatest decline in vision over 12 months, though the group size was small (small N). PhNR and pERG results showed a very high degree of variability, but PhNR did also show a decline in vision for patients with the highest BL BCVA. Beacon FPF scores show increases by percentage change over 12 months, with the greatest change seen in Temporal Inferior FPF score. That the Beacon FPF changed over a short time period of about 1 year may indicate functional deterioration in the face of little anatomical change. [0861] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS What is claimed is:

1. A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutic agent, wherein the subject has a vision test score within a reference value range and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer.

2. A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising:

(1) determining a vision test score of the subject;

(2) identifying the subject as an eligible subject for treatment when the vision test score determined in (1) is within a reference value range; and

3. A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising administering to the subject a pharmaceutical composition according to a dosing regimen selected based at least in part on a vision test score that the subject has, wherein the pharmaceutical composition comprises a therapeutic agent, and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer.

4. The method of claim 3, wherein the vision test score is measured before the subject receives administration of the pharmaceutical composition.

5. The method of claim 3, wherein the vision test score is measured after the subject receives administration of one or more prior doses of the pharmaceutical composition.

6. A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising:

(1) determining a vision test score of the subject;

(2) selecting a dosing regimen for a pharmaceutical composition for the subject based at least in part on the vision test score determined in (1), wherein the pharmaceutical composition comprises a therapeutic agent, and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer; and

7. A method of treating a subject having a disease or condition or reducing likelihood of developing the disease or condition, the method comprising:

(1) administering to the subject a pharmaceutical composition according to a dosing regimen, wherein the pharmaceutical composition comprises a therapeutic agent, and wherein the therapeutic agent comprises an antisense oligomer or a vector encoding the antisense oligomer; (2) after (1), determining a vision test score of the subject;

(3) adjusting the dosing regimen for the pharmaceutical composition based at least in part on the vision test score determined in (2); and

8. The method of claim 7, wherein the dosing regimen for the pharmaceutical composition is selected based at least in part on a vision test score measured prior to the administering in (1).

9. The method of any one of claims 3-8, wherein the dosing regimen comprises frequency of administration of the pharmaceutical composition, dose of the pharmaceutical composition per a single administration, time interval between administrations of the pharmaceutical composition, duration of treatment with the pharmaceutical composition, or administration route for the pharmaceutical composition.

10. The method of any one of claims 1-9, wherein the vision test score is within a reference value range.

11 . The method of any one of claims 1-10, wherein the vision test score is determined based at least in part on result from a Best Corrected Visual Acuity (BCVA) test of one or both eyes of the subject.

12. The method of claim 11, wherein the vision test score is determined based at least in part on a parameter selected from the group consisting of Best Corrected Visual Acuity (BCVA) letter score; Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score; high-contrast Best Corrected Visual Acuity (HC BCVA) letter score; low-contrast Best Corrected Visual Acuity (LC BCVA) letter score; and any combinations thereof.

13. The method of claim 11, wherein the vision test score is determined based at least in part on a low -contrast Best Corrected Visual Acuity (LC BCVA) letter score.

14. The method of any one of claims 11-13, wherein the vision test score is determined based at least further in part on a parameter selected from the group consisting of flavoprotein fluorescence intensity; Humphrey 10-2 Visual Field test Mean Deviation (MD); Humphrey 10-2 Visual Field test Pattern Standard Deviation (PSD); Visual Acuity Score (VAS); Quality of life questionnaires (NEI-VFQ-25, IVI- C, EQ-5D, EQ-5D-Y); Refraction test result; Minnesota Reading (MNRead) acuity chart score; Pelli- Robson chart score; slit lamp examination result; intraocular pressure using a Tonopen; perimetry; dilated fimdoscopy result; retinal nerve fiber layer (RNFL) measurement; optical coherence tomography (OCT) result; and macular ganglion cell layer/inner plexiform layer (GCL/IPL) thickness measurements; Curve Width (CW) measurement; Electroretinogram (ERG) result; fundus photography result; and any combinations thereof.

15. The method of any one of claims 11-14, wherein the BCVA comprises testing in a defined sequence for (1) ocular refraction from a fixed distance, and (2) visual acuity from a fixed distance.

16. The method of claim 15, wherein the testing comprises using at least one Original Sloan Early Treatment Diabetic Retinopathy Study (ETDRS) chart.

17. The method of claim 16, wherein ETDRS chart comprises a chart selected from the group consisting of Chart R (Precision Vision 2110) to measure refraction, Chart 1 (Pression Vision 2111) to test the right eye (OD), and Chart 2 (Precision Vision 2112) to test the left eye (OS).

18. The method of claim 17, wherein the defined sequence comprises testing for ocular refraction with Chart R before testing for visual acuity with either Chart 1 or Chart 2.

19. The method of any one of claims 16-18, wherein the fixed distance comprises a distance of about 4 meters or about 1 meter from the eyes of the subject to the front of the chart.

20. The method of any one of claims 16-18, wherein the testing for ocular refraction comprises a distance of about 4 meters from the eyes of the subject to the front of the chart.

21 . The method of any one of claims 16-18, wherein the testing for visual acuity comprises at least a first distance of about 4 meters from the eyes of the subject to the front of the chart.

22. The method of claim 20, wherein the testing for visual acuity comprises a second distance of about 1 meter from the eyes of the subject to the front of the chart.

23. The method of any one of claims 16-18, wherein the BCVA further comprises calculating a letter score comprising a sum of a total number of letters correctly identified by the subject at 4 meters, plus 30; or calculating a letter score comprising a sum of a total number of letters correctly identified by the subject at 1 meter.

24. The method of any one of claims 16-23, wherein the ETDRS chart is a High-Contrast (HC) ETDRS chart.

25. The method of any one of claims 16-23, wherein the ETDRS chart is a Low-Contrast (LC) (2.5%) ETDRS chart.

26. The method of claim 23 or 25, wherein the subject has decrease in the Low-Contrast (LC) (2.5%) ETDRS letter score of at least 5 letters after about 12 months as compared to the subject’s baseline Low- Contrast (LC) (2.5%) ETDRS letter score prior to the administering.

27. The method of any one of claims 1-10, wherein the vision test score is determined based at least in part on result from a Humphrey 10-2 Visual Field test of one or both eyes of the subject.

28. The method of claim 24, wherein the vision test score is determined based at least in part on a parameter selected from the group consisting of Humphrey 10-2 Visual Field test Mean Deviation (MD); Humphrey 10-2 Visual Field test Pattern Standard Deviation (PSD); Visual Acuity Score (VAS); flavoprotein fluorescence intensity; Quality of life questionnaires (NEI-VFQ-25, IVI-C, EQ-5D, EQ-5D- Y); Refraction test result; Minnesota Reading (MNRead) acuity chart score; Pelli-Robson chart score; slit lamp examination result; intraocular pressure using a Tonopen; perimetry; dilated fundoscopy result; retinal nerve fiber layer (RNFL) measurement; optical coherence tomography (OCT) result; and macular ganglion cell layer/inner plexiform layer (GCL/IPL) thickness measurements; Curve Width (CW) measurement; Electroretinogram (ERG) result; fundus photography result; and any combinations thereof.

29. The method of claim 28, wherein the vision test score is determined based at least further in part on a parameter selected from the group consisting of Best Corrected Visual Acuity (BCVA) letter score; Best Corrected Visual Acuity, Early Treatment Diabetic Retinopathy Study (BCVA, ETDRS) letter score; m high-contrast Best Corrected Visual Acuity (HC BCVA) letter score; low-contrast Best Corrected Visual Acuity (LC BCVA) letter score; and any combinations thereof.

30. The method of any one of claims 1-14, wherein the vision test score is indicative of a level of visual acuity in the eye of the subject.

31 . The method of any one of claims 1, 2, or 10-29, wherein the reference value range is a range lower than a vision test score of a healthy control subject.

32. The method of any one of claims 1, 2, or 10-29, wherein the reference value range is a range lower than an average vision test score measured from a population of healthy control subjects.

33. The method of any one of claims 1, 2, or 10-32, wherein when the pharmaceutical composition is tested on a population of test subjects suffering the disease or condition, a vision test score measured from the test subjects in the population is determined to have a correlation with therapeutic efficacy of the pharmaceutical composition in the test subjects, and wherein the reference value range is a range associated with the therapeutic efficacy of the pharmaceutical composition at a reference level according to the correlation.

34. The method of any one of claims 1-33, wherein the genotype of the subject is unknown prior to the administration.

35. The method of any one of claims 2 or 6-34, wherein the genotype of the subject is unknown prior to the determining.

36. The method of any one of claims 3-35, wherein the dosing regimen is not selected based on the genotype of the subject.

37. The method of any one of claims 1-36, wherein about 0.005 to about 20 mg of the antisense oligomer is administered to one eye of the subject.

38. The method of claim 37, wherein about 0.005 mg to about 15 mg, about 0.005 mg to about 10 mg, about 0.005 mg to about 5 mg, about 0.005 mg to about 1 mg, about 0.01 mg to about 15 mg, about 0.01 mg to about 10 mg, about 0.01 mg to about 5 mg, about 0.01 mg to about 2.5 mg, about 0.01 mg to about 1.0 mg, about 0.01 mg to about 0.5 mg, about 0.01 mg to about 0.25 mg, about 0.01 mg to about 0. 1 mg, about 0.01 mg to about 0.05 mg, about 0.05 mg to about 10 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 2.5 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.25 mg, about 0.05 mg to about 0.1 mg, about 0. 1 mg to about 5 mg, about 0.1 mg to about 2.5 mg, about 0.1 mg to about 1.0 mg, about 0.1 mg to about 0.5 mg, or about 0.1 mg to about 0.25 mg of the antisense oligomer is administered to one eye of the subject.

39. The method of claim 37, wherein about 0.005 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.2 mg, about 0.5 mg, about 0.75 mg, about 1.0 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2.0 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, about 5.0 mg, about 5.5 mg, about 6.0 mg, about 7.0 mg, about 8.0 mg, about 9.0 mg, about 10 mg, about 12.5 mg, about 15 mg, about 17.5 mg, or about 20 mg of the antisense oligomer is administered to one eye of the subject.

40. The method of claim 37, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0.1 mg to about 1.5 mg, about 0. 1 mg to about 1.4 mg, about 0.1 mg to about 1.2 mg, about 0.1 mg to about 1.0 mg, about 0.1 mg to about 0.8 mg, about 0. 1 mg to about 0.7 mg, about 0.1 mg to about 0.5 mg, about 0. 1 mg to about 0.3 mg, about 0.2 mg to about 1.5 mg, about 0.2 mg to about 1.4 mg, about 0.2 mg to about 1.2 mg, about 0.2 mg to about 1.0 mg, about 0.2 mg to about 0.8 mg, about 0.2 mg to about 0.7 mg, about 0.2 mg to about 0.5 mg, about 0.3 mg to about 1.5 mg, about 0.3 mg to about 1.4 mg, about 0.3 mg to about 1.2 mg, about 0.3 mg to about 1.0 mg, about 0.3 mg to about 0.8 mg, about 0.3 mg to about 0.7 mg, about 0.3 mg to about 0.5 mg, about 0.5 mg to about 1.5 mg, about 0.5 mg to about 1.4 mg, about 0.5 mg to about 1.2 mg, about 0.5 mg to about 1.0 mg, about 0.5 mg to about 0.8 mg, about 0.5 mg to about 0.7 mg, about 0.7 mg to about 1.5 mg, about 0.7 mg to about 1.4 mg, about 0.7 mg to about 1.2 mg, about 0.7 mg to about 1.0 mg, about 0.8 mg to about 1.5 mg, about 0.8 mg to about 1.4 mg, about 0.8 mg to about 1.2 mg, about 0.8 mg to about 1.0 mg, about 1.0 mg to about 1.5 mg, about 1.0 mg to about 1.4 mg, about 1.0 mg to about 1.2 mg, about 1.2 mg to about 1.5 mg, or about 1.2 mg to about 1.4 mg of the antisense oligomer.

41 . The method of claim 37, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition at a dose of about 0.1 mg, about 0.2 mg, about 0.3 mg, 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, or about 1.5 mg of the antisense oligomer.

42. The method of claim 37, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition in a volume of about 5 pl to about 250 pl, about 10 pl to about 250 pl, about 20 pl to about 250 pl, about 30 pl to about 250 pl, about 40 pl to about 250 pl, about 50 pl to about 250 pl, about 60 pl to about 250 pl, about 70 pl to about 250 pl, about 80 pl to about 250 pl, about 100 pl to about 250 pl, about 120 pl to about 250 pl, about 150 pl to about 250 pl, about 160 pl to about 250 pl, about 180 pl to about 500 pl, about 200 pl to about 250 pl, about 220 pl to about 250 pl, about 5 pl to about 220 pl, about 10 pl to about 220 pl, about 20 pl to about 220 pl, about 30 pl to about 220 pl, about 40 pl to about 220 pl, about 50 pl to about 220 pl, about 60 pl to about 220 pl, about 70 pl to about 220 pl, about 80 pl to about 220 pl, about 100 pl to about 220 pl, about 120 pl to about 220 pl, about 150 pl to about 220 pl, about 160 pl to about 220 pl, about 180 pl to about 220 pl, about 5 pl to about 200 pl, about 10 pl to about 200 pl, about 20 pl to about 200 pl, about 30 pl to about 200 pl, about 40 pl to about 200 pl, about 50 pl to about 200 pl, about 60 pl to about 200 pl, about 70 pl to about 200 pl, about 80 pl to about 200 pl, about 100 pl to about 200 pl, about 120 pl to about 200 pl, about 150 pl to about 200 pl, about 160 pl to about 200 pl, about 180 pl to about 200 pl, about 5 pl to about 180 pl, about 10 pl to about 180 pl, about 20 pl to about 180 pl, about 30 pl to about 180 pl, about 40 pl to about 180 pl, about 50 pl to about 180 pl, about 60 pl to about 180 pl, about 70 pl to about 180 pl, about 80 pl to about 180 pl, about 100 pl to about 180 pl, about 120 pl to about 180 pl, about 150 pl to about 180 pl, about 5 pl to about 150 pl, about 10 pl to about 150 pl, about 20 pl to about 150 pl, about 30 pl to about 150 pl, about 40 pl to about 150 pl, about 50 pl to about 150 pl, about 60 pl to about 150 pl, about 70 pl to about 150 pl, about 80 pl to about 150 pl, about 100 pl to about 150 pl, about 120 pl to about 150 pl, about 5 pl to about 150 pl, about 10 pl to about 120 pl, about 20 pl to about 120 pl, about 30 pl to about 120 pl, about 40 pl to about 120 pl, about 50 pl to about 120 pl, about 60 pl to about 120 pl, about 70 pl to about 120 pl, about 80 pl to about 120 pl, about 100 pl to about 120 pl, about 5 pl to about 100 pl, about 10 pl to about 100 pl, about 20 pl to about 100 pl, about 30 pl to about 100 pl, about 40 pl to about 100 pl, about 50 pl to about 100 pl, about 60 pl to about 100 pl, about 70 pl to about 100 pl, about 80 pl to about 100 pl, about 5 pl to about 80 pl, about 10 pl to about 80 pl, about 20 pl to about 80 pl, about 30 pl to about 80 pl, about 40 pl to about 80 pl, about 50 pl to about 80 pl, about 60 pl to about 80 pl, about 5 pl to about 60 pl, about 10 pl to about 60 pl, about 20 pl to about 60 pl, about 30 pl to about 60 pl, about 40 pl to about 60 pl, or about 50 pl to about 60 pl.

43. The method of any one of claims 37-42, wherein the method comprises administering to the one eye of the subject the pharmaceutical composition in a volume of about 5 pl, about 8 pl, about 10 pl, about 12 pl, about 15 pl, about 18 pl, about 20 pl, about 25 pl, about 28 pl, about 30 pl, about 35 pl, about 40 pl, about 45 pl, about 48 pl, about 50 pl, about 55 pl, about 60 pl, about 65 pl, about 70 pl, about 75 pl, about 80 pl, about 90 pl, about 100 pl, about 120 pl, about 150 pl, about 160 pl, about 180 pl, about 200 pl, about 220 pl, or about 250 pl.

44. The method of any one of claims 1-43, wherein the method comprises administering the pharmaceutical composition to both left eye and right eye of the subject.

45. The method of claim 44, wherein the method comprises administering the pharmaceutical composition at the same dose to both the left eye and the right eye of the subject.

46. The method of claim 44, wherein the method comprises administering the pharmaceutical composition at different doses to the left eye and the right eye of the subject.

47. The method of any one of claims 1-46, wherein the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NOS: 6- 275 or 280-299.

48. The method of any one of claims 1-46, wherein the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 36, 236, 242, 250, 92-96, and 166-168.

49. The method of any one of claims 1-48, wherein the therapeutic agent further comprises a gene editing molecule.

50. The method of claim 49, wherein the gene editing molecule comprises CRISPR-Cas9.

51 . The method of any one of claims 1-48, wherein the therapeutic agent comprises the antisense oligomer, and wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

52. The method of any one of claims 1-48, wherein the therapeutic agent comprises the antisense oligomer, and wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2’-O-methyl moiety, a 2 ’-Fluoro moiety, or a 2’-O-methoxyethyl moiety.

53. The method of any one of claims 1-48, wherein the therapeutic agent comprises the antisense oligomer, and wherein the antisense oligomer comprises at least one modified sugar moiety.

54. The method of claim 53, wherein each sugar moiety is a modified sugar moiety.

55. The method of any one of claims 1-54, wherein the antisense oligomer comprises a 5’- methylcytosine (5’-MeC).

56. The method of any one of claims 1-54, wherein each cytosine of the antisense oligomer is a 5’- methylcytosine (5’-MeC).

57. The method of any one of claims 1-56, wherein the antisense oligomer comprises a 5’- methyluracil (5 ’ -MeU) .

58. The method of any one of claims 1-56, wherein each cytosine or thymidine of the antisense oligomer is a 5 ’-methyluracil (5 ’-MeU).

59. The method of any one of claims 1-58, wherein the antisense oligomer comprises a phosphorothioate linkage.

60. The method of any one of claims 1-58, wherein each intemucleoside linkage of the ASO is a phosphorothioate linkage.

61 . The method of any one of claims 1-60, wherein the antisense oligomer comprises a locked nucleic acid (LNA).

62. The method of any one of claims 1-61, wherein the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to

40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

63. The method of any one of claims 1-62, wherein the therapeutic agent comprises the antisense oligomer, and the antisense oligomer has any one of the following chemical structures:

or a pharmaceutically acceptable salt thereof.

64. The method of claim 63, wherein the antisense oligomer has any of the following structures:

65. The method of any one of claims 1-48, wherein the therapeutic agent comprises the vector, and wherein the vector comprises a viral vector encoding the antisense oligomer.

66. The method of claim 65, wherein the viral vector comprises an adenoviral vector, adeno- associated viral (AAV) vector, lentiviral vector, Herpes Simplex Virus (HSV) viral vector, or retroviral vector.

67. The method of any one of claims 1-66, wherein the pharmaceutical composition is a liquid composition.

68. The method of any one of claims 1-67, wherein the method comprises administering the pharmaceutical composition as a bolus injection over 1 to 60 minutes, 1 to 50 minutes, 1 to 40 minutes, 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes, 1 to 5 minutes, or 1 to 3 minutes.

69. The method of any one of claims 1-68, wherein the method comprises administering the pharmaceutical composition as a bolus injection.

70. The method of any one of claims 1-69, wherein the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, or water for injection.

71 . The method of any one of claims 1-69, wherein the antisense oligomer is solubilized or diluted in a solution comprising one or more of sodium chloride, sodium phosphate dibasic, potassium phosphate monobasic, and water for injection.

72. The method of any one of claims 1-71, wherein the antisense oligomer is solubilized or diluted in an isotonic solution.

73. The method of any one of claims 1-72, wherein the antisense oligomer is solubilized or diluted in a phosphate-buffered solution with at least pH 5.8.

74. The method of any one of claims 1-72, wherein the antisense oligomer is solubilized or diluted in a phosphate-buffered (pH 6.6 - 7.6) solution.

75. The method of any one of claims 1-74, wherein the pharmaceutical formulation does not comprise a preservative.

76. The method of any one of claims 1-75, wherein the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml to about 200 mg/ml.

77. The method of any one of claims 1-75, wherein the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml to about 200 mg/ml, about 5 mg/ml to about 200 mg/ml, about 10 mg/ml to about 200 mg/ml, about 15 mg/ml to about 200 mg/ml, about 20 mg/ml to about 200 mg/ml, about 25 mg/ml to about 200 mg/ml, about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, 2 mg/ml to about 150 mg/ml, about 5 mg/ml to about 150 mg/ml, about 10 mg/ml to about 150 mg/ml, about 15 mg/ml to about 150 mg/ml, about 20 mg/ml to about 150 mg/ml, about 25 mg/ml to about 150 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, 2 mg/ml to about 100 mg/ml, about 5 mg/ml to about 100 mg/ml, about 10 mg/ml to about 100 mg/ml, about 15 mg/ml to about 100 mg/ml, about 20 mg/ml to about 100 mg/ml, about 25 mg/ml to about 100 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, 2 mg/ml to about 80 mg/ml, about 5 mg/ml to about 80 mg/ml, about 10 mg/ml to about 80 mg/ml, about 15 mg/ml to about 80 mg/ml, about 20 mg/ml to about 80 mg/ml, about 25 mg/ml to about 80 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, 2 mg/ml to about 60 mg/ml, about 5 mg/ml to about 60 mg/ml, about 10 mg/ml to about 60 mg/ml, about 15 mg/ml to about 60 mg/ml, about 20 mg/ml to about 60 mg/ml, about 25 mg/ml to about 60 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, about 40 mg/ml to about 60 mg/ml, 2 mg/ml to about 40 mg/ml, about 5 mg/ml to about 40 mg/ml, about 10 mg/ml to about 40 mg/ml, about 15 mg/ml to about 40 mg/ml, about 20 mg/ml to about 40 mg/ml, or about 25 mg/ml to about 40 mg/ml.

78. The method of any one of claims 1-75, wherein the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 12 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 18 mg/ml, about 20 mg/ml, about 22 mg/ml, about 24 mg/ml, about 26 mg/ml, about 28 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 80 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

79. The method of any one of claims 1-78, wherein the pharmaceutical composition is prepared by diluting a concentrate comprising the antisense oligomer.

80. The method of claim 79, wherein the antisense oligomer is present in the concentrate at a concentration of about 30 mg/ml to about 200 mg/ml, about 35 mg/ml to about 200 mg/ml, about 40 mg/ml to about 200 mg/ml, about 50 mg/ml to about 200 mg/ml, about 60 mg/ml to about 200 mg/ml, about 80 mg/ml to about 200 mg/ml, about 100 mg/ml to about 200 mg/ml, about 150 mg/ml to about 200 mg/ml, about 180 mg/ml to about 200 mg/ml, about 30 mg/ml to about 150 mg/ml, about 35 mg/ml to about 150 mg/ml, about 40 mg/ml to about 150 mg/ml, about 50 mg/ml to about 150 mg/ml, about 60 mg/ml to about 150 mg/ml, about 80 mg/ml to about 150 mg/ml, about 100 mg/ml to about 150 mg/ml, about 30 mg/ml to about 100 mg/ml, about 35 mg/ml to about 100 mg/ml, about 40 mg/ml to about 100 mg/ml, about 50 mg/ml to about 100 mg/ml, about 60 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, about 30 mg/ml to about 80 mg/ml, about 35 mg/ml to about 80 mg/ml, about 40 mg/ml to about 80 mg/ml, about 60 mg/ml to about 80 mg/ml, about 30 mg/ml to about 60 mg/ml, about 35 mg/ml to about 60 mg/ml, or about 40 mg/ml to about 60 mg/ml.

81. The method of claim 79, wherein the antisense oligomer is present in the concentrate at a concentration of about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, or about 200 mg/ml.

82. The method of any one of claims 79-81, wherein the concentrate is a phosphate-buffered solution.

83. The method of any one of claims 1-82, wherein the disease or condition is associated with a deficient amount or activity of the OPA protein.

84. The method of any one of claims 1-83, wherein the disease or condition comprises an eye disease or condition.

85. The method of any one of claims 1-83, wherein the disease or condition comprises a cardiovascular disease or condition.

86. The method of any one of claims 1-83, wherein the disease or condition comprises a neurological disease or condition.

87. The method of any one of claims 1-83, wherein the disease or condition comprises ADOA-plus; a mitochondrial disorder; glaucoma; normal tension glaucoma; Charcot-Marie-Tooth disease; mitochondria dysfunction; diabetic retinopathy; age-related macular degeneration; retinal ganglion cell death; mitochondrial fission-mediated mitochondrial dysfunction; progressive external ophthalmoplegia; deafness; ataxia; motor neuropathy; sensory neuropathy; myopathy; Behr syndrome; brain dysfunction; encephalopathy; peripheral neuropathy; fatal infantile mitochondrial encephalomyopathy; hypertrophic cardiomyopathy; spastic ataxic syndrome; sensory motor peripheral neuropathy; hypotonia; gastrointestinal dysmotility and dysphagia; optic atrophy; optic atrophy plus syndrome; Mitochondrial DNA depletion syndrome 14; late-onset cardiomyopathy; diabetic cardiomyopathy; Alzheimer’s Disease; focal segmental glomerulosclerosis; kidney disease; Huntington’s Disease; cognitive function decline in healthy aging; Prion diseases; late onset dementia and parkinsonism; mitochondrial myopathy; Leigh syndrome; Friedreich’s ataxia; Parkinson’s disease; MELAS (Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes); pyruvate dehydrogenase complex deficiency; chronic kidney disease; Leber’s hereditary optic neuropathy; obesity; age-related systemic neurodegeneration; skeletal muscle atrophy; heart and brain ischemic damage; or massive liver apoptosis.

88. The method of any one of claims 1-83, wherein the disease or condition comprises Optic atrophy type 1.

89. The method of any one of claims 1-83, wherein the disease or condition comprises autosomal dominant optic atrophy (ADOA).

90. The method of any one of claims 1-89, wherein the pharmaceutical composition is administered via intracerebroventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, implantation, or intravenous injection.

91 . The method of any one of claims 1-89, wherein the pharmaceutical composition is administered via intravitreal injection.

92. The method of any one of claims 1-91, wherein the method further comprises administering an additional therapeutic agent.

93. The method of claim 92, wherein the additional therapeutic agent comprises a small molecule.

94. The method of claim 92, wherein the additional therapeutic agent comprises an antisense oligomer.

95. The method of claim 92, wherein the additional therapeutic agent comprises an ophthalmologic drug.

96. The method of any one of claims 1-95, wherein the subject is a human subject.