[go: up one dir, main page]

WO2025090575A1 - Thérapie génique pour traitement de la dégénérescence rétinienne - Google Patents

Thérapie génique pour traitement de la dégénérescence rétinienne Download PDF

Info

Publication number
WO2025090575A1
WO2025090575A1 PCT/US2024/052518 US2024052518W WO2025090575A1 WO 2025090575 A1 WO2025090575 A1 WO 2025090575A1 US 2024052518 W US2024052518 W US 2024052518W WO 2025090575 A1 WO2025090575 A1 WO 2025090575A1
Authority
WO
WIPO (PCT)
Prior art keywords
prlδe1
nucleic acid
sequence
vector
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/052518
Other languages
English (en)
Other versions
WO2025090575A9 (fr
Inventor
Raghavi SUDHARSAN
William A. BELTRAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Pennsylvania Penn
Original Assignee
University of Pennsylvania Penn
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Pennsylvania Penn filed Critical University of Pennsylvania Penn
Publication of WO2025090575A1 publication Critical patent/WO2025090575A1/fr
Publication of WO2025090575A9 publication Critical patent/WO2025090575A9/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57554—Prolactin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes

Definitions

  • Retinitis pigmentosa is the most common form of inherited RD, with a worldwide prevalence of 1 in 4,000 individuals. Onset of RP is marked by night-blindness and peripheral vision loss, followed by a progressive shrinking of the visual field, loss of visual acuity, and eventual blindness.
  • Current efforts have focused on gene-specific therapies, with one FDA approved gene therapy (voritegene-neparvovec or Luxturna for RPE65-LCA) on the market, and therapies targeting 10 other genes currently in clinical trials.
  • Creregene-neparvovec or Luxturna for RPE65-LCA voritegene-neparvovec or Luxturna for RPE65-LCA
  • a method of treating retinal degeneration in a subject in need thereof comprising reducing PRL ⁇ E1 expression or activity in an eye of the subject.
  • the method comprises administering a nucleotide sequence that encodes a nucleic acid molecule that targets a portion of PRL ⁇ E1.
  • the nucleic acid molecule is an siRNA, miRNA, shRNA, ASO, or guide RNA.
  • the nucleic acid molecule is an siRNA that targets a sequence comprising nucleotides 277-302, 292-317, 267-317, 162-187, 622-647, 305-330, 320-345, 295-320, 188-231, 194-219, or 333-358 of human PRL ⁇ E1.
  • the nucleic acid molecule is an siRNA, miRNA, shRNA, ASO, or guide RNA that targets a sequence of canine PRL ⁇ E1.
  • the nucleic acid molecule that targets a sequence of canine PRL ⁇ E1 comprises a sequence set forth in Table 1 or 2.
  • the method further comprises administering a sequence that encodes a nuclease that targets a portion of PRL ⁇ E1.
  • the nuclease is a Cas enzyme.
  • the nuclease is a Cas9 or Cas13 enzyme.
  • the method comprises delivering a vector that encodes the nucleic acid molecule.
  • the vector is an AAV vector.
  • the method further comprises administering a vector that comprises a nucleotide sequence that encodes a functional ocular protein.
  • the nucleotide sequences that encode the nucleic acid molecule and the functional ocular protein are comprised in the same vector.
  • the nucleotide sequences that encode the nucleic acid molecule and the functional ocular protein are comprised in different vectors.
  • the AAV vector has an AAV2, AAV5, AAV7m8, or AAV8 capsid.
  • the subject has retinitis pigmentosa (autosomal dominant, autosomal recessive, X-linked), Leber's congenital amaurosis, cone or cone-rod dystrophy, Usher syndrome, macular degeneration, Stargardt's disease, age-related macular degeneration, trauma-induced retinal degeneration, or retinal detachment.
  • a composition comprising the nucleotide sequence that encodes the nucleic acid molecule is administered to an eye of the subject. In certain embodiments, the composition is administered via subretinal injection or intravitreal injection. In another aspect, provided herein is a composition comprising a nucleotide sequence that encodes a nucleic acid molecule that targets a portion of PRL ⁇ E1. In certain embodiments, the nucleic acid molecule is an siRNA, miRNA, shRNA, ASO, or guide RNA.
  • the nucleic acid molecule is an siRNA that targets a sequence comprising nucleotides 277-302, 292-317, 267-317, 162-187, 622-647, 305-330, 320-345, 295-320, 188-231, 194-219, or 333-358 of human PRL ⁇ E1.
  • the composition comprises a nucleic acid molecule that is an siRNA, miRNA, shRNA, ASO, or guide RNA that targets a sequence of canine PRL ⁇ E1.
  • the nucleic acid molecule that targets a sequence of canine PRL ⁇ E1 comprises a sequence set forth in Table 1 or 2.
  • the composition further comprises a sequence that encodes a nuclease that targets a portion of PRL ⁇ E1.
  • the nuclease is a Cas enzyme.
  • the nuclease is a Cas9 or Cas13 enzyme.
  • the composition comprises a vector that encodes the nucleic acid molecule.
  • the vector is an AAV vector.
  • the vector further comprises a nucleic acid that encodes a functional ocular protein.
  • the AAV vector has an AAV2, AAV5, AAV7m8, or AAV8 capsid.
  • the composition further comprises one or more pharmaceutically acceptable carriers, adjuvants, excipients, or diluents.
  • FIG.1A PRL ⁇ E1 mRNA identified by RNA in situ hybridization (magenta punctate staining, see black arrows) in the PRs in RPGR- XLPRA1 (left) and NPHP5-LCA (middle), but not in CNGB3-ACHM3, a cone-specific disease (right).
  • FIG.1B PRL ⁇ E1 expression observed by RNA-ISH (white) only in the degenerating NPHP5-LCA ONL containing both rods and cones and not in the adjacent cone-only ONL. Rhodopsin (RHO) staining for rods in green, Arrestin-3 (ARR3) staining for cones in red.
  • RHO Rhodopsin
  • ARR3 Arrestin-3
  • FIG.2 shows PRL ⁇ E1 expression overlaps with areas of PR degeneration in carriers of RPGR mutations. Retinal cellular layers identified by Hoechst staining of nuclei. Patchy areas of degeneration (white dashed boxes) resulting from random X-inactivation identified by rhodopsin mislocalization to PR cell bodies in RPGR-XLPRA1 carriers (age: 24 weeks) and RPGR-XLPRA2 (age: 24 weeks) carrier female dogs. PRL ⁇ E1 transcript identified by RNA-ISH.
  • FIG.3 shows PRL ⁇ E1 expression is not associated with acute PR cell death in RHO- T4R ADRP but correlates with protracted rod degeneration.
  • PRL ⁇ E1 expression is absent in acute light-induced RD in RHO-T4R retinas 24 hours and 2 weeks after light-induced damage but is observed in the more slowly degenerating PDE6 ⁇ -RCD1 retina (age: 14 weeks).
  • FIG.4A – FIG.4C show specific corrective gene augmentation therapy is associated with suppression of PRL ⁇ E1 expression in treated areas.
  • PRL ⁇ E1 RNA-ISH in a RPGR-XLPRA1 dog (treated at 28 weeks of age, assessed 49 weeks post-treatment) shows PRL ⁇ E1 expression in the untreated retina but the transcript is not expressed in AAV-RPGR treated area.
  • PRL ⁇ E1 RNA-ISH in a RPGR-XLPRA2 (treated at 5 weeks of age, assessed 33 weeks post-treatment) dogs shows PRL ⁇ E1 expression in the untreated retina but none in the AAV-RPGR treated area. Transition zone presents a clear demarcation in PRL ⁇ E1 expression between treated and untreated regions.
  • PRL ⁇ E1 RNA-ISH White punctate staining; RPGR immunostaining in green.
  • PRL ⁇ E1 RNA-ISH in a NPHP5-LCA dog shows abundant PRL ⁇ E1 in the ONL of the untreated area but markedly reduced levels in the AAV-NPHP5 treated region.
  • FIG.4C Schematic representation of the en face fundus image of the retina, dotted line corresponds to the area of treatment (bleb) injected with a corrective gene therapy AAV vector.
  • ONL Outer nuclear layer
  • INL Inner nuclear layer.
  • FIG.5 shows in vivo assessment of ONL thickness after treatment with shRNA PRL ⁇ E1 in PDE6 ⁇ -RCD1 and RPGR-XLPRA2 retinas.
  • Topographical pseudocolor maps (left) and reconstituted horizontal b-scans spanning through (red arrow) the treated (Tx) and untreated (Un-Tx) retinal areas (right) of a normal untreated retina, two RPGR-XLPRA2 treated retinas, and a PDE6 ⁇ -RCD1 treated retina. Dashed lines on maps (left) encircle the area of the subretinal bleb, dashed vertical lines on b-scans (right) demarcate the transition between Tx and Un-Tx. ONL layer thickness on b-scans is highlighted in blue. Calibration bars and pseudocolor scale shown are applicable to all.
  • FIG.6A – FIG.6E show assessment of AAV2/5- shRNA PRL ⁇ E1 -mediated PRL ⁇ E1 knockdown and effect on photoreceptors.
  • FIG.6A Percentage of PRL ⁇ E1 remaining as quantified by qPCR in RPGR-XLPRA2 retinas 5 weeks and 11 weeks post injection (PI).
  • FIG.6B Schematic representation of RPGR-XLPRA2 retinas with treated area demarcated by dotted lines and 3 mm retinal punches used for qPCR analysis shown with green (treated area) or red (untreated area) circles.
  • FIG.6C Representative RNA-ISH (magenta dots, arrows) image showing PRL ⁇ E1 expression in ONL of untreated and shRNA PRL ⁇ E1 -treated retinal areas in PDE6 ⁇ -RCD1.
  • FIG.6D Immunohistochemical staining for rods (Rhodopsin, RHO, green) and cones (Arrestin-3, ARR3, red) with PRL ⁇ E1-ISH (white) in untreated and treated regions of a RPGR-XLPRA2 retina 5-weeks PI.
  • FIG.6E Immunohistochemical staining for rods (Rhodopsin, RHO, green) and cones (Arrestin-3, ARR3, red) with PRL ⁇ E1-ISH (white) in untreated and, treated regions of a PDE6 ⁇ -RCD1 retina 9 weeks PI. Note: Only PRL and ARR3 are shown in and to emphasize the loss of cones in the retina following the shRNA treatment. Data presented is from one PDE6 ⁇ -RCD1 and 2 RPGR-XLPRA2 dogs treated with shRNA PRL ⁇ E1 .
  • ONL Outer nuclear layer
  • INL Inner nuclear layer
  • GCL Ganglion cell layer.
  • FIG.7A – FIG.7B show effects of subretinal injection of AAV2/5-shRNAPRL ⁇ E1 in normal dog retina.
  • FIG.7A cSLO en face image and OCT b-scan of the retina showing the treated (Tx) and the untreated (UnTx) areas.
  • FIG.7B Immunohistochemical staining of rods (RHO, green) and cones (ARR3, red) in untreated and treated retinal areas 8 weeks post injection. Mislocalization of rhodopsin (RHO) protein to the cell bodies and loss of cone specific Arrestin-3 (ARR3) staining is observed in the treated retina.
  • FIG.8A – FIG.8B show induction of PRL ⁇ E1 expression in NPHP5-LCA and RPGR-XLPRA1 retinas with disease progression.
  • ONL Outer nuclear layer
  • INL Inner nuclear layer
  • GCL Ganglion cell layer.
  • FIG.9 shows rods but not cone PRs express PRL ⁇ E1.
  • FIG.10A – FIG.10B show expression of PRL ⁇ E1 isoform is not observed in the rapidly degenerating RHO-T4R retinas post light exposure (LE).
  • FIG.10A Photomicrographs of H&E stained retinal cryosections labeled with RNA-ISH to visualize PRL ⁇ E1 expression at 24 hours (FIG.10A) and 2 weeks (FIG.10B) post light exposure (LE) to 3 different intensities.
  • FIG.11A – FIG.11B show identification of optimal shRNA for PRL ⁇ E1 knockdown.
  • FIG.11A PRL ⁇ E1 sequence with the three shRNA target sequences highlighted.
  • FIG.12 shows knockdown of human PRL ⁇ E1 (hPRL ⁇ E1) using DsiRNAs in HEK293 cells.
  • the bar graph illustrates the knockdown efficiencies of five different DsiRNAs tested on HEK293 cells exogenously expressing hPRL ⁇ E1.
  • Each bar represents the mean fold change in hPRL ⁇ E1 expression (represented as percentage of hPRL ⁇ E1 remaining) relative to the control group, which consisted of cells expressing hPRL ⁇ E1 without any DsiRNA treatment.
  • DsiRNA-NS Non-specific DsiRNA.
  • FIG.13 shows a vector genome for delivery of an shRNA for PRL ⁇ E1 knockdown.
  • compositions and methods for silencing or reducing expression of the PRL ⁇ E1 isoform of prolactin in the retina to promote photoreceptor survival are useful for treatment of a wide of range ocular conditions, including, for example, inherited retinal degenerations (RD) and age-related macular degeneration (AMD).
  • RD inherited retinal degenerations
  • AMD age-related macular degeneration
  • the compositions and methods are combined with gene replacement or gene augmentation therapies for treatment of an ocular disease.
  • Prolactin is a pleiotropic hormone that contributes to multiple physiologic and homeostatic functions in the body including maturation of mammary glands and initiation of lactation, metabolic regulation, angiogenesis, immunomodulation, neuroprotection and neurogenesis(1-6).
  • Full-length PRL activates the JAK/STAT pathway through its transmembrane receptor PRL-R, which is expressed on multiple cell-types(7).
  • PRL plays a protective role in light-induced retinal degeneration and in diabetic retinopathy(3, 8-12). Its anti-apoptotic and antioxidant effects promote survival of both the photoreceptors (PR) and the retinal pigment epithelium (RPE)(10, 13, 14).
  • PRL modulates neurotrophin signaling from glial cells to protect PRs from light-induced damage in the rodent retina(10).
  • elevated circulating levels of PRL have been associated with a reduced risk of proliferative diabetic retinopathy in diabetic patients, largely due to the antiangiogenic properties of vasoinhibins, a family of bioactive peptides generated from the N-terminus of PRL by proteolytic cleavage. These peptides are currently being investigated as a therapeutic target to regulate ocular angiogenesis and prevent disease progression in diabetic retinopathy and diabetic macular edema(9, 15-18).
  • PRL is also expressed in other tissues(19, 20), including normal retinas from rats, green monkeys, and baboons(15, 21, 22).
  • PRL ⁇ E1 a novel isoform of PRL that lacks the first exon, expressed in the retinas of two non-allelic, naturally occurring canine models of early-onset inherited retinal degeneration (IRD), PDE6 ⁇ -RCD1 and RPGR- XLPRA2(23). In these two models, a large number of PRs die acutely between 5 and 7 weeks of age, marking the early phase of disease(24, 25).
  • PRL ⁇ E1 expression is induced in the remaining rods and increases through the protracted phase of disease/degeneration, even as the rate of cell death declines(23). Expression of PRL ⁇ E1 is not observed in healthy young or adult dog retinas. However, this PRL isoform is found in the aged human retina(23). As described herein, PRL ⁇ E1 expression correlates with protracted RD and not acute PR cell death. The present inventors have identified a function of PRL ⁇ E1 in RD and demonstrated that PRL ⁇ E1 is a therapeutic target for promoting PR cell survival. Provided herein are compositions and methods for delivering a nucleic acid construct that reduces or inhibits expression of PRL ⁇ E1 in a target cell.
  • PRL ⁇ E1 refers to an isoform of prolactin (PRL) that lacks the first exon and, unless specified, may refer to a gene of a variety of mammals including, but not limited to, humans, canines, non- human primates, and mice.
  • “disease”, “disorder”, and “condition” are used interchangeably, to indicate an abnormal state in a subject.
  • expression is used herein in its broadest meaning and comprises the production of RNA, of protein, or of both RNA and protein. With respect to RNA, the term “expression” or “translation” relates in particular to the production of peptides or proteins. Expression may be transient or may be stable.
  • “Patient” or “subject”, as used herein interchangeably, means a male or female mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research such as canines, non-human primates, and mice.
  • the subject is a human patient.
  • the subject in a canine.
  • the subject has or is suspected of having one or more conditions, diseases, or disorders of the eye.
  • the term “a” or “an” refers to one or more. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.
  • an “expression cassette” refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence for reducing PRL ⁇ E1 expression (e.g., encodes a sequence that silences a gene via RNA interference (i.e. a nucleic acid molecule) or sequence that encodes one or more elements of a gene editing system) and regulatory sequences operably linked thereto which direct or modulate transcription, translation, and/or expression of the nucleic acid sequence.
  • RNA interference i.e. a nucleic acid molecule
  • regulatory sequences operably linked thereto which direct or modulate transcription, translation, and/or expression of the nucleic acid sequence.
  • Such an expression cassette may be administered to a subject for therapeutic purposes.
  • Expression cassettes can also be used for generating a viral vector for therapeutic delivery of the nucleotide sequences described.
  • the compositions provided reduce expression of PRL ⁇ E1 by RNA interference.
  • the nucleic acids provided include small interfering nucleic acids (e.g., RNAs) that target PRL ⁇ E1 mRNA.
  • small interfering RNAs that digest PRL ⁇ E1 mRNA are provided for the purpose of reducing or depleting endogenously produced PRL ⁇ E1.
  • Interfering RNA includes, but is not limited to, RNA enzymes (ribozymes), small interfering RNA (siRNA), small hairpin RNA (shRNA), or artificial microRNAs (miRNA).
  • the nucleic acids target a sequence comprising nucleotides 277-302, 292-317, 267-317, 162-187, 622-647, 305-330, 320-345, 295-320, 188-231, 194-219, or 333-358 of SEQ ID NO: 1.
  • the nucleic acid targets a sequence of SEQ ID NO: 1, for example a sequence comprising a 10-20 nucleotide portion of SEQ ID NO: 1.
  • the nucleic acid sequence that targets a sequence of SEQ ID NO: 1 comprises a sequence set forth below in Table 3 or 4.
  • the nucleic acid targets a sequence of SEQ ID NO: 2, for example a sequence comprising a 10-20 nucleotide portion of SEQ ID NO: 2.
  • the nucleic acid that targets a sequence of SEQ ID NO: 2 comprises a sequence set forth in Table 1 or Table 2 below.
  • one or more of the interfering RNAs are encoded by a sequence delivered by an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • the sequence encodes a short hairpin RNAs (shRNAs) driven by a promoter (e.g., an RNA polymerase III promoter or other suitable constitutive or inducible promoter), an artificial microRNAs (miRNAs) driven by a promoter (e.g., using an RNA polymerase II promoter or other suitable constitutive or inducible promoter), or an siRNA driven by a promoter (e.g., an RNA polymerase III promoter or other suitable constitutive or inducible promoter).
  • a promoter e.g., an RNA polymerase III promoter or other suitable constitutive or inducible promoter
  • miRNAs an artificial microRNAs driven by a promoter
  • siRNA driven by a promoter e.g., an RNA polymerase III promoter or other suitable constitutive or inducible promoter
  • an “Antisense Oligonucleotide” refers to a chemically synthesized, single-stranded oligomeric compound, typically comprising deoxyribonucleotides (DNA), ribonucleotides (RNA), or analogs thereof, that is designed to hybridize in a sequence-specific manner to a target nucleic acid sequence, such as mRNA, pre-mRNA, or other RNA molecules, through complementary base pairing.
  • ASOs are typically between 10 and 30 nucleotides, though other ranges may be specified.
  • siRNA small Interfering RNA
  • RNA target RNA
  • RISC RNA-induced silencing complex
  • the siRNA can be obtained using a number of techniques known to those of skill in the art.
  • the siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. published application 2002/0086356 of Tuschl et al., the entire disclosure of which is herein incorporated by reference.
  • the siRNA are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • the siRNA can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
  • siRNA can also be expressed from recombinant circular or linear DNA plasmids using any suitable promoter.
  • suitable promoters for expressing siRNA from a plasmid include, for example, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
  • the recombinant plasmids can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment.
  • the siRNA expressed from recombinant plasmids can either be isolated from cultured cell expression systems by standard techniques, or can be expressed intracellularly.
  • the siRNA or ASO may be modified to provide enhanced delivery to the subject.
  • Such modifications include, without limitation, PS-ASOs, ribose sugar modifications, nucleobase modifications, bridged nucleic acids, and alternative backbones. See, e.g., Gagliardi M, Ashizawa AT. The Challenges and Strategies of Antisense Oligonucleotide Drug Delivery. Biomedicines.2021 Apr 16;9(4):433. doi: 10.3390/biomedicines9040433. PMID: 33923688; PMCID: PMC8072990.
  • expression cassettes that include a nucleic acid sequence encoding one or more elements of a gene editing system that targets and/or disrupts PRL ⁇ E1 expression.
  • gene editing system refers to technologies or molecular machinery for modifying genetic material, typically with specificity for a particular gene or nucleic acid sequence (including, e.g., target sequences or motifs). Such gene editing systems are designed to modify a target site in the genome or introduce a mutation.
  • a “mutation” or “modification”, unless otherwise stated, can refer to any alteration of a genomic sequence, including but not limited to small nucleotide insertions or deletions (indels) or a larger deletion, insertion, or inversion.
  • the introduction of a mutation or modification is referred to as “editing” or “gene editing”.
  • Terms such as “target site” and “target sequence”, unless indicated otherwise, are used herein to refer to a sequence that is recognized by one or more elements of a gene- editing system.
  • a sgRNA includes a sequence that binds (i.e., is complementary to) a target site or target sequence in the genome.
  • the gene editing system is a Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) system that edits a PRL ⁇ E1 sequence.
  • CRISPR Clustered Regulatory Interspaced Short Palindromic Repeats
  • a suitable gene editing system includes a zinc-finger nuclease (ZFN) to induce DNA double-strand breaks, which may or may not be in conjunction with delivery of an exogenous DNA donor substrate (See, e.g., Ellis et al, Gene Therapy (epub January 2012) 20:35-42 which is incorporated herein by reference).
  • a suitable gene editing system includes a meganuclease (see, e.g., in US Patent 8,445,251; US 9,340,777; US 9,434,931; US 9,683,257, and WO 2018/195449, each of which is incorporated herein by reference) or transcription activator ⁇ like (TAL) effector nucleases (TALENs).
  • TALENs transcription activator ⁇ like effector nucleases
  • a suitable CRISPR gene editing system includes, at a minimum, a Cas9 enzyme and an sgRNA specific for a target site in a PRL ⁇ E1 sequence.
  • the gene editing vector comprises a Cas9 gene as the editing enzyme and an sgRNA which is at least 20 nucleotides in length and specifically binds to a selected site in PRL ⁇ E15 ' to a protospacer- adjacent motif (PAM) that is specifically recognized by the Cas9.
  • the expression cassette or vector genome includes a nucleic acid sequence encoding the sgRNA molecule and a nucleic acid sequence encoding a Cas9 enzyme.
  • the gene editing system also includes a donor or repair template. The expression cassette providing the donor template may be the same as the expression cassettes encoding the sgRNA and Cas9, or a different expression cassette.
  • a dual-vector system (as described for example in WO 2016/176191) is provided, wherein the gene editing system includes an expression cassette comprising a Cas9 gene under control of regulatory sequences which direct its expression and a second expression cassette comprising a sgRNA and a donor template.
  • Cas9 CRISPR associated protein 9 refers to family of RNA-guided DNA endonucleases which is characterized by two signature nuclease domains, RuvC (cleaves non-coding strand) and HNH (coding strand).
  • Suitable bacterial sources of Cas9 include Staphylococcus aureus (SaCas9), Staphylococcus pyogenes (SpCas9), and Neisseria meningitides (KM Estelt et al, Nat Meth, 10:1116-21 (2013)).
  • the wild-type coding sequences may be utilized in the constructs described herein.
  • bacterial codons are optimized for expression in humans, e.g. using any of a variety of known human codon optimizing algorithms.
  • Other endonucleases with similar properties may optionally be substituted. See, e.g., the public CRISPR database (db) accessible at crispr.u-psud.fr/crispr.
  • CRISPR/Cas9 gene targeting requires a single guide RNA (sgRNA) that contains a targeting sequence (crRNA sequence) and a Cas9 nuclease-recruiting sequence (tracrRNA).
  • sgRNA single guide RNA
  • crRNA sequence targeting sequence
  • tracrRNA Cas9 nuclease-recruiting sequence
  • the crRNA region is a 20-nucleotide sequence that is homologous to a target site and will direct Cas9 nuclease activity.
  • Strategies for identifying suitable target sites in the genome while also eliminating off target effects are known to those of skill in the art (see, e.g., ChopChop available online at chopchop.cbu.uib.no/).
  • the CRISPR nuclease may be Cpf1 (CRISPR from Prevotella and Francisella).
  • Cpf1 and LbCpf1 are particularly useful. See, www.addgene.Org/69982/sequences/#depositor-full (AsCpf1 sequences; and www.addgene.Org/69988/sequences/#depositor-full (LbCpf1 sequences), which are incorporated herein by reference. Further, Cpfl1 does not require a tracrRNA; allowing use of shorter guide RNAs (about 42 nucleotides) as compared to Cas9. Plasmids may be obtained from Addgene, a public plasmid database.
  • a gene editing system is utilized to introduce a mutation in PRL ⁇ E1 allele in target cell.
  • the target PRL ⁇ E1 polynucleotide sequence is cleaved such that a double-strand break results.
  • the target polynucleotide sequence is cleaved such that a single-strand break results.
  • the alteration is an insertion or deletion (indel), which can result in random insertion/deletion mutations at the site of junction as a result of non-homologous end joining. Indel mutations occurring within the coding region of a gene can result in frame-shift and a premature stop codon, and disrupt transcription.
  • the gene editing system one or more elements of a RNA- targeting CRISPR system, such as a member of the Cas13 enzyme family and/or crRNA construct.
  • the diverse Cas13 family contains at least four known subtypes, including Cas13a (formerly C2c2), Cas13b, Cas13c, and Cas13d.
  • the Cas13 family is the only family of class 2 Cas enzymes known to exclusively target single-stranded RNA.
  • Cas13 enzymes and systems are known in the art, see, e.g., US Patent No.10,362,616, Abudayyeh, et al, C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.
  • A. A. Smargon, et al, Cas13b is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28. Mol. Cell 65, 618–630.e7 (2017); J. S.
  • a Cas13 protein uses a short crRNA that interacts with the Cas13 molecule through a stem loop and facilitates target binding and cleavage through a series of conformational changes in the Cas13 molecule.
  • the Cas13 protein is Cas13a, Cas13b, Cas13c, or Cas13d.
  • the Cas13 comprises one or more mutations the HEPN domain(s).
  • the Cas13d protein is a Class 2, Type VI CRISPR effector guided by a crRNA.
  • Two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains have been found in the Cas13d, flanking a helical domain. See, for example, WO 2019/010384 Al, US 2019/0169595A1, Zhang C, et al. (2018). Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d.
  • Cas13d includes, e.g., a wild type or naturally occurring Cas13d protein, an ortholog of a Cas13d, a functional variant thereof, or another modified variant as disclosed.
  • the Cas13d is selected from a RfxCas13d from Ruminococcus flavefaciens strain XPD3002, an AdmCas13d from Anaerobic digester metagenome 15706, EsCas13d from Eubacterium siraeum DSM15702, P1E0Cas13d from Gut metagenome assembly P1E0-k21, UrCas13d from Uncultured Ruminoccocus sp., RffCas13d from Ruminoccocus flavefaciens FD1, and RaCas13d from Ruminoccocus albus.
  • the Cas13d protein is a RfxCas13d or a variant thereof.
  • the amino acid sequences of the Cas13d orthologs are publicly available.
  • the Cas13d has an amino acid sequence as provided by a Protein Data Bank (PDB) accession number 6OAW_B or 6OAW_A or 6E9F_A or 6E9E_A or 6IV9_A, or an amino acid sequence as provided by the UniProtKB identifier B0MS50 (B0MS50_9FIRM) or A0A1C5SD84 (A0A1C5SD84_9FIRM).
  • PDB Protein Data Bank
  • target RNA refers to an RNA polynucleotide being or comprising the target sequence, including coding and non-coding transcripts.
  • the target RNA may be an RNA polynucleotide or a part of a RNA polynucleotide to which a part of a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) is designed to have complementarity and to which the effector function mediated by the complex comprising CRISPR enzyme and a guide RNA (gRNA) is to be directed.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • gRNA guide RNA
  • a viral vector is used to deliver one more elements of a gene editing system.
  • a viral vector delivers one or more components of a genome editing system, such as CRISPR/Cas9 or CRISPR/Cas13.
  • a combination or dual AAV vector system is provided to deliver the components of the CRISPR system when co-administered to a subject (see, e.g. WO 2016/176191, which is incorporated by reference herein in its entirety).
  • the vectors may be formulated together or separately and delivered essentially simultaneously, preferably by the same route.
  • the term “regulatory sequence”, or “expression control sequence” refers to nucleic acid sequences, such as initiator sequences, enhancer sequences, and promoter sequences, which induce, repress, or otherwise control the transcription of protein encoding nucleic acid sequences to which they are operably linked.
  • “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • the regulatory control elements typically contain a promoter sequence as part of the expression control sequences.
  • the promoter is a chicken beta actin promoter with CMV enhancer elements, e.g., the CB7 promoter.
  • a cell-specific promoter for ocular cells is selected.
  • the promoter may be human G-protein-coupled receptor protein kinase 1 (GRK1) promoter (Genbank Accession number AY327580) (See also, Beltran et al, Gene Therapy 201017:1162-74, which is hereby incorporated by reference herein).
  • the promoter is human interphotoreceptor retinoid-binding protein proximal (IRBP) promoter.
  • the promoter is rod opsin (mOps) promoter, or rhodopsin-mOP500 promoter.
  • suitable promoters include, e.g., constitutive promoters, regulatable promoters [see, e.g., WO 2011/126808 and WO 2013/04943], or a promoter responsive to physiologic cues.
  • the promoter can be selected from different sources, e.g., human cytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the JC polymovirus promoter, myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoters, herpes simplex virus (HSV-1) latency associated promoter (LAP), rouse sarcoma virus (RSV) long terminal repeat (LTR) promoter, neuron-specific promoter (NSE), platelet derived growth factor (PDGF) promoter, melanin-concentrating hormone (MCH) promoter, CBA, matrix metalloprotein promoter (MPP), and the chicken beta-actin promoter.
  • CMV human cytomegalovirus
  • MBP myelin basic protein
  • GFAP glial fibrillary acidic protein
  • HSV-1 herpes simplex virus
  • LAP rouse sarcoma virus
  • LTR
  • a vector may contain one or more other appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA for example WPRE; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • RNA processing signals such as splicing and polyadenylation (poly A) signals
  • sequences that stabilize cytoplasmic mRNA for example WPRE sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • An example of a suitable enhancer is the CMV enhancer.
  • Other suitable enhancers include those that are appropriate for desired target tissue indications.
  • the expression cassette comprises one or more expression enhancers.
  • the expression cassette contains two or more expression enhancers. These enhance
  • an enhancer may include a CMV immediate early enhancer. This enhancer may be present in two copies which are located adjacent to one another. Alternatively, the dual copies of the enhancer may be separated by one or more sequences.
  • the expression cassette further contains an intron, e.g., the chicken beta-actin intron.
  • suitable introns include those known in the art, e.g., such as are described in WO 2011/126808.
  • suitable poly A sequences include, e.g., rabbit beta globin, SV40, SV50, bovine growth hormone (bGH), human growth hormone, HSV TK, and synthetic poly As.
  • one or more sequences may be selected to stabilize mRNA.
  • regulatory elements comprise but not limited to: promoter; enhancer; transcription factor; transcription terminator; efficient RNA processing signals such as splicing and polyadenylation signals (poly A); sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequence).
  • WPRE Woodchuck Hepatitis Virus
  • WPRE Woodchuck Hepatitis Virus
  • a suitable promoter may include without limitation, an elongation factor 1 alpha (EF1 alpha) promoter (see, e.g., Kim DW et al, Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene.1990 Jul 16;91(2):217-23) or a CB6 promoter (see, e.g., Large-Scale Production of Adeno- Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene, Mol Biotechnol.2016 Jan;58(1):30-6. doi: 10.1007/s12033-015-9899-5).
  • EF1 alpha elongation factor 1 alpha
  • CAG promoter which comprises (C) the cytomegalovirus (CMV) early enhancer element, (A) the promoter, the first exon and the first intron of chicken beta-actin gene, and (G) the splice acceptor of the rabbit beta-globin gene. See, e.g., Alexopoulou, Annika N., et al. BMC cell biology 9.1 (2008): 2.
  • the expression cassette includes an U6 promoter.
  • the regulatory elements comprise an enhancer.
  • the enhancer(s) is selected from one or more of an APB enhancer, an ABPS enhancer, an alpha mic/bik enhancer, a TTR enhancer, an en34 enhancer, an ApoE enhancer, a CMV enhancer, or an RSV enhancer.
  • the regulatory elements comprise an intron.
  • the intron is selected from CBA, human beta globin, IVS2, SV40, bGH, alpha-globulin, beta-globulin, collagen, ovalbumin, or p53.
  • the regulatory elements comprise a polyA.
  • the polyA is a synthetic polyA or from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit ⁇ -globin (RGB), or modified RGB (mRGB).
  • the regulatory elements may comprise a WPRE sequence.
  • the regulatory elements comprise a Kozak sequence. Expression cassettes can be delivered via any suitable delivery system.
  • Suitable non- viral delivery systems are known in the art (see, e.g., Ramamoorth and Narvekar. J Clin Diagn Res.2015 Jan; 9(1):GE01-GE06, which is incorporated herein by reference) and can be readily selected by one of skill in the art and may include, e.g., naked DNA, naked RNA, dendrimers, PLGA, polymethacrylate, an inorganic particle, a lipid particle (e.g., a lipid nanoparticle or LNP), or a chitosan-based formulation.
  • the vector is a non-viral plasmid that comprises an expression cassette described thereof, e.g., “naked DNA”, “naked plasmid DNA”, RNA, and mRNA; coupled with various compositions and nano particles, including, e.g., micelles, liposomes, cationic lipid - nucleic acid compositions, poly-glycan compositions and other polymers, lipid and/or cholesterol-based - nucleic acid conjugates, and other constructs such as are described herein. See, e.g., X. Su et al, Mol.
  • a “self-complementary nucleic acid” refers to a nucleic acid capable of hybridizing with itself (i.e., folding back upon itself) to form a single-stranded duplex structure, due to the complementarity (e.g., base-pairing) of the nucleotides within the nucleic acid strand.
  • Self-complementary nucleic acids can form a variety of secondary structures, such as hairpin loops, loops, bulges, junctions and internal bulges.
  • nucleic acids e.g., miRNA or a-miRNA (artificial miRNA) perform regulatory functions, such as gene silencing.
  • the term “substantial homology” or “substantial similarity,” when referring to a nucleic acid, or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95 to 99% of the aligned sequences.
  • the homology is over full-length sequence, or an open reading frame thereof, or another suitable fragment which is at least 15 nucleotides in length. Examples of suitable fragments are described herein.
  • sequence identity refers to the residues in the two sequences which are the same when aligned for maximum correspondence.
  • the length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g. of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired.
  • percent sequence identity may be readily determined for amino acid sequences, over the full-length of a protein, or a fragment thereof.
  • a fragment is at least about 8 amino acids in length and may be up to about 700 amino acids. Examples of suitable fragments are described herein.
  • the term “substantial homology” or “substantial similarity,” when referring to amino acids or fragments thereof, indicates that, when optimally aligned with appropriate amino acid insertions or deletions with another amino acid (or its complementary strand), there is amino acid sequence identity in at least about 95 to 99% of the aligned sequences.
  • the homology is over full-length sequence, or a protein thereof, e.g., a cap protein, a rep protein, or a fragment thereof which is at least 8 amino acids, or more desirably, at least 15 amino acids in length. Examples of suitable fragments are described herein.
  • highly conserved is meant at least 80% identity, preferably at least 90% identity, and more preferably, over 97% identity. Identity is readily determined by one of skill in the art by resort to algorithms and computer programs known by those of skill in the art. Generally, when referring to “identity”, “homology”, or “similarity” between two different adeno-associated viruses, “identity”, “homology” or “similarity” is determined in reference to “aligned” sequences.
  • Alignments refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.
  • AAV alignments are performed using the published AAV9 sequences as a reference point. Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Examples of such programs include, “Clustal Omega”, “Clustal W”, “CAP Sequence Assembly”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used.
  • nucleotide sequence identity there are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above.
  • polynucleotide sequences can be compared using FastaTM, a program in GCG Version 6.1.
  • FastaTM provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences.
  • percent sequence identity between nucleic acid sequences can be determined using FastaTM with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
  • sequence alignment programs are also available for amino acid sequences, e.g., the “Clustal Omega”, “Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).
  • one or more nucleic acid sequences provided herein are delivered to ocular cells by a vector or a viral vector, of which many are known and available in the art.
  • a vector comprising an expression cassette as described herein.
  • the vector is a non-viral vector.
  • the non-viral vector is a plasmid.
  • the vector is a viral vector.
  • Viral vectors include any virus suitable for gene therapy, including but not limited to a bocavirus, adenovirus, adeno-associated virus (AAV), herpes virus, lentivirus, retrovirus, or parvovirus.
  • a “vector” as used herein is a biological or chemical moiety comprising a nucleic acid sequence which can be introduced into an appropriate target cell for replication or expression of a nucleic acid sequence.
  • a vector examples include but are not limited to a recombinant virus, a plasmid, Lipoplexes, a Polymersome, Polyplexes, a dendrimer, a cell penetrating peptide (CPP) conjugate, a magnetic particle, or a nanoparticle.
  • a vector is a nucleic acid molecule having an exogenous or heterologous engineered nucleic acid encoding a functional gene product, which can then be introduced into an appropriate target cell.
  • Such vectors preferably have one or more origins of replication, and one or more site into which the recombinant DNA can be inserted.
  • Vectors often have means by which cells with vectors can be selected from those without, e.g., they encode drug resistance genes.
  • Common vectors include plasmids, viral genomes, and “artificial chromosomes”. Conventional methods of generation, production, characterization, or quantification of the vectors are available to one of skill in the art.
  • a recombinant viral vector is any suitable viral vector which targets the desired cell(s).
  • the recombinant viral vectors described herein preferably target one or more of the cells and tissues affected by an ocular condition, including rods and/or cones of the eye.
  • the examples provide illustrative recombinant adeno-associated viruses (rAAV).
  • viral vectors may include, e.g., a recombinant adenovirus, a recombinant parvovirus such a recombinant bocavirus, a hybrid AAV/bocavirus, a recombinant herpes simplex virus, a recombinant retrovirus, or a recombinant lentivirus.
  • these recombinant viruses are replication-defective.
  • the capsid protein is a non-naturally occurring capsid.
  • Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vp1 capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV, non-contiguous portions of the same AAV, from a non-AAV viral source, or from a non-viral source.
  • An artificial AAV may be, without limitation, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid.
  • Pseudotyped vectors wherein the capsid of one AAV is replaced with a heterologous capsid protein, are useful in the invention.
  • AAV2/5 and AAV2/8 are exemplary pseudotyped vectors.
  • the selected genetic element may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
  • a “replication-defective” virus or viral vector refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells.
  • the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be “gutless” - containing only the gene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of the viral enzyme required for replication.
  • replication-defective viruses may be adeno-associated viruses (AAV), adenoviruses, lentiviruses (integrating or non-integrating), or another suitable virus source.
  • Plasmid or “plasmid vector” generally is designated herein by a lower-case p preceded and/or followed by a vector name. Plasmids, other cloning and expression vectors, properties thereof, and constructing/manipulating methods thereof that can be used in accordance with the present invention are readily apparent to those of skill in the art.
  • the expression cassettes described herein are engineered into a suitable genetic element (a vector) useful for generating viral vectors and/or for delivery to a host cell, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the sequences carried thereon.
  • the selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
  • the methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.
  • the term “host cell” may refer to the packaging cell line in which a vector (e.g., a recombinant AAV) is produced from a production plasmid.
  • the term “host cell” may refer to any target cell in which expression an expression cassette or a miRNA or modified snRNA described herein is desired.
  • a “host cell,” refers to a prokaryotic or eukaryotic cell that contains exogenous or heterologous DNA that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
  • the term “host cell” refers to cultures of cells of various mammalian species for in vitro assessment of the compositions described herein.
  • a “vector genome” refers to the nucleic acid sequence packaged inside a viral vector.
  • a vector genome contains, at a minimum, from 5’ to 3’, a vector-specific sequence, a nucleic acid sequence as provided herein for reducing PRL ⁇ E1 expression in a target cell, where the vector-specific sequence may be a terminal repeat sequence that specifically packages the vector genome into a viral vector capsid or envelope protein.
  • the vector genome contains, at a minimum, from 5’ to 3’, a 5’ AAV ITR sequence, an expression cassette, and a 3’ AAV inverted terminal repeat (ITR) sequence, including intervening sequences.
  • AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids.
  • Lentivirus long terminal repeats may be utilized where packaging into a lentiviral vector is desired.
  • other terminal repeats e.g., a retroviral long terminal repeat, or the like may be selected.
  • An AAV vector is an AAV nuclease (e.g., DNase)-resistant particle having an AAV protein capsid into which is packaged expression cassette flanked by AAV inverted terminal repeat sequences (ITRs) for delivery to target cells.
  • a nuclease-resistant recombinant AAV indicates that the AAV capsid has fully assembled and protects these packaged vector genome sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids which may be present from the production process.
  • the rAAV described herein is DNase resistant.
  • An AAV capsid is composed of 60 capsid (cap) protein subunits, VP1, VP2, and VP3, that are arranged in an icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending upon the selected AAV.
  • Various AAVs may be selected as sources for capsids of AAV vectors as identified above. See, e.g., US Published Patent Application No.2007- 0036760-A1; US Published Patent Application No.2009-0197338-A1; EP 1310571.
  • the AAV capsid, ITRs, and other selected AAV components described herein may be readily selected from among any AAV, including, without limitation, the AAVs commonly identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7m8 and AAVAnc80. See, e.g., WO 2005/033321, which is incorporated herein by reference.
  • the rAAV particles provided herein may be of any AAV serotype, including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2/1, 2/5, 2/8, or 2/9).
  • the serotype of an rAAV viral vector refers to the serotype of the capsid proteins of the recombinant virus.
  • the rAAV particle is not AAV2.
  • the rAAV particle is AAV2.
  • the rAAV particle is AAV6.
  • the rAAV particle is an AAV6 serotype comprising an rAAV capsid protein as described herein.
  • Non-limiting examples of derivatives and pseudotypes include rAAV2/l , rAAV2/5, rAAV2/8, rAAV2/9, AAV2-AAV3 hybrid, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShHIO, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, and AAVr3.45.
  • the rAAV particle is a pseudotyped rAAV particle, which comprises (a) a nucleic acid vector comprising ITRs from one serotype (e.g., AAV2) and (b) a capsid comprised of capsid proteins derived from another serotype (e.g., AAVl, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10).
  • a pseudotyped rAAV particle which comprises (a) a nucleic acid vector comprising ITRs from one serotype (e.g., AAV2) and (b) a capsid comprised of capsid proteins derived from another serotype (e.g., AAVl, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10).
  • a “stock” of rAAV refers to a population of rAAV. Despite heterogeneity in their capsid proteins due to deamidation, rAAV in a stock are expected to share an identical vector genome.
  • a stock can include rAAV having capsids with, for example, heterogeneous deamidation patterns characteristic of the selected AAV capsid proteins and a selected production system.
  • the stock may be produced from a single production system or pooled from multiple runs of the production system.
  • a variety of production systems, including but not limited to those described herein, may be selected.
  • the term “variant” means any AAV sequence which is derived from a known AAV sequence, including those sharing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or greater sequence identity over the amino acid or nucleic acid sequence.
  • the AAV capsid includes variants which may include up to about 10% variation from any described or known AAV capsid sequence.
  • the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity or about 97% to about 98% identity to an AAV capsid provided herein and/or known in the art.
  • the AAV capsid shares at least 95% identity with an AAV capsid.
  • the comparison may be made over any of the variable proteins (e.g., vp1, vp2, or vp3).
  • the AAV capsid shares at least 95% identity with the AAV8 vp3.
  • a self-complementary AAV is used.
  • the ITR sequences or other AAV components may be readily isolated or engineered using techniques available to those of skill in the art from an AAV.
  • AAV may be isolated, engineered, or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, VA).
  • the AAV sequences may be engineered through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank, PubMed, or the like.
  • AAV vectors may be engineered by conventional molecular biology techniques, making it possible to optimize these particles for cell specific delivery of nucleic acid sequences, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivery to the nucleus, etc.
  • an AAV vector comprising an AAV capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence that encodes an RNA interference construct (e.g., siRNA, shRNA, miRNA, or ASO) for inhibiting or reducing PRL ⁇ E1 expression or activity in a target cell.
  • an AAV vector comprises an AAV capsid and an expression cassette, wherein the expression cassette comprises one or more components of a gene editing system (e.g., CRISPR/Cas enzyme, guide RNA, crRNA) for inhibiting or reducing PRL ⁇ E1 expression or activity in a target cell.
  • the nucleic acid packaged in the AAV capsid sequence includes AAV ITR sequences flanking the expression cassette.
  • the ITRs are the genetic elements responsible for the replication and packaging of the genome during vector production and are the only viral cis elements required to generate rAAV.
  • the ITRs are from an AAV different than that supplying a capsid.
  • ITRs from other AAV sources may be selected. Where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped.
  • AAV vector genome comprises an AAV 5’ ITR, the nucleic acid sequences encoding the gene product(s) and any regulatory sequences, and an AAV 3’ ITR.
  • AAV 5’ ITR the nucleic acid sequences encoding the gene product(s) and any regulatory sequences
  • AAV 3’ ITR an AAV 3’ ITR.
  • a self- complementary AAV is provided.
  • a shortened version of the 5’ ITR termed ⁇ ITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted.
  • the vector genome includes a shortened AAV2 ITR of 130 base pairs, wherein the external “a” element is deleted. The shortened ITR is reverted back to the wild-type length of 145 base pairs during vector DNA amplification using the internal A element as a template.
  • the full-length AAV 5’ and 3’ ITRs are used.
  • the regulatory sequences are selected such that the total rAAV vector genome is about 2.0 to about 5.5 kilobases in size. In certain embodiments, the regulatory sequences are selected such that the total rAAV vector genome is about 2.9 to about 5.5 kilobases in size. In certain embodiments, the regulatory sequences are selected such that the total rAAV vector genome is about 2.9 kb in size. In certain embodiments, it is desirable that the rAAV vector genome approximate the size of the native AAV genome. Thus, in certain embodiments, the regulatory sequences are selected such that the total rAAV vector genome is about 4.7 kb in size.
  • the total rAAV vector genome is less about 5.2 kb in size.
  • the size of the vector genome may be manipulated based on the size of the regulatory sequences including the promoter, enhancer, intron, poly A, etc. See, Wu et al., Mol Ther, Jan 2010, 18(1):80-6, which is incorporated herein by reference.
  • the recombinant adeno-associated virus (AAV) described herein may be generated using techniques which are known. See, e.g., WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2.
  • Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette as described herein flanked by AAV inverted terminal repeats (ITRs); and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein.
  • the host cell which contains a nucleic acid sequence encoding an AAV capsid; a functional rep gene; a vector genome as described; and sufficient helper functions to permit packaging of the vector genome into the AAV capsid protein.
  • the host cell is a HEK 293 cell.
  • Suitable methods may include without limitation, baculovirus expression system or production via yeast. See, e.g., Robert M. Kotin, Large-scale recombinant adeno-associated virus production. Hum Mol Genet.2011 Apr 15; 20(R1): R2–R6. Published online 2011 Apr 29. doi: 10.1093/hmg/ddr141; Aucoin MG et al., Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios. Biotechnol Bioeng.2006 Dec 20;95(6):1081-92; SAMI S.
  • the number of particles (pt) per 20 ⁇ L loaded is then multiplied by 50 to give particles (pt) /mL.
  • Pt/mL divided by GC/mL gives the ratio of particles to genome copies (pt/GC).
  • Pt/mL–GC/mL gives empty pt/mL.
  • Empty pt/mL divided by pt/mL and x 100 gives the percentage of empty particles.
  • methods for assaying for empty capsids and AAV vector particles with packaged genomes have been known in the art.
  • the methods include subjecting the treated AAV stock to SDS- polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in the buffer, then running the gel until sample material is separated, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon.
  • SDS- polyacrylamide gel electrophoresis consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in the buffer, then running the gel until sample material is separated, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon.
  • Anti-AAV capsid antibodies are then used as the primary antibodies that bind to denatured capsid proteins, preferably an anti-AAV capsid monoclonal antibody, most preferably the B1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Viral. (2000) 74:9281-9293).
  • a secondary antibody is then used, one that binds to the primary antibody and contains a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound to it, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase.
  • a method for detecting binding is used to semi-quantitatively determine binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit.
  • a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit.
  • samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing reducing agent (e.g., DTT), and capsid proteins were resolved on pre-cast gradient polyacrylamide gels (e.g., Novex).
  • Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions or other suitable staining method, i.e. SYPRO ruby or Coomassie stains.
  • the concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q-PCR).
  • Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using primers and a TaqManTM fluorogenic probe specific for the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing identical sequences to that contained in the AAV vector is employed to generate a standard curve in the Q-PCR reaction.
  • the cycle threshold (Ct) values obtained from the samples are used to determine vector genome titer by normalizing it to the Ct value of the plasmid standard curve.
  • End-point assays based on the digital PCR can also be used.
  • an optimized q-PCR method is used which utilizes a broad-spectrum serine protease, e.g., proteinase K (such as is commercially available from Qiagen). More particularly, the optimized qPCR genome titer assay is similar to a standard assay, except that after the DNase I digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation.
  • proteinase K buffer may be concentrated to 2-fold or higher.
  • proteinase K treatment is about 0.2 mg/mL, but may be varied from 0.1 mg/mL to about 1 mg/mL.
  • the treatment step is generally conducted at about 55 °C for about 15 minutes, but may be performed at a lower temperature (e.g., about 37 °C to about 50 °C) over a longer time period (e.g., about 20 minutes to about 30 minutes), or a higher temperature (e.g., up to about 60 °C) for a shorter time period (e.g., about 5 to 10 minutes).
  • heat inactivation is generally at about 95 °C for about 15 minutes, but the temperature may be lowered (e.g., about 70 to about 90 °C) and the time extended (e.g., about 20 minutes to about 30 minutes). Samples are then diluted (e.g., 1000 fold) and subjected to TaqMan analysis as described in the standard assay. Additionally, or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods.2014 Apr;25(2):115-25.
  • compositions provided herein are included in a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.
  • a final formulation suitable for delivery to a subject e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.
  • one or more surfactants are present in the formulation.
  • the composition may be transported as a concentrate which is diluted for administration to a subject.
  • the composition may be lyophilized and reconstituted at the time of administration.
  • the suspension further comprises a surfactant, preservative, excipients, and/or buffer dissolved in the aqueous suspending liquid.
  • the buffer is PBS.
  • suitable solutions include one or more of: buffering saline, a surfactant, and a physiologically compatible salt or mixture of salts adjusted to an ionic strength equivalent to about 100 mM sodium chloride (NaCl) to about 250 mM sodium chloride, or a physiologically compatible salt adjusted to an equivalent ionic concentration.
  • a suitable surfactant, or combination of surfactants may be selected from among Poloxamers, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol.
  • the formulation contains a poloxamer.
  • the pH may be in the range of 6.5 to 8.5, or 7 to 8.5, or 7.5 to 8.
  • a pH within this range may be desired; whereas for intravenous delivery, a pH of 6.8 to about 7.2 may be desired.
  • other pHs within the broadest ranges and these subranges may be selected for other routes of delivery.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a vector comprising a nucleotide sequence encoding a miRNA or modified snRNA operatively linked to regulatory elements therefor as described herein.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
  • Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells.
  • Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the vector is directed.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
  • Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present invention.
  • Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
  • Suitable chemical stabilizers include gelatin and albumin.
  • the term “dosage” or “amount” can refer to the total dosage or amount delivered to the subject in the course of treatment, or the dosage or amount delivered in a single unit (or multiple unit or split dosage) administration.
  • the aqueous suspension or pharmaceutical compositions described herein are designed for delivery to subjects in need thereof by any suitable route or a combination of different routes.
  • compositions described herein are designed for delivery to subjects in need thereof by any suitable route or a combination of different routes.
  • a pharmaceutical composition comprising a dosage of a vector as described herein in a formulation buffer.
  • dosage can refer to the total dosage delivered to the subject in the course of treatment, or the amount delivered in a single unit (or multiple unit or split dosage) administration.
  • the pharmaceutical virus compositions can be formulated in dosage units to contain an amount of replication- defective AAV vector carrying a nucleic acid sequence as described herein that is in the range of about 1.0 x 10 9 vg (vector genomes)/mL to about 1.0 x 10 15 vg/mL including all integers or fractional amounts within the range.
  • the compositions are formulated to contain at least 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , or 9x10 9 vg/mL including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 1x10 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 , or 9x10 10 vg/mL including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 1x10 11 , 2x10 11 , 3x10 11 , 4x10 11 , 5x10 11 , 6x10 11 , 7x10 11 , 8x10 11 , or 9x10 11 vg/mL including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 1x10 12 , 2x10 12 , 3x10 12 , 4x10 12 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 , or 9x10 12 vg/mL including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 1x10 13 , 2x10 13 , 3x10 13 , 4x10 13 , 5x10 13 , 6x10 13 , 7x10 13 , 8x10 13 , or 9x10 13 vg/mL including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 1x10 14 , 2x10 14 , 3x10 14 , 4x10 14 , 5x10 14 , 6x10 14 , 7x10 14 , 8x10 14 , or 9x10 14 vg/mL including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 1x10 15 , 2x10 15 , 3x10 15 , 4x10 15 , 5x10 15 , 6x10 15 , 7x10 15 , 8x10 15 , or 9x10 15 vg/mL including all integers or fractional amounts within the range.
  • the dose can range from 1x10 10 to about 1x10 12 vg/mL including all integers or fractional amounts within the range. All dosages may be measured by any known method, including as measured by qPCR or digital droplet PCR (ddPCR) as described in, e.g., M. Lock et al, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi: 10.1089/hgtb.2013.131, which is incorporated herein by reference.
  • ddPCR digital droplet PCR
  • the volume of carrier, excipient or buffer is at least about 25 ⁇ L. In one embodiment, the volume is about 50 ⁇ L. In another embodiment, the volume is about 75 ⁇ L. In another embodiment, the volume is about 100 ⁇ L. In another embodiment, the volume is about 125 ⁇ L. In another embodiment, the volume is about 150 ⁇ L. In another embodiment, the volume is about 175 ⁇ L. In yet another embodiment, the volume is about 200 ⁇ L.
  • the volume is about 225 ⁇ L. In yet another embodiment, the volume is about 250 ⁇ L. In yet another embodiment, the volume is about 275 ⁇ L. In yet another embodiment, the volume is about 300 ⁇ L. In yet another embodiment, the volume is about 325 ⁇ L. In another embodiment, the volume is about 350 ⁇ L. In another embodiment, the volume is about 375 ⁇ L. In another embodiment, the volume is about 400 ⁇ L. In another embodiment, the volume is about 450 ⁇ L. In another embodiment, the volume is about 500 ⁇ L. In another embodiment, the volume is about 550 ⁇ L. In another embodiment, the volume is about 600 ⁇ L. In another embodiment, the volume is about 650 ⁇ L.
  • the volume is about 700 ⁇ L. In another embodiment, the volume is about 800 ⁇ L. In another embodiment, the volume is about or at least 100 ⁇ L. In another embodiment, the volume is between about 100 to 250 ⁇ L. In another embodiment, the volume is between about 150 and 800 ⁇ L. In another embodiment, the volume is between about 700 and 1000 ⁇ L. In another embodiment, the volume is between about 250 and 500 ⁇ L.
  • quantification of the genome copies (“GC”), vector genomes (“VG”), or virus particles may be used as the measure of the dose contained in the formulation or suspension. Any method known in the art can be used to determine the genome copy (GC) number of the replication-defective virus compositions of the invention.
  • AAV GC number titration One method for performing AAV GC number titration is as follows: Purified AAV vector samples are first treated with DNase to eliminate un-encapsulated AAV genome DNA or contaminating plasmid DNA from the production process. The DNase resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genomes are then quantitated by real-time PCR using primer/probe sets targeting specific region of the viral genome (usually the transgene or the poly A signal). See, e.g.,S.K. McLaughlin et al, 1988 J. Virol., 62:1963, which is incorporated by reference in its entirety. In certain embodiments, nucleic acid compositions described herein are formulated in a nanoparticle.
  • nucleic acid compositions described herein are formulated in a lipid nanoparticle.
  • nucleic acid compositions described herein are formulated in a lipid-polycation complex, referred to as a cationic lipid nanoparticle.
  • the formation of the lipid nanoparticle may be accomplished by methods known in the art and/or as described in U.S. Pub. No.20120178702, herein incorporated by reference in its entirety.
  • the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyomithine and/or polyarginine and the cationic peptides described in International Pub. No.
  • nucleic acid compositions described herein are formulated in a lipid nanoparticle that includes a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • the siRNA or ASO can be administered to the subject once, such as by a single injection or deposition at or near a tumor.
  • the siRNA or ASO can be administered to a subject multiple times daily or weekly.
  • the siRNA or ASO can be administered to a subject once weekly for a period of from about three to about twenty-eight weeks, more preferably from about seven to about ten weeks.
  • the siRNA or ASO is injected at or near the site of neovascularization (e.g., intravitreally) once a week for seven weeks. It is understood that periodic administrations of the siRNA or ASO for an indefinite length of time may be necessary.
  • a dosage regimen comprises multiple administrations or the administration of two or more siRNA or ASO, each of which comprise a different target sequence, it is understood that the effective amount of siRNA or ASO administered to the subject can comprise the total amount of siRNA administered over the entire dosage regimen.
  • the siRNA or ASO are preferably formulated as pharmaceutical compositions prior to administering to a subject, according to techniques known in the art.
  • Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen- free.
  • pharmaceutical formulations include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
  • the composition comprises an siRNA or ASO (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a physiologically acceptable carrier medium.
  • Preferred physiologically acceptable carrier media are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
  • Pharmaceutical compositions can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • physiologically biocompatible buffers e.g., tromethamine hydrochloride
  • additions of chelants such as, for example, DTPA or DTPA-bisamide
  • calcium chelate complexes as for example calcium DTPA, CaNaDTPA-bisamide
  • calcium or sodium salts for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate.
  • Pharmaceutical compositions can be packaged for use in liquid form or can be lyophilized. It should be understood that the pharmaceutical compositions described herein are intended to be
  • an expression cassette, nucleic acid, or a viral or non-viral vector is used in preparing a medicament.
  • uses of the same for treatment of an ocular disease in a subject in need thereof are provided.
  • treatment or “treating” is defined encompassing administering to a subject one or more compositions described herein for the purposes of amelioration of one or more symptoms of an ocular disease. “Treatment” can thus include one or more of reducing onset or progression of disease, preventing disease, reducing the severity of the disease symptoms, retarding their progression, removing the disease symptoms, delaying progression of disease, or increasing efficacy of therapy in a given subject. The desired result will depend upon the active agent being administered.
  • an effective amount of a rAAV particle may be an amount of the particle that is capable of transferring a heterologous nucleic acid to a host organ, tissue, or cell.
  • a “therapeutically effective amount” of a composition provided herein is delivered to a subject to achieve a desired result or to reach a therapeutic goal.
  • administering or “route of administration” is delivery of a therapy described herein (e.g. a rAAV comprising an RNA interference sequence), with or without a pharmaceutical carrier or excipient, of the subject.
  • the administration is repeated periodically.
  • a composition is administered direct to an eye.
  • the route of administration is ocular delivery, subretinal injection, intra-retinal injection, or intravitreal injection. Routes of administration may be combined, if desired.
  • the methods provide herein include administration of nucleic acid molecules and/or vectors described herein in a single composition or multiple compositions.
  • two or more different AAV may be delivered, or multiple viruses [see, e.g., WO202011/126808 and WO 2013/049493].
  • multiple viruses may contain different replication-defective viruses (e.g., AAV and adenovirus), alone or in combination with proteins.
  • the amount of the vectors, the virus and the replication- defective virus described herein are administered in a range of about 1.0 x 10 7 vector genomes (VG) per eye to about 1.0 x 10 15 VG per eye including all integers or fractional amounts within the range.
  • the amount thereof is at least 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , or 9x10 7 VG per eye including all integers or fractional amounts within the range.
  • the amount thereof is at least 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , or 9x10 8 VG per eye including all integers or fractional amounts within the range. In one embodiment, the amount thereof is at least 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , or 9x10 9 VG per eye including all integers or fractional amounts within the range.
  • the amount thereof is at least 1x10 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 , or 9x10 10 VG per eye including all integers or fractional amounts within the range. In one embodiment, the amount thereof is at least 1x10 11 , 2x10 11 , 3x10 11 , 4x10 11 , 5x10 11 , 6x10 11 , 7x10 11 , 8x10 11 , or 9x10 11 VG per eye including all integers or fractional amounts within the range.
  • the amount thereof is at least 1x10 12 , 2x10 12 , 3x10 12 , 4x10 12 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 , or 9x10 12 VG per eye including all integers or fractional amounts within the range. In one embodiment, the amount thereof is at least 1x10 13 , 2x10 13 , 3x10 13 , 4x10 13 , 5x10 13 , 6x10 13 , 7x10 13 , 8x10 13 , or 9x10 13 VG per eye including all integers or fractional amounts within the range.
  • the amount thereof is at least 1x10 14 , 2x10 14 , 3x10 14 , 4x10 14 , 5x10 14 , 6x10 14 , 7x10 14 , 8x10 14 , or 9x10 14 VG per eye including all integers or fractional amounts within the range. In one embodiment, the amount thereof is at least 1x10 15 , 2x10 15 , 3x10 15 , 4x10 15 , 5x10 15 , 6x10 15 , 7x10 15 , 8x10 15 , or 9x10 15 VG per eye including all integers or fractional amounts within the range.
  • the method comprises delivery of a dose ranging from 1x10 9 to about 1x10 13 VG per eye per dose including all integers or fractional amounts within the range.
  • the method comprises delivery of the vector in an aqueous suspension.
  • the method comprises administering the rAAV described herein in a dosage of from about 1 x 10 12 to 1 x 10 13 VG in a volume about or at least 150 microliters.
  • the term “ocular cells” refers to any cell in, or associated with the function of, the eye. The term may refer to any one of photoreceptor cells, including rod, cone and photosensitive ganglion cells or retinal pigment epithelium (RPE) cells.
  • RPE retinal pigment epithelium
  • the ocular cells are the photoreceptor cells. In other embodiments, the ocular cells are cones, rods, or cones and rods. In certain embodiments, the compositions provided herein are administered to a subject in need of treatment. In certain embodiments, the subject has or is suspected of having one or more conditions, diseases, or disorders of the eye.
  • the subject has, or is at risk of developing retinitis pigmentosa (autosomal dominant, autosomal recessive, or X-linked), Leber's congenital amaurosis, cone or cone-rod dystrophy, Usher syndrome, macular degeneration, Stargardt's disease, age-related macular degeneration, trauma-induced retinal degeneration, or retinal detachment.
  • the subject has been identified as having expression of PRL ⁇ E1 in cells of the eye.
  • the subject has been identified as having a mutation that causes retinitis pigmentosa, including, but not limited to, a mutation in RPGR, RPE65, PDE6 ⁇ , or NPHP5.
  • the composition is administered before disease onset. In another embodiment, the composition is administered prior to the initiation of vision impairment or loss. In another embodiment, the composition is administered after initiation of vision impairment or loss. In certain embodiments, the composition is readministered at a later date. Optionally, more than one readministration is permitted. Such readministration may be with the same type of vector, a different viral vector, or via non-viral delivery as described herein. In certain embodiments, the vector is readministered to the patient to a different portion of the initially injected retina. In one embodiment, the vector is readministered to the patient to the same portion of the initially injected retina. In certain embodiments, the subject has about 10% or more photoreceptor damage/loss.
  • the subject has about 20% or more photoreceptor damage/loss. In another embodiment, the subject has about 30% or more photoreceptor damage/loss. In another embodiment, the subject has about 40% or more photoreceptor damage/loss. In another embodiment, the subject has about 50% or more photoreceptor damage/loss. In another embodiment, the subject has about 60% or more photoreceptor damage/loss. In another embodiment, the subject has about 70% or more photoreceptor damage/loss. In another embodiment, the subject has about 80% or more photoreceptor damage/loss. In another embodiment, the subject has about 90% or more photoreceptor damage/loss. In certain embodiments, the subject has, or is at risk of developing, RP and more particularly, XLRP.
  • the subject is a “carrier” for XLRP, i.e., has at least one RPGR mutation in at least one X chromosome.
  • XLRP is an X-linked disease
  • females which normally have two X chromosomes, may be homozygous or heterozygous for a specific mutation in the RPGR gene, or compound heterozygotes, which have a different mutation in the RPGR gene on each X chromosome.
  • Normal males, having only one X chromosome, with a mutation in the RPGR gene are termed hemizygous.
  • the subject having, or at risk of developing XLRP is a hemizygous male.
  • the subject having, or at risk of developing XLRP is a homozygous female or a heterozygous female.
  • subjects at risk of developing XLRP include those with a family history of XLRP, those with one or more confirmed mutations in the RPGR gene, offspring of female carriers of an RPGR mutation (heterozygous females), or offspring of females carrying an RPGR mutation on both X chromosomes.
  • the subject has shown clinical signs of XLRP.
  • Clinical signs of XLRP include, but are not limited to, decreased peripheral vision, decreased central (reading) vision, decreased night vision, loss of color perception, reduction in visual acuity, decreased photoreceptor function, pigmentary changes.
  • the subject has been diagnosed with XLRP. In yet another embodiment, the subject has not yet shown clinical signs of XLRP.
  • the retinitis pigmentosa is an X-linked retinitis pigmentosa (XLRP).
  • XLRP is one of the most severe forms of RP, demonstrating an early age of onset (usually within the first decade) and rapid progression of disease. Because the disease is X- linked, homozygous females are rare, usually only manifesting in small, isolated populations. Thus, the disease primarily affects males, although carrier (heterozygous) females are also affected, demonstrating various levels of retinal degeneration. The disease demonstrates a broad spectrum of disease severity, between and within families.
  • RPGR retinitis pigmentosa GTPase regulator
  • the protein contains a RCC-1 like domain, characteristic of the highly conserved guanine nucleotide exchange factors.
  • the constitutive transcript of RPGR containing 19 exons, is expressed in a wide variety of tissues (Hong and Li, Invest Opthalmology Vis Sci, 43(11):3373-82, incorporated by reference herein).
  • An RPGR variant terminates in intron 15 of the RPGR gene.
  • the alternative terminal exon consists of the constitutive exon 15 and part of intron 15, and is termed ORF15.
  • ORF15 This protein isoform that is encoded by exons 1 through ORF15 is used prevalently in photoreceptors and a large number of disease causing mutations have been found in ORF15 (Vervoort and Wright, Hum Mutat.2002 May, 19(5):486-500; Aguirre et al, Exp Eye Res, 2002, 75:431-43; and Neidhardt et al, Hum Mutat.2007, 28(8):797-807, each of which is hereby incorporated by reference herein).
  • RPGR refers to the full length gene itself or a functional fragment, as further defined below.
  • the nucleic acid sequence encoding a normal RPGR gene may be derived from any mammal which natively expresses the RPGR gene, or homolog thereof.
  • the RPGR gene sequence is derived from the same mammal that the composition is intended to treat.
  • the RPGR is derived from a human.
  • the RPGR sequence is the sequence of the full length human RPGRORF15 clone, which includes exons 1 though ORF15 (Vervoort R, et al. (2000), Nat Genet 25:462-466, which is incorporated by reference herein). See also, WO 2014/011210 A1, which is incorporated herein in its entirety.
  • the subject has, or is at risk of developing, Leber congenital amaurosis (LCA). In certain embodiments, the subject has or is at risk of developing LCA caused by a mutation in RPE65. In certain embodiments, the subject has or is at risk of developing LCA caused by a mutation in IQCB1/NPHP5 (nephrocystin-5). In certain embodiments, the subject has, or is at risk of developing, Senior-L ⁇ ken syndrome. In certain embodiments, the subject has or is at risk of developing Senior-L ⁇ ken syndrome caused by a mutation in IQCB1/NPHP5. In certain embodiments, the subject has shown clinical signs of LCA-ciliopathy.
  • LCA Leber congenital amaurosis
  • Clinical signs of LCA-ciliopathy include, but are not limited to, nystagmus, decreased peripheral vision, decreased central (reading) vision, decreased night vision, loss of color perception, reduction in visual acuity, decreased photoreceptor function, pigmentary changes.
  • the subject has been diagnosed with LCA-ciliopathy.
  • the method includes delivery of a nucleic acid sequence that reduces or inhibits PRL ⁇ E1 expression and a nucleic acid sequence encoding a functional gene product, or fragment thereof.
  • the nucleic acid sequences may be delivered in a single construct (e.g., a vector genome wherein the sequences are operably linked) or in separated constructs that may be delivered using more than one vector delivered by one or more routes of administration.
  • the method includes delivery of a functional gene product, or fragment thereof, that the gene RPGR, RPE65, PDE6 ⁇ , or NPHP5.
  • the subject has been previously treated with a gene therapy, specifically a gene augmentation therapy, prior to administration of a composition for delivery of a nucleic acid sequence that reduces or inhibits PRL ⁇ E1 expression.
  • the subject is administered of a composition for delivery of a nucleic acid sequence that reduces or inhibits PRL ⁇ E1 expression prior to administration of a gene augmentation therapy.
  • the gene augmentation therapy is LUXTURNA® (voretigene neparvovec- rzyl).
  • the methods of treatment for treatment of a subject having an ocular disease with a composition described herein to reducing or inhibiting PRL ⁇ E1 expression or activity does not require re-administration.
  • the methods of treatment described herein may be used in conjunction with other treatments (secondary therapy), i.e., the standard of care for the subject’s diagnosis and condition.
  • secondary therapy refers to the therapy that could be combined with the gene therapy described herein for the treatment of an ocular disease.
  • the methods of treatment described herein may be used in conjunction with other gene therapies, including, for example those for choroideremia (REP- 1), retinitis pigmentosa (RPGR, RPE65, Abca4, Ar12bp, Arl6, Bbs2, Best1, C2orf71, C8orf37, Ca4, Cc2d2a, Cerkl, Clrn1, Cnga1, Cngb1, crb1, Crx, Cyp4v2, Dhdds, Dhx38, Emc1, Eys, Fam161a, Flvcr1, Fscn2, Gnptg, Gpr125, Guca1b, Idh3b, Ifg140, Impdh1, Impg2, Kiaa1549, Klh17, Lrat, Mak, Mertk, Myk, Nek2, Nr2e3, Nrl, Ofc1, Pde6a, Pde6b, Pde6g, Pex1, Pex7, Pgk1, Phyh, Pro
  • the method of treatment reduces COS elongation, thinning, and/or curving. In another embodiment, the treatment reduces ROS elongation, thinning, and/or curving. In another embodiment, the treatment reduces glial activation. In another embodiment, the treatment reduces ELM-RPE distance, in another embodiment, treatment reduces accumulation of retinal debris. In another embodiment, treatment reduces abnormal POS-RPE apposition and microarchitecture of RPE-PR interface. In another embodiment, treatment reduces subretinal debris at RPE apical surface, or within subretinal space. In another embodiment, treatment reduces compromised IPM and defective ELM. In another embodiment, treatment reduces fluctuation of ONL thickness associated with reactive gliosis and cell migration.
  • treatment reduces schistic changes in the inner/outer retina.
  • treatment reduces formation of subretinal & intraretinal scars.
  • treatment reduces RPE monolayer hypertrophy.
  • treatment reduces occasional severe deformation of individual RPE cells associated with ONL & INL thickness fluctuations.
  • treatment reduces and Muller Glial trunks/projections penetrating ONL layer.
  • treatment reduces gross macular lesion.
  • treatment reduces bullous detachment.
  • the non-invasive retinal imaging and functional studies may include electroretinography (ERG), perimetry, topographical mapping of the layers of the retina and measurement of the thickness of its layers by means of confocal scanning laser ophthalmoscopy (cSLO) and optical coherence tomography (OCT), topographical mapping of cone density via adaptive optics (AO), functional eye exam, etc., depending upon the species of the subject being treated, their physical status and health and treatment.
  • ERG electroretinography
  • perimetry topographical mapping of the layers of the retina and measurement of the thickness of its layers by means of confocal scanning laser ophthalmoscopy (cSLO) and optical coherence tomography (OCT), topographical mapping of cone density via adaptive optics (AO), functional eye exam, etc.
  • AO adaptive optics
  • functional eye exam etc.
  • the volume and viral titer of each injection is determined individually, as further described herein, and may be the same or different from other injections performed in the same, or contralateral, eye. In another embodiment, a single, larger volume injection is made in order to treat the entire eye. In one embodiment, the volume and concentration of the rAAV composition is selected so that only the region of damaged ocular cells is impacted. In another embodiment, the volume and/or concentration of the rAAV composition is a greater amount, in order reach larger portions of the eye, including non-damaged ocular cells. In another embodiment, the method includes performing additional studies, e.g., functional and imaging studies to determine the efficacy of the treatment.
  • Such tests include retinal and visual function assessment via electroretinograms (ERGs) looking at rod and cone photoreceptor function, optokinetic nystagmus, pupillometry, water maze testing, light-dark preference, optical coherence tomography (to measure thickness of various layers of the retina), histology (retinal thickness, rows of nuclei in the outer nuclear layer, immunofluorescence to document transgene expression, cone photoreceptor counting, staining of retinal sections with peanut agglutinin - which identifies cone photoreceptor sheaths).
  • ERPs electroretinograms
  • EMGs electroretinograms
  • the subject is tested for efficacy of treatment using electroretinograms (ERGs) to examine rod and cone photoreceptor function, pupillometry visual acuity, contrast sensitivity color vision testing, visual field testing (Humphrey visual fields/Goldmann visual fields), perimetry mobility test (obstacle course), and reading speed test.
  • EMGs electroretinograms
  • fMRI functional magnetic resonance imaging
  • full-field light sensitivity testing retinal structure studies including optical coherence tomography
  • fundus photography fundus autofluorescence
  • adaptive optics laser scanning ophthalmoscopy mobility testing
  • test of reading speed and accuracy microperimetry and/or ophthalmoscopy.
  • kits for generating a recombinant rAAV comprises obtaining a plasmid containing an AAV expression cassette as described above and culturing a packaging cell carrying the plasmid in the presence of sufficient viral sequences to permit packaging of the AAV viral genome into an infectious AAV envelope or capsid.
  • Specific methods of rAAV vector generation are described above and may be employed in generating an rAAV vector that can deliver the sequences of the expression cassettes and genomes described above and in the examples below. It should be understood that the methods of treatment described herein are intended to be applied to other compositions and methods described across the Specification. The invention is now described with reference to the following examples.
  • HEK293 cells were seeded in 12 well plates and co-transfected the following day at 70-90% confluence with a PRL ⁇ E1-mCherry plasmid and one of the three PRL ⁇ E1-targetting shRNAs, or a non-specific shRNA. Transfections were performed in triplicates using Lipofectamine 2000. The cells were incubated for 24 hours at 37°C in a 5% CO 2 incubator. Following incubation, the cells were collected, total RNA was extracted using the Qiagen RNeasy Plus Mini kit and reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, ThermoFisher Scientific).
  • qPCR analysis was performed using PowerUp SYBR Green Mastermix (Applied Biosystems, ThermoFisher Scientific) in either Applied Biosystems 7500 Real Time PCR System or Applied Biosystems ViiA 7 Real Time PCR System in a 96-well or 384-well format.
  • Subretinal AAV-shRNA PRLdE1 injections AAV vector was delivered subretinally in the supertemporal quadrant using a 25/38G PolyTip cannula (MedOne ® Sarasota, FL, U.S.A.) attached to a 1mL Luer-LokTM syringe (Beckton, Dickinson and Company, NJ, U.S.A.) under general anesthesia.
  • Injections were performed using a Stellaris PC injection system (Bausch and Lomb, Rochester, NY, USA) and visualized through a Zeiss operating microscope (Carl Zeiss Meditec, Inc., Dublin, CA, U.S.A.) with the image projected on a NGenuity ® 3D screen (Alcon, Geneva, Switzerland).
  • Optimal dose of AAV2/5-shRNA PRL ⁇ E1 was determined in a pilot study by subretinally injecting 150 ⁇ L of two different doses (5 x10 11 and 5 x 10 12 vg/ml) of the vector in each eye of one RPGR-XLPRA2 dog (age ⁇ 40 weeks) when ample PRL ⁇ E1 expression in the PRs is observed.
  • AAV2/5-shRNA PRL ⁇ E1 was delivered at a dose of 5 x 10 12 vg/ml in one PDE6 ⁇ -RCD1 (age: 5 weeks) and two RPGR-XLPRA2 (age: 7 weeks) dogs (subretinal, volume: 70 ⁇ Ls), and in one normal adult (age: 4 years and 6 months) dog (subretinal, volume: 150 ⁇ Ls).
  • OCT optical coherence tomography
  • cSLO confocal scanning laser ophthalmoscopy
  • Autofluorescence cSLO images were taken using a 130 o field-of-view lens, 55 o infra-red and blue-light. Sequential OCT scans of the bleb and bleb-adjacent areas were obtained to check for retinal changes over time.
  • Ocular tissue collection Ocular tissue was collected after euthanasia with intravenous sodium pentobarbital injection. After enucleation, 3 mm diameter retinal punches were obtained within and outside the area injection bleb. The posterior eyecup was fixed in 4% paraformaldehyde (PFA) for 3 hours, followed by 2% PFA for 24 hours.
  • PFA paraformaldehyde
  • RNA in situ hybridization, immunohistochemistry, and TUNEL Assay PRL ⁇ E1 mRNA was visualized by RNA-in situ hybridization (RNA-ISH) using the RNAscope assay 2.5 HD Assay-Red (Advanced Cell Diagnostics, ACD-Bio, BioTechne, Newark, CA, USA) as described previously 23 . Briefly, 10 ⁇ m thick sections were cut from PFA-fixed OCT-embedded canine retinas.
  • Target retrieval was performed by heating the slides at 88°C for 10 min in Target Retrieval buffer, followed by 15 min of protease treatment.
  • PRL ⁇ E1 probe RNAscope cat no.535781 binding and visualization were performed as described in the kit protocol Slides were then either counterstained with hematoxylin or used for immunohistochemistry/TUNEL assay.
  • RNAscope 2.5 HD Duplex Assay was performed following kit recommendations for co-visualization of PRL ⁇ E1 and NPHP5 (RNAscope probe cat no.462841) RNAs.
  • PRL ⁇ E1 was visualized using Fast Red dye and NPHP5 was stained with HRP-Green.
  • RNA-ISH immunohistochemistry
  • the retinal sections were treated with a blocking buffer for 1h (5% BSA and 4.5% fish gelatin in PBS) immediately following RNA-ISH, and then incubated overnight with the primary antibodies (RHO, Millipore MAB5316, 1:200; ARR3, C.Craft, Univ. of Southern California, LUMIF; RPGR, Sigma HPA001593, 1:100) at 4°C.
  • Antigen antibody complexes were visualized with Alexa-stained secondary antibodies.
  • Hoechst 33342 stain was used to label cell nuclei. TUNEL assay was performed to assess cell death in the retinal sections as previously reported 37 .
  • PRL ⁇ E1 expression is common to other forms of IRD.
  • RPGR-XLPRA1 X-Linked Progressive Retinal Atrophy 1, caused by a five-nucleotide deletion in the RPGR exon ORF15)(29, 30)
  • NPHP5-LCA Leber Congenital Amaurosis, caused by a single nucleotide insertion in exon 10 of the IQCB1/NPHP5 gene)(31, 32) (FIG.1A). Both diseases affect rods and cones, however, with varying age of onset and disease severity.
  • PRL ⁇ E1 transcript was detected at 14 weeks of age. This time point is characterized by early stages of PR loss and moderate thinning of the ONL, consistent with the early phase of degeneration in this model(31). In contrast, in the RPGR- XLPRA1 model, degenerative changes in the PR are observed before onset of cell death(33). PRL ⁇ E1 expression was observed starting at 26 weeks of age, which notably precedes the onset of noticeable PR loss. This early upregulation of PRL ⁇ E1 expression prior to extensive PR cell death, indicates a potential role in the initial response to degenerative stress (FIG.8A and FIG.8B).
  • RNA- in situ hybridization within the outer nuclear layer (ONL, the cellular layer containing the PR cell bodies) is challenging due to the dense packing of rod and cone nuclei in this area.
  • PRL ⁇ E1 RNA-ISH staining was found in regions containing both PR cell types but not in cone only areas (FIG.1B), supporting rod-specific expression in degenerating retina.
  • PRL ⁇ E1 expression in rods, and not cones using data from single cell RNAseq analysis. (FIG.9).
  • PRL ⁇ E1 expression colocalizes with areas of PR disease in RPGR-XLPRA carriers.
  • PRL ⁇ E1 expression colocalizes with areas of PR disease in RPGR-XLPRA carriers.
  • PRL ⁇ E1 expression was restricted to degenerating rods, we examined its expression in the two RPGR-XLPRA carriers, XLPRA1 and XLPRA2. In female dogs with a single mutant copy of RPGR, random X-chromosome inactivation creates a mosaic of normal and mutant PRs(36).
  • PRL ⁇ E1 expression overlapped with the patchy areas of degeneration identified by rhodopsin mislocalization and was not observed in the intervening patches with normal PRs (FIG.2).
  • PRL ⁇ E1 expression is limited to mutant rods with active disease.
  • PRL ⁇ E1 expression is not directly associated with acute PR cell death.
  • PRL ⁇ E1 expression is not associated with the acute rod cell death that can be experimentally triggered in this model but is instead linked to the chronic degenerative state observed in models with prolonged disease.
  • PRL ⁇ E1 expression is suppressed in RPGR and NPHP5 mutant retinas after corrective gene augmentation therapy. Since we found that PRL ⁇ E1 expression was restricted to unhealthy/diseased rods, we predicted that PRL ⁇ E1 expression would be downregulated or abrogated if degeneration was halted, and PR homeostasis restored therapeutically. To test this hypothesis, we examined PRL ⁇ E1 expression in two non-allelic diseases (RPGR-XLPRA and NPHP5-LCA) that have been previously successfully treated by AAV mediated gene augmentation therapy(33, 41, 42).
  • PRL ⁇ E1 was not expressed within the AAV-RPGR treated area (treated at 28- and 5-weeks of age, assessed at 49- and 33- weeks post-treatment, respectively) (FIG.4A).
  • PRL ⁇ E1 expression was remarkably reduced within the AAV-NPHP5 treated (at 6 weeks of age) retinal area when assessed 27 weeks post-treatment, compared to the untreated retina (FIG. 4B).
  • PRL ⁇ E1 As expression of PRL ⁇ E1 is associated with rod disease in the protracted phase of IRDs and can be suppressed by restoring PR to normal state following gene therapy, it raised the intriguing possibility that PRL ⁇ E1 itself could be contributing to disease. Knockdown of PRL ⁇ E1 expression confers transient protection to photoreceptors. To determine if PRL ⁇ E1 expression is a cause or an effect of the disease process, we used RNA interference to knockdown PRL ⁇ E1 expression in PDE6 ⁇ -RCD1 and RPGR- XLPRA2 retinas.
  • FIG.11A and FIG.11B Three different targeting shRNAs driven by the ubiquitous H1 promoter were tested in vitro in HEK293 cells co-transfected with a plasmid expressing PRL ⁇ E1 (FIG.11A and FIG.11B).
  • the shRNA showing the highest efficacy (shRNA2, ⁇ 70% knockdown) was selected for packaging in AAV2/5 vector for sustained expression after subretinal delivery in canine retinas.
  • AAV2/5-shRNA PRL ⁇ E1 was delivered subretinally in one PDE6 ⁇ -RCD1 (age: 5 weeks) and two RPGR-XLPRA2 (age: 7 weeks) dogs at ages corresponding to the early acute phase of cell death and start of PRL ⁇ E1 expression in surviving PRs in these models(23-25).
  • Quantitative-PCR analysis showed approximately 80% knockdown at 5-weeks PI and nearly 95% reduction in PRL ⁇ E1 mRNA at 11-weeks PI in the shRNA treated area in the two RPGR-XLPRA2 dogs (FIG.6A). This reduction was confirmed by RNA-ISH in the PDE6 ⁇ - RCD1 dog (FIG.6C). Immunohistochemistry performed in the RPGR-XLPRA2 dog terminated at 5 weeks PI confirmed that the ONL was thicker in the shRNA-treated area compared to the untreated areas. PRL-ISH indicated a qualitative reduction in PRL ⁇ E1 expression in the treated area where the ONL thickness was maintained.
  • PRL ⁇ E1 One of the top upregulated transcripts in both diseases was PRL(46) which we subsequently showed to be the novel PRL ⁇ E1 isoform(23). Furthermore, our previous studies showed that neither the full-length PRL nor the short isoform is expressed in normal retinas (Sudharsan et al., 2021, FIG.8A and FIG.8B) (23). In the current study, PRL ⁇ E1 was consistently upregulated in rods during chronic stages of disease progression across several IRDs. This expression pattern implies that PRL ⁇ E1 may play a role in the cellular response to prolonged stress and degeneration rather than being a direct mediator of cell death.
  • PRL ⁇ E1 is not merely a byproduct of degeneration but may actively contribute to the progression of the disease. Its expression during the chronic phase of degeneration hints at a role in sustaining or exacerbating the degenerative process, potentially through mechanisms related to cellular stress responses or inflammatory pathways. Understanding the exact function of PRL ⁇ E1 in these contexts could provide valuable insights into its potential as a therapeutic target for IRDs. While the precise modifications and transcription factors involved in PRL ⁇ E1 expression are currently under investigation, alternative Transcription Start Sites (aTSS) have been identified in 52% of human protein-coding genes(48).
  • aTSS alternative Transcription Start Sites
  • aTSS significantly increase transcriptomic diversity, allowing for dynamic fine-tuning of the cellular transcriptome in response to various physiological and pathological stimuli(49-55).
  • Epigenetic alterations such as DNA methylation and histone acetylation/deacetylation are common to many neurodegenerative diseases, including IRDs, and change the chromosomal accessibility to direct transcription from aTSS(56-58).
  • IRDs neurodegenerative diseases
  • PRL ⁇ E1 expression was associated with disease, it remained to be determined if it played an active role in IRD or its expression was a consequence of the disease process.
  • Our PRL ⁇ E1 knockdown studies support a role for this transcript in promoting disease in the retina. This is in contrast with the anti-apoptotic role identified for the full length PRL in retina(10, 13), and the neurogenic and neuroprotective roles in the CNS(59).
  • shRNA PRL ⁇ E1 Given the limitations of our currently used shRNA PRL ⁇ E1 with demonstrated off-target effects even in normal retinas, we could not address in the present study the long-term effects that silencing PRL ⁇ E1 expression may have on PR preservation.
  • PRL ⁇ E1 Efforts to develop a PRL ⁇ E1 targeting shRNA without off target /non-specific deleterious effects are ongoing. It is currently unclear whether PRL ⁇ E1 exerts its effect as RNA or protein. In our earlier publication(23), we showed that very small amounts of PRL protein could be identified in extracts from PDE6 ⁇ -RCD1 and RPGR-XLPRA2 retinas using mass spectrometry. However, the low protein abundance and resulting poor peptide coverage precluded identification of the complete PRL ⁇ E1 protein sequence. PRL ⁇ E1 mRNA lacks a consensus Kozak sequence(60) at the first in-frame AUG codon, potentially explaining the extremely low levels of PRL ⁇ E1 protein in extracts from mutant retinas.
  • the protein structure prediction algorithm I-TASSER(61, 62) estimates that the short protein isoform can form a 4 helix bundle similar to the full length PRL, and we predict that, if expressed, the protein should be able to bind the PRL receptor, albeit with lower affinity(23).
  • PRL ⁇ E1 protein was present at extremely low levels if present at all, it is also worth considering the potential role of PRL ⁇ E1 mRNA as a regulatory non-coding RNA.
  • lncRNA Long non-coding RNAs
  • lncRNA Long non-coding RNAs
  • the PRL ⁇ E1 transcript exhibits most attributes of mRNAs. As we cannot currently rule out the potential for this transcript to have both protein coding and regulatory functions(68, 69), our ongoing studies are aimed at defining the active PRL ⁇ E1 entity. We acknowledge two limitations in this study: the small number of dogs in which ONL rescue was demonstrated by PRL ⁇ E1 silencing and the lack of elucidation of the biological pathways through which PRL ⁇ E1 may drive rod degeneration and loss. We intentionally limited the number of dogs recruited for this study after we observed off-target effects with the currently used shRNA.
  • PRL ⁇ E1 isoform is not expressed in the rodent retinas (23), making dogs affected with IRDs valuable animal models in which the function of PRL ⁇ E1 can be investigated. Future studies employing more specific knockdown of this isoform will aim to identify the pathways that are activated by PRL ⁇ E1 in mutant retinas, and explore the translational potential of modulating its expression.
  • examination of PRL ⁇ E1 mRNA expression in four different forms of canine IRD with different degeneration paradigms reveals its consistent expression in IRDs, regardless of the causative gene mutation.
  • PRL ⁇ E1 knockdown/silencing represents an exciting novel “gene agnostic” therapeutic strategy for IRD. Future studies will include assessing the long-term outcome of PRL ⁇ E1 knockdown on preservation of PRs and identification of the specific pathways through which PRL ⁇ E1 modulates PR survival.
  • Example 3 Knockdown of human PRL ⁇ E1 (hPRL ⁇ E1) using DsiRNAs in HEK293 Cells DsiRNAs (Dicer siRNAs) were evaluated for their ability to knock down human PRL ⁇ E1 (hPRL ⁇ E1) in vitro.
  • HEK293 cells were transfected with pCMVTag5a-hPRL ⁇ E1- mCherry to exogenously express hPRL ⁇ E1.
  • Five different siRNAs were introduced to these cells, and knockdown efficiency was assessed 24 hours post-transfection using qPCR analysis. The experiment was conducted independently in four separate trials to confirm the knockdown efficiencies. The results are presented in FIG.12.
  • Front Endocrinol (Lausanne).2022;13:994898. 6. Torner L. Actions of Prolactin in the Brain: From Physiological Adaptations to Stress and Neurogenesis to Psychopathology. Front Endocrinol (Lausanne).2016;7:25. 7. Bole-Feysot C, Goffin V, Edery M, Binart N, Kelly PA. Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr Rev.1998;19(3):225-68. 8. O'Steen WK, Kraeer SL.
  • the hormone prolactin is a novel, endogenous trophic factor able to regulate reactive glia and to limit retinal degeneration.
  • Froland A Hagen C, Lauritzen E. Diabetic retinopathy and prolactin. Lancet. 1977;1(8001):45. 13.
  • Irish setter dogs affected with rod/cone dysplasia contain a nonsense mutation in the rod cGMP phosphodiesterase beta-subunit gene.
  • Aguirre GD Rubin LF. Rod-cone dysplasia (progressive retinal atrophy) in Irish setters. J Am Vet Med Assoc.1975;166(2):157-64. 29.
  • Zhang Q Acland GM, Wu WX, Johnson JL, Pearce-Kelling S, Tulloch B, et al.
  • Different RPGR exon ORF15 mutations in Canids provide insights into photoreceptor cell degeneration.
  • Hum Mol Genet.2002 11(9):993-1003.
  • Zeiss CJ, Acland GM, Aguirre GD Retinal pathology of canine X-linked progressive retinal atrophy, the locus homologue of RP3.
  • Age-dependent disease expression determines remodeling of the retinal mosaic in carriers of RPGR exon ORF15 mutations. Invest Ophthalmol Vis Sci.2009;50(8):3985-95. 37. Kijas JW, Cideciyan AV, Aleman TS, Pianta MJ, Pearce-Kelling SE, Miller BJ, et al. Naturally occurring rhodopsin mutation in the dog causes retinal dysfunction and degeneration mimicking human dominant retinitis pigmentosa. Proc Natl Acad Sci U S A. 2002;99(9):6328-33. 38. Cideciyan AV, Jacobson SG, Aleman TS, Gu D, Pearce-Kelling SE, Sumaroka A, et al.
  • Aguirre GD Cideciyan AV, Dufour VL, Ripolles-Garcia A, Sudharsan R, Swider M, et al. Gene therapy reforms photoreceptor structure and restores vision in NPHP5-associated Leber congenital amaurosis. Mol Ther.2021;29(8):2456-68. 43. Arango-Gonzalez B, Trifunovic D, Sahaboglu A, Kranz K, Michalakis S, Farinelli P, et al. Identification of a common non-apoptotic cell death mechanism in hereditary retinal degeneration. PLoS One.2014;9(11):e112142. 44.
  • I-TASSER a unified platform for automated protein structure and function prediction. Nat Protoc.2010;5(4):725-38. 62. Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. The I-TASSER Suite: protein structure and function prediction. Nat Methods.2015;12(1):7-8. 63. Carrella S, Banfi S, Karali M. Sophisticated Gene Regulation for a Complex Physiological System: The Role of Non-coding RNAs in Photoreceptor Cells. Front Cell Dev Biol.2020;8:629158. 64. Salta E, De Strooper B. Noncoding RNAs in neurodegeneration. Nat Rev Neurosci. 2017;18(10):627-40. 65.
  • Salvatori B Biscarini S, Morlando M. Non-coding RNAs in Nervous System Development and Disease. Front Cell Dev Biol.2020;8:273.
  • Song J, Kim YK Targeting non-coding RNAs for the treatment of retinal diseases. Mol Ther Nucleic Acids.2021;24:284-93.
  • Mattick JS Amaral PP, Carninci P, Carpenter S, Chang HY, Chen LL, et al.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Biochemistry (AREA)
  • Endocrinology (AREA)
  • Microbiology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des méthodes et des compositions pour traiter une maladie oculaire, y compris des affections associées à la dégénérescence rétinienne.
PCT/US2024/052518 2023-10-23 2024-10-23 Thérapie génique pour traitement de la dégénérescence rétinienne Pending WO2025090575A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363592346P 2023-10-23 2023-10-23
US63/592,346 2023-10-23

Publications (2)

Publication Number Publication Date
WO2025090575A1 true WO2025090575A1 (fr) 2025-05-01
WO2025090575A9 WO2025090575A9 (fr) 2025-06-12

Family

ID=95516405

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/052518 Pending WO2025090575A1 (fr) 2023-10-23 2024-10-23 Thérapie génique pour traitement de la dégénérescence rétinienne

Country Status (1)

Country Link
WO (1) WO2025090575A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100203060A1 (en) * 2007-05-30 2010-08-12 Lobie Peter E Inhibitors for growth hormone and related hormones, and methods of use thereof
US20140127284A1 (en) * 2012-10-18 2014-05-08 The Regents Of The University Of California Micro-rnas and micro-rna inhibitors to modulate blood vessel growth, patterning, tumor growth and malignant disease and method for making and using them

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100203060A1 (en) * 2007-05-30 2010-08-12 Lobie Peter E Inhibitors for growth hormone and related hormones, and methods of use thereof
US20140127284A1 (en) * 2012-10-18 2014-05-08 The Regents Of The University Of California Micro-rnas and micro-rna inhibitors to modulate blood vessel growth, patterning, tumor growth and malignant disease and method for making and using them

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SUDHARSAN RAGHAVI, MURGIANO LEONARDO, TANG HSIN-YAO, OLSEN TIMOTHY W., CHAVALI VENKATA R. M., AGUIRRE GUSTAVO D., BELTRAN WILLIAM : "Short prolactin isoforms are expressed in photoreceptors of canine retinas undergoing retinal degeneration", SCIENTIFIC REPORTS, NATURE PUBLISHING GROUP, US, vol. 11, no. 1, US , XP093312579, ISSN: 2045-2322, DOI: 10.1038/s41598-020-80691-6 *

Also Published As

Publication number Publication date
WO2025090575A9 (fr) 2025-06-12

Similar Documents

Publication Publication Date Title
ES2856403T3 (es) Composiciones a base de rAAV y procedimientos para el tratamiento de la esclerosis lateral amiotrofica
JP7642211B2 (ja) 優性網膜色素変性の治療のためのａａｖベクター
JP2023011865A (ja) ＭｅＣＰ２発現カセット
EP3189142B1 (fr) Oligonucléotides antisens pour le traitement de l'amaurose congénitale de leber
CA3018076A1 (fr) Agent therapeutique pour le traitement de maladies, notamment les maladies touchant le systeme nerveux central
WO2023122804A1 (fr) Compositions et méthodes comprenant un promoteur spécifique du coeur
JP2023532864A (ja) 導入遺伝子発現系
WO2020176747A1 (fr) Vecteurs virus adéno-associé et procédés d'administration d'agents thérapeutiques
US20200248204A1 (en) Methods of treating genetic hearing loss
WO2018218359A1 (fr) Thérapie génique destinée au traitement de troubles des peroxysomes
WO2025090575A1 (fr) Thérapie génique pour traitement de la dégénérescence rétinienne
WO2024069144A1 (fr) Vecteur d'édition d'arn
Chekuri et al. AAV2-mediated intravitreal delivery of exon-specific U1 snRNA rescues optic neuropathy in a mouse model of familial dysautonomia
WO2023030541A1 (fr) Traitement de maladies et de troubles oculaires associés à rpe65
WO2025231487A1 (fr) Promoteurs spécifiques des photorécepteurs destinés à être utilisés dans des thérapies géniques pour le traitement de la dégénérescence rétinienne
US20200352977A1 (en) Antisense oligonucleotides for the treatment of leber congenital amaurosis
HK40088240A (en) Aav vectors for treatment of dominant retinitis pigmentosa
WO2025091025A1 (fr) Thérapie génique pour troubles oculaires liés à sirt1
JP2024515612A (ja) 球脊髄性筋萎縮症（ｓｂｍａ）の治療に有用な組成物
WO2025102034A1 (fr) Thérapie génique pour le syndrome de barth
HK40002351B (en) Aav vectors for treatment of dominant retinitis pigmentosa
HK40002351A (en) Aav vectors for treatment of dominant retinitis pigmentosa

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24883227

Country of ref document: EP

Kind code of ref document: A1