WO2024221049A1 - Agents and method of treatment for optic conditions - Google Patents

Abstract

The disclosure provides an antisense oligonucleotide that binds within a targeted region of intron 7 of an OPA1 transcript and reduces expression of OPA1 gene transcript lacking exon 7x and uses of such an antisense oligonucleotide.

Description

AGENTS AND METHOD OF TREATMENT FOR OPTIC CONDITIONS

RELATED APPLICATION DATA

The present application claims priority from Australian Patent Application No. 2023901265 filed on 28 April 2023 entitled “Agents and method of treatment for optic conditions”. The entire contents of which is hereby incorporated by reference.

SEQUENCE LISTING

The present application is filed together with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to antisense oligonucleotides binding to target regions in OPA1 and their use to treat, prevent or ameliorate the effects caused by mutations in the gene OPAl.

BACKGROUND

Autosomal dominant optic atrophy (ADOA) is the world’s most common hereditary optic neuropathy, with an estimated disease prevalence ranging from 1 in 10,000 to 1 in 50,000. OPAl is the major causative gene in most families with ADOA.

ADOA classically presents in early childhood with progressive visual failure, with selective retinal ganglion cell loss as the defining pathological characteristic. The mean age of onset of visual failure is 7 years, with a range of 1 to 16 years. Eighty percent of affected individuals are symptomatic before the age of 10 years. In up to 20% of cases, ADOA is associated with other clinical manifestations, most commonly sensorineural deafness, ataxia, myopathy and progressive external ophthalmoplegia. The significance of these syndromal ADOA variants has been highlighted by the identification of cytochrome c oxidase (COX)-deficient muscle fibres and multiple mitochondrial DNA deletions in skeletal muscle biopsies from these patients, implicating a role of OPAl in mitochondrial DNA maintenance.

OPAl consists of 30 coding exons spanning 100 kb of genomic DNA on the long arm of chromosome 3 (3q28-q29), and the protein product is a 1,015 amino acid polypeptide that co-localizes to the inner mitochondrial membrane. OPAl contains a highly conserved functional GTPase domain shared by members of the dynamin superfamily of mechanoenzymes and regulates several important cellular processes including the stability of the mitochondrial network. Over 400 different OPAl mutations have been reported to be responsible for optic nerve degeneration and visual loss, ranging from isolated ‘Dominant Optic Atrophy’ (DOA; OMIM #165500) to more severe multi- systemic syndromes named ‘DOAplus’ (OMIM #125250), including some bi-allelic cases with Behr Syndrome (OMIM #210000).

OPA1 is a ubiquitously expressed mitochondrial GTPase that is indispensable for mitochondrial function. In humans, OPA1 generates at least eight isoforms via differential splicing of exons 4, 4b and 5b or equivalent to exon 4, 5 and 7 according to Figure 1A. OPA1 precursor proteins are targeted and mobilised to the mitochondria by their mitochondrial targeting sequence (MTS). In the mitochondria, the OPA1 precursor proteins are cleaved into either long forms (1 forms) that are anchored to the inner mitochondrial membrane, or into short, soluble forms (s forms).

The coding sequence of the full OPA1 gene is beyond the packaging capacity of AAV vectors, which have a limit of less than 5 kb. Furthermore, certain forms of CRISPR/Cas9 gene correction will require a different product for each unique OPA1 mutation, and splicing switching strategies using antisense oligomers targeted to the OPA1 pre-mRNA may only target regions of mutations (e.g., within an exon, as with antisense oligomer drugs like Eteplirsen) and may not provide a functional protein product. In addition, both the gene replacement and gene editing approaches require subretinal injection of viral vectors to achieve adequate transfection. The procedure carries risks of retinal trauma.

There is a need to provide new treatments or preventative measures for ADOA, or at least the provision of methods to compliment the previously known treatments. The present invention seeks to provide an improved or alternative method for treating, preventing or ameliorating the effects of ADOA.

The previous discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

SUMMARY OF INVENTION

In producing the present invention, the inventors identified antisense oligonucleotides (ASOs) that increase expression of OPA1 expression that are useful for the treatment or prevention of conditions associated with the OPA1 gene, e.g., ocular conditions, such as ADOA or glaucoma. The inventors have identified ASOs that bind to an OPA1 gene pre-mRNA in a cell to promote exclusion of a nonsense-mediated RNA decay- inducing (NMD) exon during splicing of the OPAl pre-mRNA to increase the level of OPA1 mRNA transcripts encoding full length, functional OPAl.

The inventors identified antisense oligonucleotides that reduce expression of OPAl gene transcript lacking exon 7x (a NMD exon) do so substantially without changing the proportion of other transcripts of the OPAl gene. For example, antisense oligonucleotides of the disclosure reduce expression of OPAl gene transcript lacking exon 7x without substantially affecting the relative expression levels of transcripts comprising exon 7 or lacking exon 7. The data herein suggest that antisense oligonucleotides of the disclosure reduce expression of OPAl gene transcript lacking exon 7x without changing normal physiological ratios of transcripts of the OPAl gene comprising exon 7 or lacking exon 7. Such antisense oligonucleotides are of interest therapeutically since transcripts lacking exon 7 are thought to increase the sensitivity of cells to apoptosis and, as a consequence, oligonucleotides that increase relative levels of transcripts lacking exon 7 are considered to be of less therapeutic benefit.

The present disclosure provides an antisense oligonucleotide that binds within a targeted region of intron 7 of an OPAl transcript and reduces expression of OPAl gene transcript lacking exon 7x but does not substantially affect the relative expression levels of OPAl gene transcripts comprising exon 7 or lacking exon 7, wherein the antisense oligonucleotide comprises one or more mismatches relative to the sequence set forth in SEQ ID NO: 1.

The present disclosure provides an antisense oligonucleotide that binds within a targeted region of intron 7 of an OPAl transcript and reduces expression of OPAl gene transcript lacking exon 7x but does not substantially affect the relative expression levels of OPAl gene transcripts lacking exon 7, wherein the antisense oligonucleotide comprises one or more mismatches relative to the sequence set forth in SEQ ID NO: 1.

In one example, the antisense oligonucleotide significantly increases the level of a transcript comprising exons 3, 4, 6, 7 and 8 in a cell to which the antisense oligonucleotide is contacted relative to the level of the transcript in a cell to which the antisense oligonucleotide has not been contacted. In one example, the level of expression is determined using quantitative RT-PCR, digital droplet PCR or RNA sequencing.

In one example, the antisense oligonucleotide comprises two mismatches relative to the sequence set forth in SEQ ID NO: 1.

In one example, the antisense oligonucleotide comprises one or more G residue mismatches corresponding to an A residue in SEQ ID NO: 1.

In one example, the mismatch is not at a terminus of the antisense oligonucleotide. The present disclosure provides an antisense oligonucleotide comprising a sequence set forth in any one of SEQ ID NOs: 2-14 or a sequence having at least about 50% or 60% or 70% or 80% or 95% identity thereto.

The present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 2-14 or a sequence having at least about 80% identity thereto.

In one example, the present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 2.

In one example, the present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 3.

In one example, the present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 4.

In one example, the present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 5.

In one example, the present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 6.

In one example, the present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 7.

In one example, the present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 8.

In one example, the present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 9.

In one example, the present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 10.

In one example, the present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 11.

In one example, the present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 12.

In one example, the present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 13.

In one example, the present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 14.

In one example, the disclosure provides an oligonucleotide comprising 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 consecutive nucleotides of any one of the foregoing nucleotides. In one example, the nucleotide sequence of the ASO consists of 10 to 50 nucleotides, 15 to 40 nucleotides, 18 to 40 nucleotides, 17 to 25 nucleotides, 20 to 35 nucleotides, 20 to 30 nucleotides, 22 to 30 nucleotides, 22 to 28 nucleotides, 24 to 30 nucleotides, 25 to 30 nucleotides, or 26 to 30 nucleotides. In one example, the nucleotide sequence of the ASO consists of 20 to 30 nucleotides. For example, the nucleotide sequence of the ASO consists of 17 nucleotides. In one example, the nucleotide sequence of the ASO consists of 19 nucleotides. In another example, the nucleotide sequence of the ASO consists of 21 nucleotides. In a further example, the nucleotide sequence of the ASO consists of 22 nucleotides. In one example, the nucleotide sequence of the ASO consists of 23 nucleotides. In another example, the nucleotide sequence of the ASO consists of 24 nucleotides. In another example, the nucleotide sequence of the ASO consists of 25 nucleotides. In another example, the nucleotide sequence of the ASO consists of 26 nucleotides. In another example, the nucleotide sequence of the ASO consists of 27 nucleotides. In another example, the nucleotide sequence of the ASO consists of 28 nucleotides. In another example, the nucleotide sequence of the ASO consists of 29 nucleotides. In another example, the nucleotide sequence of the ASO consists of 30 nucleotides.

In other examples the ASO comprises at least 10 contiguous nucleotides of an ASO sequence described herein. In some examples ASO comprises at least 10 contiguous nucleotides (subsequence) from each of two or more ASO sequences described herein or one sequence described herein and another sequence known in the art, where the two or more subsequences are not contiguous in a OPA1 mRNA sequence.

In some examples for each occurrence of “G” in an ASO sequence disclosed herein, the “G” is guanosine or inosine. In some examples for each occurrence of “T” in an ASO sequence disclosed herein, the “T” is any one of: thymidine, inosine, uracil, or an isomeric or modified form of uracil (e.g. , pseudouridine or N1 -methyl-pseudo uridine). In some examples for each occurrence of “C” in an ASO sequence disclosed herein, the C is cytosine or a modified form of cytosine (e.g., 5-methylcytosine).

In one example, the ASO comprises a backbone modification. For example, the backbone modification comprises a phosphoro thio ate linkage or a phosphorodiamidate linkage. In one example, the ASO comprises a phosphoro thio ate linkage. In another example, the ASO comprises a phosphorodiamidate linkage.

In one example, the ASO comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxyethyl moiety. For example, the ASO comprises a phosphorodiamidate morpholino moiety. In another example, the ASO comprises a locked nucleic acid. In a further example, the ASO comprises a 2'-O-methyl moiety. In one example, the ASO comprises a 2'-Fluoro moiety. In another example, the ASO comprises a 2'-O-methoxyethyl moiety.

In one example, the ASO comprises at least one modified sugar moiety. For example, each sugar moiety in the antisense oligonucleotide is a modified sugar moiety.

In one example, the ASO comprises a 2'-O-methoxyethyl moiety. For example, each nucleotide of the ASO comprises a 2'-O-methoxyethyl moiety.

In one example, the ASO comprises one or more phosphorodiamidate morpholino moieties.

In one example of any method described herein, the ASO is linked to a functional moiety. The functional moiety can be covalently linked or non-covalently linked to the ASO. The functional moiety can be at the 5' end and/or 3' end of the ASO.

In some examples, the functional moiety comprises a delivery moiety. For example, the delivery moiety is selected from the group consisting of lipids, peptides, carbohydrates, and antibodies. An exemplary delivery moiety comprises a cellpenetrating peptide (CPP). The present disclosure additionally contemplates delivery moieties such as a N-acetylgalactosamine (GalNAc) moiety, a fatty acid moiety, or a lipid moiety.

In some examples, the functional moiety comprises a stabilising moiety.

The present disclosure additionally provides a pharmaceutical composition comprising an ASO of the disclosure, and a pharmaceutically acceptable excipient, for use in any method of the disclosure.

In one example, the ASO is complexed with a delivery nanocarrier. For example, the delivery nanocarrier is selected from the group consisting of: lipoplexes, liposomes, exosomes, inorganic nanoparticles, and DNA nanostructures. In one example, the delivery nanocarrier comprises a lipid nanoparticle (LNP) encapsulating the antisense oligonucleotide.

In one example, the ASO is formulated for a route of administration selected from the group consisting of intravitreal, suprachoroidal, subretinal, ciliary intramuscular, intravenous, intra-arterial, subcutaneous, and topical routes.

The disclosure also provides a modified cell comprising an ASO of the disclosure for use in any method described herein. For example, the modified cell is a mammalian cell, such as a human cell.

The present disclosure additionally provides a method of treating a condition, the method comprising administering an ASO of the disclosure. In one example, the condition is associated with OPA1 expression, e.g., reduced OPA1 expression. In one example, the condition is glaucoma. In another example, the condition is autosomal dominant optic atrophy.

The present disclosure additionally provides a method for reducing expression of an isoform of 0PA1 comprising exon 7x and/or increasing expression of functional 0PA1 comprising administering an ASO of the disclosure to a subject. In one example, administration of the ASO reduces expression of 0PA1 gene transcript lacking exon 7x without changing normal physiological ratios of transcripts of the 0PA1 gene comprising exon 7 or lacking exon 7.

The present disclosure further provides a method of improving mitochondrial function and/or mitochondrial fusion in a subject suffering from ADO A, wherein the method comprises administering an ASO of the disclosure to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic showing antisense oligonucleotides targeting intron 7 of 0PA1 to mediate the exclusion of 0PA1 exon 7x. (A) Illustration showing 0PA1 transcript consisting of 31 exons and the location of exon 7x (if present) in the transcript. (B) Illustration showing enlargement of a partial intron 7 and exon 7x in 0PA1 ; SEQ ID NO: 1. (C) Antisense oligonucleotides were designed to target the region of intron 7 upstream of 0PA1 exon 7x.

Figure 2 shows results of screening of PMOs (25 and/or 50 pM) in ADO A patient fibroblasts. Patient fibroblasts were transfected for 48 hours with PMOs targeting removal of the OPA1 exon 7x as indicated. OPA1 transcript expression was assessed by digital droplet PCR (ddPCR) and normalised to HPRT1 transcript levels. The OPA1 expression in untreated cells was set to 1. Data represent mean+S.D. n=l biological replicate, n=3 technical replicates.

Figure 3 shows an illustration of OPA1 mRNA isoforms analysed for changes in expression. Amplification primers were designed to target the amplification of OPA1 exon 3 and exon 6 with the expected size for RT-PCR amplicons for individual OP Al mRNA isoforms. F; Forward RT-PCR primer 5’- ACTTTTGATCAGTGGAAAGATATGATACCGG-3’ (SEQ ID NO: 17); R; Reverse RT-PCR primer 5’-CTGAGTGTGCAGAAGTTCTTCCTGAAGTT-3’ (SEQ ID NO: 18).

Figure 4 is a graphical representation showing the balance of major OPA1 mRNA isoforms following PMO treatment. Assessment of OPA1 isoforms in PMO- treated (black bars) and untreated (white bars) fibroblasts derived from an ADOA patient was obtained using PCR-based Illumina MiSeq. Data represent Mean+S.D. of relative 0PA1 mRNA isoforms. Total 0PA1 mRNA was set to 100%. n=l biological replicate, n=3 technical replicates. Two-way ANOVA.

Figure 5 is a graphical representation showing a quantitative assessment of OPA1 mRNA isoform comprising exon 7 upregulation following PMO treatment. Assessment of OPA1 isoforms in PMO-treated (black bars) and untreated (white bars) fibroblasts derived from an ADOA patient was obtained using PCR-based Illumina MiSeq. Data represent Median+S.D. of the transcript per million reads (TPM) for indicated OP Al isoforms. n=l biological replicate, n=3 technical replicates. Two-way ANOVA. *p=0.0222.

Figure 6 is a graphical representation showing a quantitative assessment of OPA1 mRNA isoform lacking exon 7 following PMO treatment. Assessment of OPA1 isoforms in PMO-treated (black bars) and untreated (white bars) fibroblasts derived from an ADOA patient was obtained using PCR-based Illumina MiSeq. Data represent Median+S.D. of the transcript per million reads (TPM) for indicated OPA1 isoforms. n=l biological replicate, n=3 technical replicates. Two-way ANOVA.

Figure 7 is a graphical representation showing PPMO mediated upregulation of total OPA1 protein compared to untreated patient fibroblasts (UT). Experiments were conducted with n=l biological replicate, n=3 technical replicates. Patientl#1422 derived fibroblast harbouring OPA1 c.2708delTTAG mutation, Patient2#3825 derived fibroblast harbouring OPA1 c.985-lG>C mutation. Patient3#3674 derived fibroblast harbouring OPA1 c.2708delTTAG mutation, Patient4#1464 derived fibroblast harbouring OPA1 c.2608delA mutation, Patient5#2167 derived fibroblast harbouring OPA1 c.642+2T>G. Data represent mean + S.D. n=l biological replicate, n=3 technical replicates. Statistical analysis was performed using Student’s z-test. ***p<0.001, ****p<0.0001

Figure 8 is a graphical representation showing PPMO-mediated OPA1 protein upregulation improves the mitochondrial network of ADOA cells as shown by (A) reduced mitochondrial number and (B) increased branch length. Patient #1422 harboring OPA1 c.2708delTTAG mutation; Experiments were conducted in an 8-well chamber slide format. TOMM20; translocase of outer mitochondrial membrane 20, The bar graphs depict the mean + S.D. of mitochondrial network analysis. Data obtained from n=l biological replicate per patient line with 3 technical replicates. Statistical analysis was performed using Student’s z-test.

DESCRIPTION OF INVENTION

Detailed Description of the Invention General

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (z.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to "an" includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.

Each example of the present disclosure described herein is to be applied mutatis mutandis to each and every other example unless specifically stated otherwise.

Those skilled in the art will appreciate that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such techniques are described and explained throughout the literature in sources such as Perbal 1984, Sambrook et al., 2001, Brown (editor) 1991, Glover and Hames (editors) 1995 and 1996, Ausubel et al. including all updates until present, Coligan et al. (editors) (including all updates until present), Maniatis et aL 1982, Gait (editor) 1984, Hames and Higgins (editors) 1984, Freshney (editor) 1986.

The term “and/or”, e.g, “X and/or Y” shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

The term “about”, unless stated to the contrary, refers to +/- 20%, more preferably +/- 10%, of the designated value. For the avoidance of doubt, the term “about” followed by a designated value is to be interpreted as also encompassing the exact designated value itself (for example, “about 10” also encompasses 10 exactly).

Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The term “antisense oligonucleotide” “antisense oligomer” or “ASO,” as used herein, encompasses oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridizing to a complementary sequence on a target RNA transcript, including, but not limited to, those that do not comprise a sugar moiety, such as in the case of a peptide nucleic acid (PNA). Preferably, the ASO is an ASO that is resistant to nuclease cleavage or degradation.

The phrase “binds to a targeted portion” or “binds within a targeted portion,” in reference to an ASO, as used herein, refers to specific hybridization between the ASO nucleotide sequence and a target nucleotide sequence that is complementary within the ranges set forth herein. In some examples, specific hybridization occurs where, under ex vivo conditions, the hybridization occurs under high stringency conditions. By "high stringency conditions" is meant that the ASO, under such ex vivo conditions, hybridize to a target sequence in an amount that is detectably stronger than non-specific hybridization. High stringency conditions, then, are conditions that distinguish a polynucleotide with an exact complementary sequence, or one containing only a few scattered mismatches (e.g., two mismatches) from a random sequence that happened to have a few small regions (e.g., 1-5 bases) that matched the probe. Such small regions of complementarity are more easily melted than a full-length complement of 12-17 or more bases, and moderate stringency hybridization makes them easily distinguishable. In one example, high stringency conditions include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or the equivalent, at temperatures of about 50-70 °C. The skilled person will appreciate that under in vivo conditions, the specificity of hybridization between an ASO and its target sequence is defined in terms of the level of complementarity between the ASO and the target sequence to which it hybridizes within a cell.

The term “peptide” is intended to include compounds composed of amino acid residues linked by amide bonds. A peptide may be natural or unnatural, ribosome encoded or synthetically derived. Typically, a peptide will consist of between 2 and 200 amino acids. For example, the peptide may have a length in the range of 10 to 20 amino acids or 10 to 30 amino acids or 10 to 40 amino acids or 10 to 50 amino acids or 10 to 60 amino acids or 10 to 70 amino acids or 10 to 80 amino acids or 10 to 90 amino acids or 10 to 100 amino acids, including any length within said range(s). The peptide may comprise or consist of fewer than about 150 amino acids or fewer than about 125 amino acids or fewer than about 100 amino acids or fewer than about 90 amino acids or fewer than about 80 amino acids or fewer than about 70 amino acids or fewer than about 60 amino acids or fewer than about 50 amino acids.

Peptides, as referred to herein, include "inverso" peptides in which all L-amino acids are substituted with the corresponding D-amino acids, "retro -inverso" peptides in which the sequence of amino acids is reversed and all L-amino acids are replaced with D-amino acids.

Peptides may comprise amino acids in both L- and/or D-form. For example, both L- and D-forms may be used for different amino acids within the same peptide sequence. In some examples the amino acids within the peptide sequence are in L-form, such as natural amino acids. In some examples the amino acids within the peptide sequence are a combination of L- and D-form. Further, peptides may comprise unusual, but naturally occurring, amino acids including, but not limited to, hydroxyproline (Hyp), beta-alanine, citrulline (Cit), ornithine (Orn), norleucine (Nle), 3 -nitrotyrosine, nitroarginine, pyroglutamic acid (Pyr). Peptides may also incorporate unnatural amino acids including, but not limited to, homo amino acids, N-methyl amino acids, alpha-methyl amino acids, beta (homo) amino acids, gamma amino acids, and N-substituted glycines. Peptides may be linear peptides or cyclic peptides.

The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical bond or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

Percentage amino acid sequence identity with respect to a given amino acid sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Amino acid sequence identity may be determined using the EMBOSS Pairwise Alignment Algorithms tool available from The European Bio informatics Institute (EMBL-EBI), which is part of the European Molecular Biology Laboratory. This tool is accessible at the website located at www.ebi.ac.uk/Tools/emboss/align/. This tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). Default settings are utilized which include Gap Open: 10.0 and Gap Extend 0.5. The default matrix “Blosum62” is utilized for amino acid sequences and the default matrix. Percent (%) or percentage “nucleic acid sequence identity" with respect to the nucleotide sequences disclosed herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are known in the art, for instance, using publicly available computer software such as BLAST or ALIGN. The skilled person can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The term “cell penetrating peptide” (CPP) refers to a peptide that is capable of crossing a cellular membrane. In one example, a CPP is capable of translocating across a mammalian cell membrane and entering into a cell. In another example, a CPP may direct a conjugate to a desired subcellular compartment. Thus, a CPP may direct or facilitate penetration of a molecule of interest across a phospholipid, mitochondrial, endosomal, lysosomal, vesicular, or nuclear membrane. A CPP may be translocated across the membrane with its amino acid sequence complete and intact, or alternatively partially degraded.

A CPP may direct a molecule of interest, such as an ASO disclosed herein, from outside a cell through the plasma membrane, and into the cytoplasm or a desired subcellular compartment. Alternatively, or in addition, a CPP may direct a molecule of interest across the blood-brain, trans-mucosal, hematoretinal, skin, gastrointestinal and/or pulmonary barriers.

The term “peptide ligand” or “receptor binding domain” refers to a peptide that is capable of binding to a membrane surface receptor to enable translocation of the peptide across a cellular membrane. In one example a peptide ligand may enable translocation across the cellular membrane via the natural endocytosis of the targeted receptor. In another example the peptide ligand may utilise a complementary mechanism of translocation across the cellular membrane including utilising a conjugated CPP. In one example, a peptide ligand is capable of translocating across a mammalian cell membrane and to enter a cell. In another example, a peptide ligand may direct a conjugate to a desired subcellular compartment. Thus, a peptide ligand may direct or facilitate cellular uptake of a molecule of interest across a phospholipid, mitochondrial, endosomal, lysosomal, vesicular, or nuclear membrane. A peptide ligand may be translocated across the membrane with its amino acid sequence complete and intact, or alternatively partially degraded.

A peptide ligand via its binding to a target receptor may direct a molecule of interest, such as an ASO disclosed herein, from outside a cell through the plasma membrane, and into the cytoplasm or a desired subcellular compartment. Alternatively, or in addition, a peptide ligand via its binding to a target receptor may direct a molecule of interest across a relevant biological barrier, e.g., the blood-brain, trans-mucosal, hematoretinal, skin, gastrointestinal, and/or pulmonary barriers.

Methods of Treating or Preventing Ocular Conditions

The present disclosure provides, for example, a method of treating, preventing and/or delaying progression of an ocular condition, e.g., ADOA or glaucoma in a subject. The methods described herein include a method for treating, preventing and/or delaying progression of an ocular condition in a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising any of the ASOs disclosed herein. Likewise, in some examples, any of the ASOs herein are used in the manufacture of a medicament for treating, preventing and/or delaying progression of an ocular condition. In one example, the ASO comprises SEQ ID NO: 9.

In one example, the subject to be treated is suffering from an ocular condition, such as ADOA or glaucoma. For example, the subject has been diagnosed as having or suffering from an ocular condition, such as ADOA or glaucoma. In one example, the subject suffers from an ocular condition, such as ADOA or glaucoma. For example, the subject is in need of treatment. Such subjects can be administered the ASOs as described here to treat or prevent the progression of an ocular condition, such as ADOA or glaucoma.

In one example, administration of an ASO as described herein slows progression of an ocular condition, such as ADOA or glaucoma. In one example, the subject is at risk of developing an ocular condition, such as ADOA or glaucoma. Such subjects can be administered the ASOs as described here to prevent onset of an ocular condition, such as ADOA or glaucoma.

As used herein, the term “at risk” means that the subject has an increased chance of developing an ocular condition, such as ADOA or glaucoma compared to a normal individual. Subjects can be identified as at risk of developing an ocular condition, such as ADOA or glaucoma using any method known in the art and/or those described herein. For example, the subject may be identified at risk of developing an ocular condition, such as ADOA or glaucoma if that subject has one or more common risk factors including family history, high eye pressure, diabetes, high or low blood pressure and prolonged use of steroidal medication.

Also provided herein is a method for increasing the OPA1 transcript or OPA1 protein in a cell, the method comprising contacting the cell with a composition or pharmaceutical composition, as disclosed herein, whereby the amount of OPA1 transcript or OPA1 protein in the cell is increased. Also provided herein is a method for increasing the level of OPA1 transcript or OP Al protein in a cell, ex vivo or in a tissue in vivo, the method comprising contacting the cell with an ASO or pharmaceutical composition, as disclosed herein, whereby the amount of OPA1 transcript or OPA1 protein in the cell is increased. In some examples, the cell is a retinal cell. In some examples, the tissue is a retinal tissue, e.g., retina and/or retinal pigment epithelium.

In some examples, administration to a subject or contact with cells with any of the ASOs or pharmaceutical compositions disclosed herein increases the level of OPA1 transcript or OPA1 protein about 1.1 to about 10-fold, e.g., 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10- fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold compared to the level in the tissue prior to the administration or contact. In one example, administration to a subject or contact with cells with any of the ASOs or pharmaceutical compositions disclosed herein increases the level of OPA1 protein about 1.2 to about 1.6-fold, compared to the level in the tissue prior to the administration or contact. For example, about 1.2-fold, or about 1.3-fold, or about 1.35-fold, or about 1.4-fold, or about 1.5-fold, or about 1.55-fold, or about 1.6-fold.

Methods of determining OPA1 protein concentration will be apparent to the skilled person and/or described herein and include, for example, Western blot assay (e.g., with an anti-OPAl antibody) normalized to total protein staining.

It will be apparent to the skilled person from the disclosure herein that antisense oligonucleotides of the disclosure that reduce expression of OPA1 gene transcript lacking exon 7x do so substantially without changing the proportion of other transcripts of the OPA1 gene. For example, antisense oligonucleotides of the disclosure reduce expression of OP Al gene transcript lacking exon 7x without substantially affecting the relative expression levels of transcripts comprising exon 7 or lacking exon 7. In one example, the antisense oligonucleotides of the disclosure reduce expression of OP Al gene transcript lacking exon 7x without reducing the expression level of transcripts lacking exon 7. The data herein suggest that antisense oligonucleotides of the disclosure reduce expression of OPA1 gene transcript lacking exon 7x without changing normal physiological ratios of transcripts of the OPA1 gene comprising exon 7 or lacking exon 7. Such antisense oligonucleotides are of interest therapeutically since transcripts lacking exon 7 are thought to increase the sensitivity of cells to apoptosis and, as a consequence, oligonucleotides that increase relative levels of transcripts lacking exon 7 are considered to be of less therapeutic benefit.

In one example, antisense oligonucleotides of the disclosure that reduce expression of OPA1 gene transcript lacking exon 7x do not affect the relative expression levels of OP Al gene transcripts comprising exon 7 or lacking exon 7. In one example, antisense oligonucleotides of the disclosure that reduce expression of OPA1 gene transcript lacking exon 7x do not affect the relative expression levels of OP Al gene transcripts comprising exon 7 or lacking exon 7 when administered to a cell compared to the level or ratio of the transcripts in a cell to which the antisense oligonucleotide has not been administered. For example, antisense oligonucleotides of the disclosure reduce expression of OPA1 gene transcript lacking exon 7x without changing normal physiological ratios of transcripts comprising exon 7 or lacking exon 7 when administered to a cell compared to the level or ratio of the transcripts in a cell to which the antisense oligonucleotide has not been administered. Normal physiological ratios of transcripts comprising exon 7 or lacking exon 7 will be apparent to the skilled person and/or disclosed herein. For example, normal physiological ratios of OPA1 gene transcripts comprising exon 7 or lacking exon 7 are approximately 1:4 of OPA1 gene transcript comprising exon 7 to OP Al gene transcript lacking exon 7 (i.e., approximately 20% OPA1 gene transcript comprising exon 7 and approximately 80% OPA1 gene transcript lacking exon 7). Levels and/or ratios of the transcripts are determined using methods known in the art, such as, RT-PCR, digital droplet PCR or RNA sequencing.

In one example, the relative expression levels of OPA1 gene transcripts comprising exon 7 or lacking exon 7 are at a ratio of approximately 20% OPA1 gene transcript comprising exon 7 and approximately 80% OPA1 gene transcript lacking exon 7. For example, the OP Al gene transcripts comprising exon 7 or lacking exon 7 are at a ratio of between 15-25% OPA1 gene transcript comprising exon 7 and between 75-85% OP Al gene transcript lacking exon 7.

In one example, the relative expression levels of OPA1 gene transcripts comprising exon 7 or lacking exon 7 are at a ratio of approximately 1:4 of OPA1 gene transcript comprising exon 7 to OP Al gene transcript lacking exon 7.

The increase in functional OP Al will preferably lead to a reduction in the quantity, duration or severity of the symptoms of an OPA1 -related disease, such as ADOA.

Suitable routes of administration for treatment with the compositions, pharmaceutical compositions, or medicaments disclosed herein include, but are not limited to, intravitreal, suprachoroidal, subretinal, ciliary intramuscular, intravenous, intra-arterial, subcutaneous, and topical.

In some examples administration is into the eye by an intravitreal, suprachoroidal, or sub-retinal route. For example, administration to the eye is by intravitreal administration. In another example, administration to the eye is by suprachoroidal administration. In a further example, administration to the eye is by sub-retinal administration. In one example, administration to the eye is by a topical administration.

As the skilled person will understand, the treatment methods disclosed herein include administration of the compositions and pharmaceutical compositions disclosed herein in a therapeutically effective amount to a subject (e.g., a human subject). The terms "effective amount" or "therapeutically effective amount," as used herein, refer to a sufficient amount of a disclosed ASO being administered to relieve to some extent one or more of the symptoms and/or clinical indicia associated with a particular disease or health condition. In some examples, an "effective amount" for therapeutic uses is the amount of one of the foregoing agents required to provide a clinically significant decrease in disease symptoms and/or inflammatory markers or to prevent disease symptoms without undue adverse side effects. An appropriate "effective amount" in any individual case may be determined using techniques, such as a dose escalation study. The term "therapeutically effective amount" includes, for example, a prophylactically effective amount. It is understood that "an effective amount" or "a therapeutically effective amount" can vary from subject to subject, due to variation in metabolism of the compound of any age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. By way of example only, therapeutically effective amounts may be determined by routine experimentation, including but not limited to a dose escalation clinical trial. Where more than one therapeutic agent is used in combination, a “therapeutically effective amount” of each therapeutic agent can refer to an amount of the therapeutic agent that would be therapeutically effective when used on its own, or may refer to a reduced amount that is therapeutically effective by virtue of its combination with one or more additional therapeutic agents.

Compositions for Increasing OPA1 mRNA and/or Protein Levels

Ocular conditions, such as ADOA and glaucoma are characterized by degeneration of retinal ganglion cells, and at least 75% of cases are caused by mutations in the OPA1 gene (in the case of ADOA). OP Al mitochondrial dynamin like GTPase gene (also known as OP Al, FLJ 12460, KIAA0567, MGM1, NPG and NIG', referred to herein as OPAL) is composed of 30 coding exons distributed across approximately 100 kb of genomic DNA. It is located on chromosome 3q28-q29 and encodes for a ubiquitously expressed dynamic -related GTPase, which is imported into mitochondria by an N-terminal import sequence and localizes to the inner membrane facing the intermembrane space. OPA1 contains a highly conserved functional GTPase domain shared by members of the dynamin superfamily of mechanoenzymes and regulates several important cellular processes including the stability of the mitochondrial network. In humans, OPA1 generates at least eight isoforms via differential splicing of exons 4, 4b and 5b. For the purposes of nomenclature only and not limitation the sequence of the entire human OPA1 gene sequence and known transcript maps and sequences are publicly available through the online Ensembl database under record ENSG00000198836. An exemplary gene sequence of human OPA1 is set out in NCBI Reference Sequence NM_130837, and UniProt ID 060313.

The unique distribution of mitochondria in retinal ganglion cells points to a critical function of this network in retinal ganglion cells and may underlie the sensitivity of these cells to OPA1 loss (also potentially precipitated by exposure of these cells to photo-oxidative stress). The majority of OP Al mutations (-27% missense, 27% splice variant, 23.5% frameshift, 16.5% nonsense, 6% deletion or duplication) lead to degradation of the transcript by mRNA decay, supporting the hypothesis that haploinsufficiency is a major mechanism underlying ADOA pathogenesis. OPA1 mutations leading to haploinsufficiency cause impaired mitochondrial function including defective complex I-driven ATP synthesis. As ATP is the principal energy currency in cells, insufficient ATP ultimately leads to cellular malfunction and death.

OPA1 haploinsufficiency accounts for a significant proportion of ADOA. OPA1 haploinsufficiency causing ADOA is amenable to treatment by upregulating wild type OPA1.

The OPA1 gene contains an intron with a premature termination codon (PTC) in intron 7 (located between exons 7 and 8). In some subjects, a proportion of the OPA1 RNA transcripts from wild-type OPA1 genes retain a section of intron 7 containing this PTC; this retained intron section is called exon 7x in the transcribed RNA. The RNA transcripts that contain exon 7x (the retained intron segment containing the PTC) are subject to nonsense-mediated RNA decay. Therefore, a proportion of OPA1 RNA that is translated to mature wild-type protein, and a portion of OPA1 RNA that is degraded by RNase almost immediately due to the presence of the PTC. Recently, exon 7 of OPA1 has also been referred to as exon 5b and exon 7x has been referred to as exon 5x. Thus, reference herein to exon 7 will also be a reference to exon 5 and reference to exon 7x will also be a reference to exon 5x.

The present disclosure seeks to address this insufficiency by ensuring that an increased amount of the OPA1 transcript produced by the wild-type OPA1 gene lacks exon 7x. This would mean that an increased amount of the RNA transcribed from the wild-type OPA1 gene is able to be translated into a functional protein, as additional RNA is “rescued” from conversion into an RNA form subject to nonsense-mediated RNA decay (NMD) by the presence of the PTC in exon 7x. This additional functional protein is then able to compensate for the inability of the mutant allele to produce functional OPA1 protein.

As described herein, the ASOs according to any example bind to a targeted portion of human OPA1 pre-mRNA and which increase expression of OPA1 protein by promoting the exclusion of exon 7x in splicing of OPA1 in mammalian cells.

Without being bound by theory or mode of action, the ASOs that bind to targeted portions of human OPA1 pre-mRNA in mammalian cells and which result in the exclusion of NMD exon 7x, are thought to increase expression of OPA1 protein by preventing the translation of NMD exon 7x.

In one example, the ASOs of the disclosure improve mitochondrial function and/or mitochondrial fusion. In one example, the ASOs of the disclosure improve mitochondrial function. In another example, the ASOs of the disclosure improve mitochondrial fusion. Methods of determining mitochondrial function and/or fusion will be apparent to the skilled person and include, for example, assessing mitochondrial network connectivity and/or mitochondrial number.

In one example, the ASOs of the disclosure improve mitochondrial network connectivity. For example, the ASOs of the disclosure increase mitochondrial branch length. In one example, the ASOs of the disclosure increase the average length of branches within a mitochondrial network.

In one example, the ASOs of the disclosure improve mitochondrial fusion. For example, the ASOs of the disclosure reduce total mitochondrial number. It will be apparent to the skilled person that reducing total mitochondrial number is indicative of increased mitochondrial fusion.

Antisense Oligonucleotides (ASOs)

In some examples of the compositions and methods described herein, ASOs have a sequence that is completely complementary across its length to the target sequence or a sequence near complementarity (e.g., sufficient complementarity to bind the target sequence to promote exon splicing). ASOs are designed so that they bind (hybridize) to a target RNA sequence (e.g., a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Selection of suitable sequences for ASOs generally avoids, where possible, similar nucleic acid sequences in other (i.e., off-target) locations in the genome or in cellular mRNAs or miRNAs, such that the likelihood the ASO will hybridize at such sites is limited.

In some examples, ASOs “specifically hybridize” to or are “specific” to a target nucleic acid or a targeted portion of the OPA1 mRNA. In some examples, ASOs “specifically hybridize” to or are “specific” to a target nucleic acid or a targeted portion of the OPA1 pre-mRNA. At a given ionic strength and pH, the T_m is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.

ASO sequences are “complementary” to their target sequences when hybridization occurs in an antiparallel configuration between two single- stranded polynucleotides. Complementarity is quantifiable in terms of the proportion (e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules. The nucleotide sequence of an ASO need not be 100% complementary to that of its target nucleic acid to hybridize and the present disclosure specifically contemplates ASOs having mismatches to the target sequence. In certain examples, the nucleotide sequences of ASOs in the compositions disclosed herein can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the nucleotide sequence of the targeted portion of an RNA transcript over the length of the ASO nucleotide sequence. For example, an ASO in which 18 of 20 nucleotides of ASO sequence are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In another example, an ASO of 25-30 nucleotides may comprise 1 or 2 or 3 mismatches, such as 1 mismatch or 2 mismatches. In such an example, the remaining non-complementary nucleotides of the ASO could be clustered together or interspersed with complementary nucleotides and need not be contiguous. Complementarity of an ASO sequence to a target nucleotide sequence (expressed as “percent complementarity” to its target sequence; or “percent identity” to its reverse complement sequence) can be determined routinely using algorithms known in the art, as exemplified in the BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul, et al., 1990, J. Mol. Biol., 215:403-410; Zhang et al., 1997, Genome Res., 7:649-656).

In some examples, an ASO does not hybridize to all nucleotides in a target sequence and the nucleotide positions at which it does hybridize may be contiguous or noncontiguous. ASOs may hybridize over one or more segments of a intron 7 of the OPA1 pre-mRNA, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure may be formed).

In some examples the nucleotide sequences of ASOs described herein are complementary to a targeted portion of intron 7 of the OPA1 pre-mRNA. In some examples, the ASOs are complementary to a targeted portion within sufficient proximity to an acceptor site of exon 7x to promote exclusion of exon 7x in splicing of OP Al mRNA e.g. the antisense oligonucleotide comprises any one of SEQ ID NOs: 2-14. In some examples, the ASOs are complementary to a targeted portion within sufficient proximity to an acceptor site of exon 7x to promote exclusion of exon 7x in splicing of OPA1 mRNA e.g. the antisense oligonucleotide comprises any one of SEQ ID NOs: 2- 14. In one example, the antisense oligonucleotide comprises SEQ ID NO: 9.

The ASOs described herein may be of any length suitable for specific hybridization to a target sequence. In some examples, the nucleotide sequence of the ASOs consist of 8 to 50 nucleotides. For example, the ASO sequence can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleotides in length. In some examples, the ASOs consist of more than 50 nucleotides, but no more than 100 nucleotides in length.

In some examples, the ASO nucleotide sequence is from 8 to 50 nucleotides, 8 to 40 nucleotides, 8 to 35 nucleotides, 8 to 30 nucleotides, 8 to 25 nucleotides, 8 to 20 nucleotides, 8 to 15 nucleotides, 9 to 50 nucleotides, 9 to 40 nucleotides, 9 to 35 nucleotides, 9 to 30 nucleotides, 9 to 25 nucleotides, 9 to 20 nucleotides, 9 to 15 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 35 nucleotides, 10 to 30 nucleotides, 11 to 50 nucleotides, 11 to 40 nucleotides, 11 to 35 nucleotides, 11 to 30 nucleotides, 11 to 25 nucleotides, 12 to 50 nucleotides, 12 to 40 nucleotides, 12 to 35 nucleotides, 12 to 30 nucleotides, 12 to 25 nucleotides, 13 to 50 nucleotides, 13 to 40 nucleotides, 13 to 35 nucleotides, 13 to 30 nucleotides, 13 to 25 nucleotides, 14 to 50 nucleotides, 14 to 40 nucleotides, 14 to 35 nucleotides, 14 to 30 nucleotides, 14 to 25 nucleotides, 15 to 50 nucleotides, 15 to 40 nucleotides, 15 to 35 nucleotides, 15 to 30 nucleotides, 15 to 25 nucleotides, 20 to 50 nucleotides, 20 to 40 nucleotides, 20 to 35 nucleotides, 20 to 30 nucleotides, 20 to 25 nucleotides, 25 to 50 nucleotides, 25 to 40 nucleotides, 25 to 35 nucleotides, or 25 to 30 nucleotides in length. In some preferred examples, the nucleotide sequence of the ASO nucleotide is 25-30 nucleotides in length.

ASO Chemistry and Modifications

The ASOs used in the compositions described herein may comprise naturally- occurring nucleotides, nucleotide analogues, modified nucleotides, or any combination thereof. The term “naturally occurring nucleotides” includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” includes nucleotides with modified or substituted sugar groups and/or having a modified backbone. In some examples, all the nucleotides of an ASO are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the compositions and methods described herein are known in the art as disclosed in, e.g., in U.S. Patent No. 8,258,109, U.S. Patent No. 5,656,612, U.S. Patent Publication No. 2012/0190728, and Roberts et al., 2020, Nature Rev. Drug Disc., 19:673-694.

One or more nucleotides of an ASO may be any naturally occurring, unmodified nucleobase such as adenine, guanine, cytosine, thymine, uracil and inosine, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target RNA transcript. Examples of suitable modified nucleobases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5, 6-dihydrouracil, 5-methylcytosine, and 5 hydro xymethoylcytosine.

ASOs include a “backbone” structure that refers to the connection between nucleotides/monomers of the ASO. In naturally occurring oligonucleotides, the backbone comprises a 3'-5' phosphodiester linkage connecting sugar moieties of adjacent nucleotides. Suitable types of backbone linkages for the ASOs described herein include, but are not limited to, phosphodiester, phosphorothioate, phosphorodithioate, phosphorodiamidate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the like. In some examples, the backbone modification is a phosphorothioate linkage. In other examples, the backbone modification is a phosphorodiamidate linkage. See, e.g., Roberts el al. supra; and Agrawal (2021), Biomedicines, 9:503. In some examples, the backbone structure of the ASO does not contain phosphorous-based linkages, but rather contains peptide bonds, for example in a peptide nucleic acid (PNA), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups.

In some examples, the stereochemistry at each of the phosphorus internucleotide linkages of the ASO backbone is random. In other examples, the stereochemistry at each of the phosphorus internucleotide linkages of the ASO backbone is controlled and is not random. For example, U.S. Pat. No. 9,605,019 describes methods for independently selecting the handedness of chirality at each phosphorous atom in an oligonucleotide. In some examples, a composition or composition used in the methods disclosed herein comprises a pure diastereomeric ASO. In other examples, the composition comprises an ASO that has diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

In some examples, the ASO has a non-random mixture of Rp and Sp configurations at its phosphorus internucleotide linkages. In some examples, an ASO used in the compositions and methods disclosed herein, comprises about 5-100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, with the remainder Sp, or about 100% Rp.

In some examples, the ASOs described herein contain a sugar moiety that comprises ribose or deoxyribose, or a modified sugar moiety or sugar analog, including a morpholine ring. Suitable examples of modified sugar moieties include, but are not limited to, 2' substitutions such as 2'-O-modifications, 2'-O-methyl (2'-0-Me), l'-O- methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F, N3'->P5' phosphoramidate, 2 'dimethylaminooxy ethoxy, 2 'dimethylaminoethoxy ethoxy, 2'-guanidinidium, 1-0- guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. In some examples, the sugar moiety modification is selected from among 2'-O-Me, 2'F, and 2'MOE. In other examples, the sugar moiety modification is an extra bridge bond, such as in a locked nucleic acid (LN A). In some examples the sugar analogue contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some examples, the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification. In some examples, the sugar moiety comprises 2'4'-constrained 2'-O-methyloxyethyl (cMOE) modifications. In some examples, the sugar moiety comprises cEt 2', 4' constrained 2'- O-ethyl DNA modifications. In other examples, the sugar moiety comprises tricycloDNA (tcDNA) modifications. In some examples, the sugar moiety comprises ethylene nucleic acid (ENA) modifications. In some examples, the sugar moiety comprises 2'-O-(2-A-methylcarbamoylethyl) (MCE). Modifications are known in the art as exemplified in Jarver, et al., 2014, Nucleic Acid Therapeutics, 24(1): 37 47.

In some examples, each constituent nucleotide of the ASO is modified in the same way, e.g., every linkage of the backbone of the ASO comprises a phosphoro thio ate linkage, or each ribose sugar moiety comprises a 2'-O-methyl modification. In other examples, a combination of different modifications is used, e.g., an ASO comprising a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholinos).

In some examples, the ASO comprises one or more backbone modifications. In some examples, the ASO comprises one or more sugar moiety modification. In some examples, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some examples, the ASO comprises a 2'MOE modification and a phosphoro thio ate backbone. In some examples, the ASO comprises a peptide nucleic acid (PNA).

In some examples, the ASO comprises a phosphorodiamidate morpholino (PMO).

The skilled person in the art will appreciate that ASOs may be modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO. In some examples, an ASO is modified to alter one or more properties. For example, such modifications can: enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (e.g., RNase H); improve uptake of an ASO into a cell and/or particular subcellular compartments; alter the pharmacokinetics or pharmacodynamics of the ASO; and/or modulate the half-life of the ASO in vivo. In some examples, the ASOs comprise one or more 2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides, which have been shown to confer significantly enhanced resistance of ASOs to nuclease degradation and increased bioavailability.

Methods for synthesis and chemical modification of ASOs, as well as synthesis of ASO conjugates is well known in the art, and such ASOs are available commercially.

In some examples, a composition (e.g., a pharmaceutical composition) provided here includes two or more ASOs with different chemistries but complementary to the same targeted portion of intron 7 of the OPA1 pre-mRNA. In other examples, two or more ASOs that are complementary to different targeted portions of intron 7 of the OPA1 pre-mRNA.

In some examples, the compositions disclosed herein include ASOs that are linked to a functional moiety. In some examples, the functional moiety is a delivery moiety, a targeting moiety, a detection moiety, a stabilizing moiety, or a therapeutic moiety. In some examples the functional moiety includes a delivery moiety or a targeting moiety. In some examples the functional moiety includes a stabilizing moiety. In some examples the functional moiety is a delivery moiety.

Suitable delivery moieties include, but are not limited to, lipids, peptides, carbohydrates, and antibodies.

In some examples, the delivery moiety includes a cell-penetrating peptide (CPP). Suitable examples of CPPs are described in, e.g., PCT/AU2020/051397. In some examples the amino acid sequence of the CPP comprises or consists of: RRSRTARAGRPGRNSSRPSAPRGASGGASG (SEQ ID NO: 15). In one example, the CPP comprises the sequence RRSRTARAGRPGRNSSRPSAPRGASGGASG (SEQ ID NO: 16), optionally wherein any amino acid other than glycine is a D amino acid. In other examples, the delivery moiety includes a receptor binding domain.

In other examples, the delivery moiety includes a carbohydrate. In some examples, a carbohydrate delivery moiety is selected from among N-acetylgalactosamine (GalNAc), N-Ac-Glucosamine (GluNAc), and a mannose. In one example, the carbohydrate delivery moiety is GalNAc.

In other examples, the delivery moiety includes a lipid. Examples of suitable lipids as delivery moieties include, but are not limited to, cholesterol moiety, a cholesteryl moiety, and aliphatic lipids. In some examples the delivery moiety includes a fatty acid or lipid moiety. In some embodiments the fatty acid chain length is about C8 to C20. Examples of suitable fatty acid moieties and their conjugation to oligonucleotides are found in, e.g., International Patent Publication WO 2019232255 and in Prakash et al., (2019).

In further examples, the delivery moiety includes an antibody, as described in, e.g., Dugal-Tessier et al., (2021), J Clin Med., 10(4):838.

Suitable examples of stabilizing moieties include, but are not limited to, polyethylene glycol (PEG), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), and Poly(2-oxazoline)s (POx).

In some examples, where an ASO is linked to a functional moiety, the functional moiety is covalently linked to the ASO. In other examples, the functional moiety is non- covalently linked to the ASO.

Functional moieties can be linked to one or more of any nucleotides in an ASO at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, e.g., using a linker. Linkers can include a bivalent or trivalent branched linker. In some examples, the functional moiety is linked to the 5' end of the ASO. In other examples, the functional moiety is linked to the 3' end of the ASO. In further examples, the functional moiety is linked to the 5' end and the 3' of the ASO.

In some examples compositions comprising any of the ASOs disclosed herein also include a delivery nanocarrier complexed with ASO. In some examples, a delivery nanocarrier is selected from among lipoplexes, liposomes, exosomes, inorganic nanoparticles, and DNA nanostructures. In other examples the delivery nanocarrier includes a lipid nanoparticle encapsulating the ASO. Various delivery ASO -nanocarrier complex formats are known in the art, as reviewed in, e.g., Roberts et al., supra.

Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions comprising any of the foregoing ASOs, and formulated with at least a pharmaceutically acceptable excipient, including a carrier, filler, preservative, adjuvant, solubilizer and/or diluent.

Pharmaceutical compositions containing any of the ASOs compositions described herein, for use in the methods disclosed herein, can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the published literature. In some examples, a pharmaceutical composition for treating a subject comprises a therapeutically effective amount of any ASO disclosed herein.

Pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit/risk ratio. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.

Exemplary salts useful in a composition of the present disclosure include calcium chloride, magnesium chloride or sodium chloride.

In one example, a composition comprises a buffer. Exemplary buffers useful in a composition of the present disclosure include sodium phosphate.

In some examples, pharmaceutical compositions are formulated into any of a number of possible dosage forms including, but not limited to, ocular emulsions, topical ointments, solutions for intravitreal injection, intravenous administration, intrathecal administration, intracisterna magna administration, tablets, capsules, gel capsules, liquid syrups, and soft gels. In some examples, the compositions are formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carbo xymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. In some examples, a pharmaceutical formulation disclosed herein is provided in a form including, but not limited to, a solution, emulsion, microemulsion, foam or liposome-containing formulation (e.g., cationic or noncationic liposomes).

In some examples, pharmaceutical formulations comprising any of the ASOs described herein may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients as appropriate and known to the skilled person. In some examples, where a pharmaceutical composition includes liposomes, such liposomes can also include sterically stabilized liposomes, e.g., liposomes comprising one or more specialized lipids. These specialized lipids result in liposomes with enhanced circulation lifetimes. In some examples, a sterically stabilized liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as PEG moiety. In some examples, a surfactant is included in the pharmaceutical formulation.

In some examples, a pharmaceutical composition also includes a penetration enhancer to enhance the delivery of ASOs, e.g., to aid diffusion across cell membranes and /or enhance the permeability of a lipophilic drug. In some examples, the penetration enhancers include a surfactant, a fatty acid, a bile salt, or a chelating agent.

In some examples, a pharmaceutical composition comprises a dose of ASOs ranging from about 0.01 mg/kg to 20 mg/kg, e.g., 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, 8 mg/kg, 10 mg/kg, 15 mg/kg, or another dose ranging from about 0.01 mg/kg to 20 mg/kg.

In some examples, a pharmaceutical composition comprises multiple ASOs. In some examples, a pharmaceutical composition comprises, in addition to ASOs, another drug or therapeutic agent suitable for treatment of a subject suffering from glaucoma.

Combination Therapies

The pharmaceutical compositions comprising any of the ASOs disclosed herein, can also be used in combination with other agents of therapeutic value in the treatment of glaucoma. In general, other agents do not necessarily have to be administered in the same pharmaceutical composition, and may, because of different physical and chemical characteristics, be administered by different routes. The determination of the mode of administration and the advisability of administration, where possible, in the same pharmaceutical composition, is well within the knowledge of the skilled clinician. The initial administration can be made according to established protocols known in the art, and then, based upon the observed effects, the dosage, modes of administration and times of administration can be modified by the skilled clinician.

Compositions and pharmaceutical compositions comprising ASOs and an additional therapeutic agent may be administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, depending upon the stage and progression of the glaucoma to be treated, the condition of the patient, and the choice of specific therapeutic agents used. The determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol, is well within the knowledge of the skilled physician after evaluation of the glaucoma being treated and the condition of the patient.

It is known to those of skill in the art that therapeutically-effective dosages can vary when the drugs are used in treatment combinations. Methods for experimentally determining therapeutically-effective dosages of drugs and other agents for use in combination treatment regimens are described in the literature. For example, the use of metronomic dosing, i.e., providing more frequent, lower doses in order to minimize toxic side effects, has been described extensively in the literature. Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the patient.

For combination therapies, dosages of co -administered therapeutic agents will of course vary depending on the type of co-agents employed, ASO, and the disease stage of the patient to be treated.

Pharmaceutical compositions comprising ASOs and an additional therapeutic agent which make up a combination therapy disclosed herein may be a combined dosage form or in separate dosage forms intended for substantially simultaneous administration. The pharmaceutical compositions that make up the combination therapy may also be administered sequentially, with either therapeutic agent being administered by a regimen calling for two-step administration. The two-step administration regimen may call for sequential administration of the active agents or spaced-apart administration of the separate active agents. The time period between the multiple administration steps may range from, a few minutes to several hours, depending upon the properties of each pharmaceutical agent, such as potency, solubility, bio availability, plasma half-life and kinetic profile of the pharmaceutical agent. Circadian variation of various physiological parameters may also be evaluated to determine the optimal dose interval.

Examples of suitable therapeutic agents for co-administration with a composition or a pharmaceutical composition disclosed herein include, but are not limited to, prostaglandins (e.g., latanoprost (Xalatan®), travoprost (Travatan Z®), tafluprost (Zioptan®), bimatoprost (Lumigan®) and latanoprostene bunod (Vyzulta®)), beta blockers (e.g., timolol (Betimol®, Istalol®, Timoptic®) and betaxolol (Betoptic®)), alpha-adrenergic agonists (e.g., apraclonidine (lopidine®) and brimonidine (Alphagan P, Qoliana®)), carbonic anhydrase inhibitors (e.g., dorzolamide (Trusopt®) and brinzolamide (Azopt®)), a rho kinase inhibitor (e.g., netarsudil (Rhopressa®)) and miotic or cholinergic agents (e.g., pilocarpine (Isopto Carpine®)).

The present disclosure is not to be limited by the following non-limiting examples. EXAMPLES

Example 1

Identification of OPA1 Target Sequences and Phosphorodiamidate Morpholino Oligonucleotides (PM Os) design

The region and nucleotide sequences of OPA1 intron 7 and exon 7 are illustrated in Figure 1A and shown in SEQ ID NO: 1, respectively. Antisense oligonucleotides with 26-30 nucleotides in length (Table 2, Figure IB) were designed to target the intronic splice enhancer motifs in intron 7 to mediate exclusion of exon 7x and generate productive OPA1 transcripts. A single base pair microwalk and engineered mismatch oligos were applied to PMO sequences to identify the most efficacious PMO sequences capable of inducing exclusion of exon 7x. To maximise the coverage of ESE, PMO length was designed and optimised. The efficacy of PMOs in inducing exon 7x exclusion and improving total OPA1 transcript was assessed using ddPCR and protein assays.

Example 2

Screening of PMOs targeting Intron 7 of OPA1 in Autosomal Dominant Optic Atrophy (ADOA) Patient Cells

The above-identified antisense oligonucleotide sequences were synthesized as PMOs for initial screening. Antisense PMOs targeting intron 7 of OPA1 (as described in Example 1) were nucleofected into ADOA patient fibroblasts carrying the OPA1 mutation (c.958-lG>A) using the NEON® electroporation system (ThermoFisher) and the nucleofected cells were cultured for 48 hours. Gene Tools Control PMO sequence; GTC: CCTCTTACCTCAGTTACAATTTATA (SEQ ID NO: 19), predicted to not hybridize with OPA1 mRNA, was used as a sham treatment. Total RNA was extracted using the MagMAX™-96 Total RNA Isolation kit and the level of OPA1 transcript was assessed by ddPCR (Qiagen; probe catalog number: dHsaCPE5043545). OPA1 transcript expression was normalized to the HPRT1 level (Qiagen; probe catalog number: dHsaCPE5192872). Several PMOs (SEQ ID NOs: 2, and 8-14) showed upregulation of OPA1 transcript expression level compared to untreated and sham controls at 48 hours post-treatment. The PMO OPAl_H7xA(-134-105)2mmA>G_V2 (SEQ ID NO: 9) exhibited the highest OPA1 transcript upregulation; up to 1.42-fold at a concentration of 50 pM as compared to untreated patient fibroblasts as seen in Figure 2.

Example 3 PMO mediated OPA1 upregulation and maintaining the balance of OPA1 mRNA isoforms

The PMO OPAl_H7xA(-134-105)2mmA>G_V2 (SEQ ID NO: 9) was further analysed for the ability to maintain the balance of OPA1 mRNA isoforms essential for mitochondrial function. The PMO was transfected to ADOA patient fibroblasts carrying an OPA1 mutation (c.2708_2711delTTAG) and incubated for 48 hours. Total RNA was extracted using the MagMAX™-96 Total RNA Isolation kit according to the manufacturer’s instructions. OPA1 spliced isoform expression was tested by selecting the region between exon 3 and exon 8 of the OPA1 gene 3:193615674-193631665. The design of RT-PCR primers and expected amplicon sizes for OPA1 mRNA isoforms is shown in Figure 3. The selected OP Al region was amplified using RT-PCR to capture spliced RNA fragments. The samples were then sequenced using 2x 300 bp paired-end Illumina MiSeq. The captured spliced region length was less than 600 bp, hence, overlapping reads were merged to generate an intact fragment, representing the region of interest with a given exon composition. These merged reads were then used for OPA1 isoform quantitation analysis. Results in Figure 4 demonstrate the ability of PMO to maintain the balance of OPA1 mRNA isoforms. Figure 5 and Figure 6 show the quantitative number of transcript reads for OPA1 isoforms with and without exon 5b, respectively, following PMO treatment in patient fibroblasts.

Example 4

PPMO mediated OPA1 protein upregulation in a mutation agnostic manner

The PMO OPAl_H7xA(-134-105)2mmA>G_V2 (SEQ ID NO: 9) was conjugated with a cell penetrating peptide resulting in a PPMO. The PPMO was directly incubated with skin fibroblasts derived from five ADOA patients with confirmed OPA1 mutations and analysed for the ability to upregulate OPA1 protein expression. Total protein was harvested from the transfected cells using the CytoBuster protein extraction reagent (Merck Millipore) following the manufacturer’s instruction and assessed by western blot assay using rabbit anti-OPAl monoclonal antibody (Cell Signaling 25 Technology, catalogue number 67589) at a dilution of 1:250 in 5% BSA in TBST buffer followed by goat anti-rabbit IgG H&L antibody (Abeam, catalogue number ab216773, IRDye® 800CW). The expression of OPA1 protein was evaluated through a Western blot assay (rabbit anti-OPAl monoclonal antibody) and normalized to total protein staining (Revert™ 700 Total Protein Stain, LI-COR).

Figure 7 demonstrates the PPMO mediated upregulation of total OPA1 protein by between 1.21 and 1.57-fold (mean + S.D.) as compared to untreated patient fibroblasts (UT). In all experimental conditions, cell viability exceeding 85% was confirmed through the utilization of a CellTitreGlo assay (Promega). The findings revealed that the PPMO (SEQ ID NO: 9) has the capability to increase 0PA1 protein levels in cells of ADOA patients, despite the type of mutations.

Example 5

PPMO-mediated OPA1 protein upregulation improve the mitochondrial network of ADOA cells

The PPMO OPAl_H7xA(-134-105)2mmA>G_V2 (SEQ ID NO: 9) was further assessed for its capability to restore mitochondrial structure, key subcellular phenotype associated with 0PA1 mutation. The PPMO was direct incubated with skin fibroblasts derived from two ADOA patients for 5-7 days. Cells were stained with TOMM20 and analysed for the ability to upregulate 0PA1 protein expression. Total protein was harvested from the transfected cells using the CytoBuster protein extraction reagent (Merck Millipore) following the manufacturer’s instruction and assessed by western blot assay using rabbit anti-OPAl monoclonal antibody (Cell Signaling Technology, catalogue number 67589) at a dilution of 1:250 in 5% BSA in TBST buffer followed by goat anti-rabbit IgG H&L antibody (Abeam, catalogue number ab216773, IRDye® 800CW). The expression of OPA1 protein was evaluated through a Western blot assay (rabbit anti-OPAl monoclonal antibody) and normalized to total protein staining (Revert™ 700 Total Protein Stain, LI-COR).

Patient fibroblasts were incubated with the PPMO (SEQ ID NO:9) for 5 days, and half media change was performed on day 3. Cells were stained with mouse anti- TOMM20 monoclonal antibody and Hoechst to determine mitochondria and nuclei, respectively. Mitochondria were counted and assessed of network connectivity using ImageJ/Fiji (NIH, USA) with the Mitochondria Analyzer plug-in version 2.1.0.

Figure 8 illustrates an improvement in mitochondrial fusion is observed by reduced mitochondrial number (Figure 8A). In addition, figure 8B further reveals an improvement of mitochondrial network connectivity with increased branch length that represents the average length of branches within a mitochondrial network. The findings demonstrate the ability of PPMO-mediated OPA1 protein upregulation to enhance mitochondrial fusion, thereby restoring the key cellular phenotype of ADOA. Table 1: OP Al intron 7 (lowercase) and exon 7x (uppercase) cDNA sequence

(GRCh38/hg38: chr3 193626203-193628616)

Table 2: List of antisense oligonucleotide sequences

Table 3: List of CPPs

Table 4: List of Primers

Claims

Claims

1. An antisense oligonucleotide that binds within a targeted region of intron 7 of an 0PA1 transcript and reduces expression of 0PA1 gene transcript lacking exon 7x but does not substantially affect the relative expression levels of 0PA1 gene transcripts comprising exon 7 or lacking exon 7, wherein the antisense oligonucleotide comprises one or more mismatches relative to the sequence set forth in SEQ ID NO: 1.

2. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide significantly increases the level of a transcript comprising exons 3, 4, 6, 7 and 8 in a cell to which the antisense oligonucleotide is contacted relative to the level of the transcript in a cell to which the antisense oligonucleotide has not been contacted.

3. The antisense oligonucleotide of claim 1 which comprises two mismatches relative to the sequence set forth in SEQ ID NO: 1.

4. An antisense oligonucleotide comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 2-14 or a sequence having at least about 50% identity thereto.

5. An antisense oligonucleotide comprising or consisting of a sequence set forth SEQ ID NO: 9.

6. The antisense oligonucleotide of any one of claims 1 to 5 comprising a backbone modification.

7. The antisense oligonucleotide of claim 6, wherein the backbone modification comprises a phosphoro thio ate linkage or a phosphorodiamidate linkage.

8. The antisense oligonucleotide of claim 6 or 7 comprising a phosphorodiamidate morpholino moiety.

9. The antisense oligonucleotide of any one of claim 1 to 8 comprising at least one modified sugar moiety.

10. The antisense oligonucleotide of any one of claims 1 to 9 comprising a l'-O- methoxyethyl moiety.

11. The antisense oligonucleotide of any one of claims 1 to 10 linked to a functional moiety.

12. The antisense oligonucleotide of claim 11, wherein the functional moiety comprises a delivery moiety.

13. The antisense oligonucleotide of claim 12, wherein the delivery moiety is selected from the group consisting of lipids, peptides, carbohydrates, and antibodies.

14. The antisense oligonucleotide of claim 12 or 13 wherein the delivery moiety comprises a cell-penetrating peptide (CPP).

15. A pharmaceutical composition comprising the antisense oligonucleotide of any one of claims 1 to 14, and a pharmaceutically acceptable excipient.

16. A method of treating a condition in a subject in need thereof, the method comprising administering the antisense oligonucleotide of any one of claims 1 to 14 or the pharmaceutical formulation of claim 15.

17. The method of claim 16, wherein the condition is associated with OPA1 expression.

18. The method of claim 17, wherein the condition is glaucoma or autosomal dominant optic atrophy.

19. A method for reducing expression of an isoform of OPA1 comprising exon 7x and/or increasing expression of functional OPA1 comprising administering the antisense oligonucleotide of any one of claims 1 to 14 or the pharmaceutical formulation of claim 15 to a subject.

20. The method of claim 19, wherein administration of the ASO reduces expression of OPA1 gene transcript lacking exon 7x without changing normal physiological ratios of transcripts of the OP Al gene comprising exon 7 or lacking exon 7.