Agents for treatment of conditions associated with optic atrophy 1

Abstract

The present disclosure generally relates to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504 and conjugates thereof. The present invention also relates to antisense oligonucleotides that modulates mRNA productive transcript, stability and / or translation of OPA1 gene transcript or part thereof linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

Description

[0001] AGENTS FOR TREATMENT OF CONDITIONS ASSOCIATED WITH OPTIC

[0002] ATROPHY 1

[0003] RELATED APPLICATION DATA

[0004] This application claims priority from Australian Patent Application No. 2024901550 filed 24 May 2024 and entitled “Agents for Treatment of Conditions Associated with Optic Atrophy 1”, the entire contents of which is hereby incorporated by reference.

[0005] SEQUENCE LISTING

[0006] 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.

[0007] TECHNICAL FIELD

[0008] The present invention relates to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504 and conjugates thereof. The present invention also relates to antisense oligonucleotides that modulates mRNA productive transcript, stability and / or translation of OPA1 gene transcript or part thereof linked to a cellpenetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

[0009] BACKGROUND

[0010] OPA1 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. 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. Over 400 different OPA1 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 ‘ADOAplus’ (OMIM #125250), including some bi-allelic cases with Behr Syndrome (OMIM #210000).

[0011] 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 1 A. 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).

[0012] 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, for ocular conditions, subretinal injection of viral vectors to achieve adequate transfection. The procedure carries risks of retinal trauma.

[0013] There is a need to provide new treatments or preventative measures for conditions associated with mutations in the OP Al gene. The present invention seeks to provide an improved or alternative method for treating, preventing or ameliorating the effects of conditions associated with mutations in the OPA1 gene.

[0014] 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.

[0015] SUMMARY

[0016] In producing the present invention, the inventors identified a sequence defined cell penetrating peptide (CPP) useful for intracellular delivery of a CPP-linked cargo, e.g., a polynucleotide such as an oligonucleotide. The inventors further identified that conjugates comprising antisense oligonucleotides (ASOs) that increase expression of OPA1 expression linked to the sequence defined CPP are useful for pharmacological intervention for treating or preventing conditions associated with the OPA1 gene, e.g., ocular conditions, such as autosomal dominant optic atrophy (ADOA), glaucoma, geographic atrophy, acute ischemic optic neuritis and acute ischemic optic neuropathy. The present inventors identified that ASOs linked to the sequence defined CPP (i.e., as set forth in SEQ ID NO: 2504) were capable of upregulating OPA1 protein expression. Thus, the findings by the inventors provide the basis for a sequence defined CPP, as well as conjugates comprising ASOs that increase expression of OPA1 expression linked to the sequence defined CPP. These findings additionally provide the basis for methods of treating, preventing and / or delaying progression of an ocular condition. For example, ADOA, glaucoma, geographic atrophy, acute ischemic optic neuritis and acute ischemic optic neuropathy.

[0017] Accordingly, the present disclosure provides a CPP comprising a sequence set forth in SEQ ID NO: 2504. The present disclosure also provides a CPP, wherein the sequence of the CPP consists of a sequence set forth in SEQ ID NO: 2504.

[0018] The present disclosure also provides a CPP comprising a sequence set forth in SEQ ID NO: 2504 for targeting retinal cells in a subject in need thereof. For example, retinal ganglion cells. The present disclosure further provides a CPP, wherein the sequence of the CPP consists of a sequence set forth in SEQ ID NO: 2504, for targeting retinal cells in a subject in need thereof.

[0019] Accordingly, the present disclosure provides an antisense oligonucleotide that modulates mRNA productive transcript, stability and / or translation of OPA1 gene transcript or part thereof, wherein the antisense oligonucleotide is linked to a CPP comprising a sequence set forth in SEQ ID NO: 2504.

[0020] The present disclosure further provides an antisense oligonucleotide that modulates mRNA productive transcript, stability and / or translation of OPA1 gene transcript or part thereof, wherein the antisense oligonucleotide is linked to a CPP, wherein the CPP consists of a sequence set forth in SEQ ID NO: 2504.

[0021] The present disclosure provides an antisense oligonucleotide that increases functional OPA1 protein levels in the subject, wherein the antisense oligonucleotide is linked to a CPP comprising a sequence set forth in SEQ ID NO: 2504. For example, the level of OPA1 protein is increased in the subject compared to the level in the subject prior to administration of the ASO.

[0022] The present disclosure also provides an antisense oligonucleotide that increases functional OPA1 protein levels in the subject, wherein the antisense oligonucleotide is linked to a CPP comprising a sequence set forth in SEQ ID NO: 2504. The present disclosure further provides an antisense oligonucleotide that increases functional OPA1 protein levels in the subject, wherein the antisense oligonucleotide is linked to a CPP, wherein the CPP consists of a sequence set forth in SEQ ID NO: 2504.

[0023] In one example, the ASO increases the level of OP Al mRNA and the amount of functional OPA1 protein in a cell and / or a tissue of the subject. For example, the ASO increases the level of OPA1 mRNA in a cell and / or a tissue of the subject. In another example, the ASO increases the amount of functional OPA1 protein in a cell and / or a tissue of the subject. In one example, the amount of functional OPA1 protein in the cell and / or the tissue is increased by about 1.1 to about 10-fold. For example, the amount of functional OPA1 protein in the cell and / or the tissue is increased by about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8- fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold. For example, the amount of functional OPA1 protein in the cell and / or the tissue is increased by 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. For example, the amount of functional OPA1 protein in the cell and / or the tissue is increased 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 the ASO disclosed herein increases the level of OPA1 protein about 1.1 to about 2.5-fold compared to the level in the tissue prior to the administration or contact.

[0024] In one example, the cell and / or tissue is selected from the group consisting of an ocular tissue, retinal pigment epithelium (RPE) cells, Muller glial cells, retinal ganglion cells, endothelial cells, glial cells, astrocytes, photoreceptors. For example, the cell and / or tissue is selected from the group consisting of the retina, RPE cells and combinations thereof.

[0025] In one example, the ASOs bind to a targeted portion of: an OP Al gene pre-mRNA in a cell to promote exclusion of a nonsense-mediated RNA decay-inducing (NMD) exon during splicing of the OPA1 pre-mRNA to increase the level of OPA1 mRNA transcripts encoding full length, functional OPA1; i. the 5' untranslated region (UTR) of an OPA1 gene transcript in a cell to increase translation efficiency of an OP Al mRNA; ii. the 5' UTR of an OP Al gene transcript in a cell to increase transcript stability, e.g., by inhibiting the activity of a decapping enzyme; and / or iii. the 3' UTR of an OP Al gene transcript in a cell to increase transcript stability, e.g., by preventing binding of a miRNA to the 3' UTR.

[0026] In one example, the ASO binds to a targeted portion of an OP Al pre-mRNA in a cell to promote exclusion of a NMD exon during splicing of the OP Al pre-mRNA to increase the level of OPA1 mRNA transcripts encoding full length, functional OPA1. In one example, the ASO binds to a targeted portion of intron 7 OP Al pre-mRNA. Exemplary ASOs bind within a targeted portion of OP Al pre-mRNA nucleotide sequence corresponding to one or more of SEQ ID NO: 1.

[0027] In one example, the ASO binds to intron 7 of an OPA1 gene pre-mRNA in a cell and increases the level of OPA1 gene transcripts encoding full length, functional OPA1 by exclusion of NMD exon 7x. For example, the ASO is within sufficient proximity to an acceptor site of exon 7x to promote exclusion of exon 7x in splicing of OP Al mRNA.

[0028] In one example, the ASO binds to intron 7 of an OPA1 transcript and reduces expression of OPA1 gene transcript lacking exon 7x but does not substantially affect the relative expression levels of OPA1 gene transcripts comprising exon 7 or lacking exon 7.

[0029] In one example, the ASO binds within a targeted portion of the OPA1 pre-mRNA nucleotide sequence corresponding to SEQ ID NOs: 1, 55, 139.

[0030] In one example, the ASO comprises one or more mismatches relative to the sequence set forth in SEQ ID NO: 1.

[0031] 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.

[0032] In one example, the antisense oligonucleotide comprises two mismatches relative to the sequence set forth in SEQ ID NO: 1.

[0033] In one example, the ASO comprises one or more G residue mismatches corresponding to an A residue in SEQ ID NO: 1.

[0034] In one example, the mismatch is not at a terminus of the antisense oligonucleotide.

[0035] In one example, the nucleotide sequence of the ASO is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the nucleotide sequence of the targeted portion over the length of the ASO.

[0036] The present disclosure provides an antisense oligonucleotide comprising a sequence set forth in any one of SEQ ID NOs: 2505-2517 or a sequence having at least about 50% or 60% or 70% or 80% or 95% identity thereto, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

[0037] The present disclosure provides an antisense oligonucleotide comprising a sequence set forth in any one of SEQ ID NOs: 2505-2517 or a sequence having at least about 50% or 60% or 70% or 80% or 95% identity thereto, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP), wherein the CPP consists of a sequence set forth in SEQ ID NO: 2504.

[0038] The present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 2505-2517 or a sequence having at least about 80% identity thereto, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

[0039] The present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 2505-2517 or a sequence having at least about 80% identity thereto, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP), wherein the CPP consists of a sequence set forth in SEQ ID NO: 2504.

[0040] In one example, the ASO comprises or consists of a sequence set forth in SEQ ID NO: 2505.

[0041] In one example, the ASO comprises or consists of a sequence set forth in SEQ ID NO: 2506.

[0042] In one example, the ASO comprises or consists of a sequence set forth in SEQ ID NO: 2507.

[0043] In one example, the ASO comprises or consists of a sequence set forth in SEQ ID NO: 2508.

[0044] In one example, the ASO comprises or consists of a sequence set forth in SEQ ID NO: 2509.

[0045] In one example, the ASO comprises or consists of a sequence set forth in SEQ ID NO: 2510.

[0046] In one example, the ASO comprises or consists of a sequence set forth in SEQ ID NO: 2511.

[0047] In one example, the ASO comprises or consists of a sequence set forth in SEQ ID NO: 2512.

[0048] In one example, the ASO comprises or consists of a sequence set forth in SEQ ID NO: 2513.

[0049] In one example, the ASO comprises or consists of a sequence set forth in SEQ ID NO: 2514.

[0050] In one example, the ASO comprises or consists of a sequence set forth in SEQ ID NO: 2515.

[0051] In one example, the ASO comprises or consists of a sequence set forth in SEQ ID NO: 2516. In one example, the ASO comprises or consists of a sequence set forth in SEQ ID NO: 2517.

[0052] The present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 2512 linked to a cell-penetrating peptide (CPP), wherein the CPP consists of a sequence set forth in SEQ ID NO: 2504.

[0053] In one example, the ASO binds to intron 7 of an OPA1 gene pre-mRNA in a cell and increases the level of OPA1 gene transcripts encoding full length, functional OPA1 by exclusion of NMD exon 7x.

[0054] The present disclosure further provides an antisense oligonucleotide that binds to a targeted portion of the intron 7x of an OPA1 gene transcript in a cell and increases the level of OPA1 gene transcripts encoding full length, functional OPA1 by exclusion of NMD exon 7x, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

[0055] The present disclosure further provides an antisense oligonucleotide that binds to a targeted portion of the intron 7x of an OPA1 gene transcript in a cell and increases the level of OPA1 gene transcripts encoding full length, functional OPA1 by exclusion of NMD exon 7x, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP), wherein the CPP consists of a sequence set forth in SEQ ID NO: 2504.

[0056] For example, the ASO is within sufficient proximity to an acceptor site of exon 7x to promote exclusion of exon 7x in splicing of OP Al mRNA.

[0057] In one example, the ASO that binds to a targeted portion of intron 7 OP Al pre- mRNA comprises or consists of any one of SEQ ID NOs: 2-54. For example, the ASO comprises or consists of any one of SEQ ID NOs: 2-54.

[0058] In one example, the ASO that binds to a targeted portion of intron 7 OP Al pre- mRNA comprises or consists of any one of SEQ ID NOs: 2-54 or SEQ ID NOs: 2491- 2499 or SEQ ID NOs: 2505-2517. In one example, the ASO comprises or consists of any one of SEQ ID NOs: 2-54 or SEQ ID NOs: 2491-2499 or SEQ ID NOs: 2505-2517.

[0059] In one example, the ASO that binds to a targeted portion of intron 7 OP Al pre- mRNA comprises or consists of any one of SEQ ID NOs: 2491-2499. In another example, the ASO comprises or consists of any one of SEQ ID NOs: 2491-2499.

[0060] In one example, the ASO that binds to a targeted portion of intron 7 OP Al pre- mRNA comprises or consists of any one of SEQ ID NOs: 2505-2517. In another example, the ASO comprises or consists of any one of SEQ ID NOs: 2505-2517.

[0061] In one example, the ASO binds to a targeted portion of the 5' UTR of an OPA1 gene transcript in a cell to increase translation efficiency or transcript stability of an OPA1 mRNA. For example, the ASO increases expression of OPA1 protein. Without being bound by theory or mode of action, such ASOs may sterically inhibit translation from upstream Open Reading Frames (uORF) start site and / or sterically inhibit secondary structure in the 5' UTR and / or inhibiting the activity of a decapping enzyme.

[0062] In one example, the ASO binds within a targeted portion of the 5' UTR of OPA1 mRNA, wherein the targeted portion is within the nucleotide sequence corresponding to SEQ ID NO: 55.

[0063] The present disclosure also provides an antisense oligonucleotide that binds to a targeted portion of the 5' UTR of an OPA1 gene transcript in a cell and increases transcript stability of an OPA1 mRNA, wherein the targeted portion is within the nucleotide sequence corresponding to SEQ ID NO: 55, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

[0064] The present disclosure also provides an antisense oligonucleotide that binds to a targeted portion of the 5' UTR of an OPA1 gene transcript in a cell and increases transcript stability of an OPA1 mRNA, wherein the targeted portion is within the nucleotide sequence corresponding to SEQ ID NO: 55, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP), wherein the CPP consists of a sequence set forth in SEQ ID NO: 2504.

[0065] In one example, the ASO that binds to a targeted portion of the 5' UTR of OPA1 mRNA comprises or consists of any one of SEQ ID NOs: 56-138 or SEQ ID NOs: 2500- 2503. For example, the ASO comprises or consists of any one of SEQ ID NOs: 56-138. In another example, the ASO comprises or consists of any one of SEQ ID NOs: 2500- 2503. In a further example, the ASO comprises or consists of any one of SEQ ID NOs: 56-138 or 2500-2503.

[0066] In one example, the ASO that binds to a targeted portion of the 5' UTR of OPA1 mRNA comprises or consists of SEQ ID NO: 112. For example, the ASO comprises or consists of SEQ ID NO: 112.

[0067] The present disclosure provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 112, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

[0068] The present disclosure also provides an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 112, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP), wherein the CPP consists of a sequence set forth in SEQ ID NO: 2504. The present disclosure provides a pharmaceutical composition comprising an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 112, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

[0069] The present disclosure further provides a pharmaceutical composition comprising an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 112, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP), wherein the CPP consists of a sequence set forth in SEQ ID NO: 2504.

[0070] In one example, the ASO binds to a targeted portion of the 3' UTR of an OPA1 gene transcript in a cell to increase transcript stability of an OPA1 mRNA. For example, the ASO increases expression of OPA1 protein. Without being bound by theory or mode of action, such ASOs may sterically inhibit binding of a miRNA to the 3' UTR.

[0071] The present disclosure provides an antisense oligonucleotide that binds to a targeted portion of the 3' UTR of an OPA1 gene transcript in a cell and increases transcript stability of an OPA1 mRNA, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

[0072] The present disclosure provides an antisense oligonucleotide that binds to a targeted portion of the 3' UTR of an OPA1 gene transcript in a cell and increases transcript stability of an OPA1 mRNA, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP), wherein the CPP consists of a sequence set forth in SEQ ID NO: 2504.

[0073] In one example, the ASO binds within a targeted portion of the 3' UTR of OP Al mRNA, wherein the targeted portion is within the nucleotide sequence corresponding to SEQ ID NO: 139.

[0074] The present disclosure provides an antisense oligonucleotide that binds to a targeted portion of the 3' UTR of an OPA1 gene transcript in a cell and increases transcript stability of an OPA1 mRNA, wherein the targeted portion is within the nucleotide sequence corresponding to SEQ ID NO: 139, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

[0075] The present disclosure also provides an antisense oligonucleotide that binds to a targeted portion of the 3' UTR of an OPA1 gene transcript in a cell and increases transcript stability of an OPA1 mRNA, wherein the targeted portion is within the nucleotide sequence corresponding to SEQ ID NO: 139, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP), wherein the CPP consists of a sequence set forth in SEQ ID NO: 2504. In one example, the ASO that binds to a targeted portion of the 3' UTR of OP Al mRNA comprises or consists of any one of SEQ ID NOs: 140-2488.

[0076] In one example, the nucleotide sequence of the ASO is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the nucleotide sequence of the targeted portion over the length of the ASO.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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 orNl-methyl-pseudouridine). 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 phosphorothioate linkage or a phosphorodiamidate linkage. In one example, the ASO comprises a phosphorothioate linkage. In another example, the ASO comprises a phosphorodiamidate linkage.

[0081] In one example, the ASO comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a bridged nucleic acid, a 2'-( -methyl, a 2'-Fluoro, or a 2'-(9-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'-(9-methyl moiety. In one example, the ASO comprises a 2'-Fluoro moiety. In another example, the ASO comprises a -O- methoxyethyl moiety. In a further example, the ASO comprises a bridged nucleic acid.

[0082] In one example, the ASO comprises at least one modified sugar moiety. In one example, one or more sugar moieties in the antisense oligonucleotide is a modified sugar moiety. For example, each sugar moiety in the antisense oligonucleotide is a modified sugar moiety.

[0083] In one example, the ASO comprises a 2'-(9-methoxy ethyl moiety. In one example, one or more nucleotides of the ASO comprises a 2'-(9-methoxyethyl moiety. For example, each nucleotide of the ASO comprises a 2'-(9-methoxy ethyl moiety.

[0084] In one example, the ASO comprises one or more phosphorodiamidate morpholino moieties.

[0085] In one example of any composition described herein, the CPP can be covalently linked or non-covalently linked to the ASO. The CPP can be at the 5' end and / or 3' end of the ASO. In one example, the CPP is at the 3' end of the ASO. For example, the C terminus of the CPP is at the 3' end of the ASO. In one example, the carboxylic acid C terminus of the CPP is linked to the 3' end of the ASO.

[0086] 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.

[0087] 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.

[0088] The present disclosure further provides a pharmaceutical composition comprising the antisense oligonucleotide of the present disclosure and a pharmaceutically acceptable excipient. The present disclosure additionally provides a method of treating a condition, the method comprising administering an antisense oligonucleotide or pharmaceutical composition of the disclosure. In one example, the condition is associated with OPA1 expression, e.g., reduced OPA1 expression. In one example, the condition is an ocular condition. For example, the ocular condition is ADOA, glaucoma, geographic atrophy, acute ischemic optic neuritis or acute ischemic optic neuropathy. In one example, the condition is glaucoma. In another example, the condition is autosomal dominant optic atrophy. In a further example, the condition is geographic atrophy. In one example, the condition is acute ischemic optic neuritis. In another example, the condition is acute ischemic optic neuropathy.

[0089] BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 is a series of graphical representations showing the binding sites for oligonucleotides to mediate increasing OPA1 protein levels. (A) Schematic illustration of exon structure of OPA1 (Transcript ID: ENST00000361510), indicating start and stop codons and the regions of 5' UTR, NMD exon 7x (in case of unspliced) and 3' UTR. (B) Prediction for secondary structure of the 5' UTR of OPA1 transcript (corresponding to SEQ ID NO: 55) using RNAfold web tool (http: / / rna.tbi.univie.ac.at / cgi- bin / RNAWebSuite / RNAfold.cgi). The free energy of the thermodynamic ensemble is - 137.26 kcal / mol. Oligonucleotides SEQ ID NOs: 56-138, 2500-2503 were designed, which target start codons of upstream open reading frames (uORFs), regulatory binding sites, inhibitory 5' UTR secondary structures and / or G-quadruplexes. (C) Exon 7x (black box) containing the premature termination codon (PTC), is located between exons 7 and 8 (not drawn to scale). PMOs (SEQ ID NOs: 2-54, 2491-2499, 2505-2512) were designed to target splicing regulatory elements within intron 7 (dash line, corresponding to SEQ ID NO: 1) to mediate exclusion of exon 7x during pre-mRNA splicing to increase productive OP Al transcript. (D) Schematic illustration of the 3' UTR (not drawn to scale) located in exon 31. Oligonucleotides (SEQ ID NOs: 140-2488) were designed to hybridize with the transcript and mask / inhibit binding of miRNA(s) to prevent mRNA degradation and increase in OPA1 protein levels.

[0091] Figure 2 shows the effect of a PMO (SEQ ID NO: 2512) targeting exon 7x exclusion in ADOA derived iPSC-RGCs harboring an OPA1 c.2708 271 IdelTTAG mutation, conjugated to a CPP (SEQ ID NO: 2504). Patient iPSC-RGCs were transfected in triplicates for 5 days with the CPP -PMO targeting removal of the OPA1 exon 7x as indicated. OPA1 transcript expression was assessed by ddPCR and normalised to the HPRT1 transcript level. The OPA1 expression in untreated cells was set to 1. A non-targeting control PMO, conjugated with the same CPP, was employed as a negative control. Statistical analysis was conducted using Two-Way ANOVA. The CPP -PMO targeting exon 7x showed a significant upregulation of OPA1 mRNA in iPSC-RGCs derived from an ADOA patient in a dose dependent manner.

[0092] Figure 3 shows the effect of a PMO (SEQ ID NO: 2512) targeting exon 7x exclusion in ADOA derived iPSC-RGCs to increase OPA1 protein, conjugated to a CPP (SEQ ID NO: 2504) Patient iPSC-RGCs were incubated at indicated concentrations in triplicates for 5-7 days with CPP -PMO targeting removal of the OPA1 exon 7x as indicated. OPA1 transcript expression was assessed by ddPCR and normalised to the HPRT1 transcript level. The OPA1 expression in untreated cells was set to 1. A nontargeting control PMO, conjugated with the same CPP, was employed as a negative control. Statistical analysis was conducted using Student’s / -test. The CPP -PMO targeting exon 7x showed a significant upregulation of OPA1 protein in multiple ADOA patient iPSC-RGCs. Patient#l harboring OPA1 c.985-lG>C mutation, Patient #2 harboring OPA1 c.2608delA mutation and Patient#4 harboring OPA1 c.2708_271 IdelTTAG mutation.

[0093] DETAILED DESCRIPTION

[0094] General

[0095] 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 (i.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.

[0096] Those skilled in the art will appreciate that the present disclosure 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 present disclosure.

[0097] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive).

[0098] Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.

[0099] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology).

[0100] Unless otherwise indicated, the conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0101] 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.

[0102] 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).

[0103] 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.

[0104] Selected Definitions

[0105] 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, but may or may 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.

[0106] 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 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.

[0107] The term “nonsense-mediated RNA decay-inducing (NMD) exon” or “NMD exon” refers to an exon or a pseudo-exon that is a region within an intron and can activate the NMD pathway if included in a mature RNA transcript. In the constitutive splicing events, the intron containing an NMD exon is usually spliced out, but the intron or a portion of it can be retained during alternative or aberrant splicing events. Mature mRNA transcripts containing such an NMD exon can be non-productive due to a frame shift which induces the NMD pathway. Inclusion of an NMD exon in mature OPA1 RNA transcripts can downregulate overall OPA1 mRNA and OPA1 protein expression.

[0108] The term “precursor mRNA” or “pre-mRNA” refers to the primary transcript and is the single-stranded RNA product synthesized by transcription of the genomic DNA sequence of the transcription unit for a particular gene, which generally encompasses the nucleotide sequence between a transcription start site and a termination signal.

[0109] 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.

[0110] 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.

[0111] 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 (Om), 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.

[0112] 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 Bioinformatics 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.

[0113] 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.

[0114] 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.

[0115] Cell Penetrating Peptides (CPP)

[0116] The present disclosure provides a CPP comprising a sequence set forth in SEQ ID NO: 2504. The present disclosure further provides a CPP, wherein the CPP consists of a sequence set forth in SEQ ID NO: 2504.

[0117] The present disclosure provides a CPP comprising a sequence RASARRASARRANARRANARRSGGR (SEQ ID NO: 2504).

[0118] The present disclosure also provides a CPP, wherein the CPP consists of a sequence RASARRASARRANARRANARRSGGR (SEQ ID NO: 2504).

[0119] 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.

[0120] In some examples the amino acid sequence of a CPP provided herein includes at least one D-amino acid. In some examples the amino acid sequence of a CPP includes only D-amino acids. For example, the amino acid sequence of the CPP is D-arginyl-D- alanyl-D-seryl-D-alanyl-D-arginyl-D-arginyl-D-alanyl-D-seryl-D-alanyl-D-arginyl-D- arginyl-D-alanyl-D-asparagyl-D-alanyl-D-arginyl-D-arginyl-D-alanyl-D-asparagyl-D- alanyl-D-arginyl-D-arginyl-D-seryl-glycyl-glycyl-D-arginyl. In other examples, a CPP includes at least one L-amino acid. In some examples, the CPP includes only L-amino acids. In some examples the amino acid sequence of a CPP provided herein is the retro- inverso sequence of any of the CPP amino acid sequence enumerated herein.

[0121] In one example, the CPP consists of a sequence D-arginyl-D-alanyl-D-seryl-D- alanyl-D-arginyl-D-arginyl-D-alanyl-D-seryl-D-alanyl-D-arginyl-D-arginyl-D-alanyl- D-asparagyl-D-alanyl-D-arginyl-D-arginyl-D-alanyl-D-asparagyl-D-alanyl-D-arginyl- D-arginyl-D-seryl-glycyl-glycyl-D-arginyl.

[0122] A CPP may direct a molecule of interest, such as an antisense oligonucleotide 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.

[0123] Modified CPPs

[0124] In some examples, the CPP is a modified CPP. In some examples, a CPP provided herein is a modified CPP by virtue of comprising a moiety other than a canonical amino acid. Such modifications include, but are not limited to, non-peptide linkers, and detectable labels, as described in further detail herein. In some examples a CPP is a modified CPP comprising a moiety other than a canonical amino acid. Such modified CPPs may confer additional functionalities to a CPP, such as facilitating detection of CPP entry, localisation within cells, enhanced cell entry, and / or reduced CPP degradation in vitro or in vivo. Suitable moieties for a modified CPP include, but are not limited to, any moiety selected from the group consisting of: a detectable label, a non-canonical amino acid, a reactive group, a fatty acid, cholesterol, a bioactive carbohydrate, a lipid, a nanoparticle, a small molecule drug, and a polynucleotide. In some examples the moiety in a modified CPP is a detectable label.

[0125] The term “detectable label” refers to any type of molecule which can be detected by optical, fluorescent, isotopic imaging or by mass spectroscopic techniques, or by performing simple enzymatic assays. Any detectable label known in the art may be used. In some examples the detectable label is selected from among a fluorophore, a fluorogenic substrate, a luminogenic substrate, and a biotin.

[0126] A fluorescent tag may be a fluorophore. For example, a fluorophore may be fluorescein isothiocyanate, fluorescein thiosemicarbazide, rhodamine, Texas Red, a CyDye such as Cy3, Cy5 and Cy5.5, a Alexa Fluor such as Alexa488, Alexa555, Alexa594 and Alexa647) or a near infrared fluorescent dye. A fluorophore may be a pH-sensitive fluorescent probe. For example, a pH-sensitive fluorescent probe may be naphthofluorescein, A fluorescent tag may be a fluorescent protein. For example, a fluorescent protein may be green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), AcGFP or TurboGFP, Emerald, Azami Green, ZsGreen, EBFP, Sapphire, T-Sapphire, ECFP, mCFP, Cerulean, CyPet, AmCyanl, Midori -Ishi Cyan, mTFPl (Teal), enhanced yellow fluorescent protein (EYFP), Topaz, Venus, mCitrine, YPet, PhiYFP, ZsYellowl, mBanana, Kusabira,ange, mOrange, dTomato, dTomato- Tandem, AsRed2, mRFPl, Jred, mCherry, HcRedl, mRaspberry, HcRedl, HcRed- Tandem, mPlum, AQ 143. A fluorescent tag may be a quantum dot. In some examples, where the detectable label is a fluorophore, the fluorophore is a pH-sensitive fluorescent probe. Suitable pH-sensitive fluorescent probes include, but are not limited to, naphthofluorescein, pHrodo® Green (ThermoFisher), and pHrodo® Red (ThermoFisher). Fluorescent tags may be detected using fluorescent microscopes such as epifluorescence or confocal microscopes, fluorescence scanners such as microarray readers, spectrofluorometers, microplate readers and / or flow cytometers.

[0127] In some examples the detectable label is a fluorogenic substrate. Suitable fluorogenic substrates include fluorogenic substrates of P lactamase (e.g, CCF-2-AM, CCF4-AM, and any of those described in U.S. Patent No. 7,427,680) and P-gal (e.g, HMRef-PGal described in Asanuma et al 2015, Nature Comm., 6:6463).

[0128] In some examples the detectable label is a luminogenic substrate. Suitable luminogenic substrates include, but are not limited to, D-Luciferin, L-Luciferin, Coelenterazine,

[0129] An epitope tag may be a poly-histidine tag such as a hexahistidine tag or a dodecahistidine, a FLAG tag, a Myc tag, a HA tag, a GST tag or a V5 tag. Epitope tags are routinely detected with commercially available antibodies. A person skilled in the art will be aware that an epitope tag may facilitate purification and / or detection. For example, a conjugate containing a hexahistidine tag may be purified using methods known in the art, such as, by contacting a sample comprising the protein with nickelnitrilotriacetic acid (Ni-NTA) that specifically binds a hexahistidine tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. Alternatively, or in addition a ligand or antibody that binds to an epitope tag may be used in an affinity purification method.

[0130] An isobaric tag may be a mass tag or an isobaric tag for relative absolute quantification (iTRAQ). A mass tag is a chemical label used for mass spectrometry based quantification of proteins and peptides. In such methods mass spectrometers recognise the mass difference between the labeled and unlabeled forms of a protein or peptide, and quantification is achieved by comparing their respective signal intensities as described, for example, in Bantscheff et al. 2007. Examples of mass tags include TMTzero, TMTduplex, TMTsixplex and TMT 10-plex. An isobaric tag for relative absolute quantification (iTRAQ) is a chemical tag used in quantitative proteomics by tandem mass spectrometry to determine the amount of proteins from different sources in a single experiment as described, for example, in Wiese et al. 2007.

[0131] In some examples the moiety is a non-canonical amino acid. Suitable non- canonical amino acids include, but are not limited to, ornithine, citrulline (Cit), diaminobutyric acid (Dab), diaminopropionic acid (Dap), 2-Aminoisobutyric acid a Amino-n-butyric acid, Norvaline, Norleucine, Alloisoleucine, t-leucine, Ornithine, Allothreonine, P-Alanine, P -Amino-n-butyric acid, N-isopropyl glycine, Isoserine, and Sarcosine.

[0132] In other examples a moiety in a modified CPP is a reactive group. Suitable reactive groups include, but are not limited to, azide groups, amine-reactive groups, thiolreactive groups, and carbonyl -reactive groups. In some examples the reactive groups are part of a chemical tag. Suitable chemical tags include, but are not limited to, a SNAP tag, a CLIP tag, a HaloTag or a TMP-tag. In one example, the chemical tag is a SNAP- tag or a CLIP -tag. SNAP and CLIP fusion proteins enable the specific, covalent attachment of virtually any molecule to a protein or peptide of interest as described, for example, in Correa 2015 (Methods Mol Biol, 1266:55-79). In another example, the chemical tag is a HaloTag. HaloTag involves a modular protein tagging system that allows different molecules to be covalently linked, either in solution, in living cells, or in chemically fixed cells. In another example, the chemical tag is a TMP-tag. TMP-tags are able to label intracellular, as opposed to cell-surface, proteins with high selectivity.

[0133] In some examples the moiety in a modified CPP is a fatty acid. Suitable fatty acids for modified peptides include, but are not limited to, palmitic acid, myristic acid, caprylic acid, lauric acid, n-octanoic acid, and n-decanoic acid.

[0134] In other examples the moiety in a modified CPP is cholesterol.

[0135] CPP Conjugates

[0136] The term “CPP conjugate,” as used herein, refers to a CPP that is linked (covalently or non-covalently) to a polynucleotide as described herein.

[0137] In some examples, a CPP conjugate protein comprises a flexible linker linking the CPP and a heterologous amino acid sequence such a peptide or protein. Examples of flexible linkers include, but are not limited to, GGGGS (SEQ ID NO: 2518), GGGGSGGGGS (SEQ ID NO: 2519), GAS, GGG, GSG, GTG, GGTAGSTGG (SEQ ID NO: 2520), GASGGASG (SEQ ID NO: 2521) and GASG (SEQ ID NO: 2522). Other examples of such flexible linkers are known in the art as described in, e.g., Chen et al (2013), Adv Drug Deliv Rev., 65(10): 1357-1369. In one example, the amino acid sequence of the linker only includes D-amino acids.

[0138] In one example, the carboxylic acid C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide via a linker.

[0139] The present disclosure provides a CPP conjugate comprising a sequence set forth in SEQ ID NO: 2504 linked to an antisense oligonucleotide that modulates mRNA productive transcript, stability and / or translation of OPA1 gene transcript or part thereof, The present disclosure also provides a CPP conjugate comprising a sequence set forth in SEQ ID NO: 2504, linked to an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 2512.

[0140] The present disclosure further provides a CPP conjugate comprising a sequence set forth in SEQ ID NO: 2504, linked to an antisense oligonucleotide that binds to a targeted portion of the intron 7x of an OP Al gene transcript in a cell and increases the level of OPA1 gene transcripts encoding full length, functional OPA1 by exclusion of NMD exon 7x.

[0141] The present disclosure provides a CPP conjugate comprising a sequence set forth in SEQ ID NO: 2504, linked to an antisense oligonucleotide that binds to a targeted portion of the 5' UTR of an OPA1 gene transcript in a cell and increases transcript stability of an OPA1 mRNA, wherein the targeted portion is within the nucleotide sequence corresponding to SEQ ID NO: 55.

[0142] The present disclosure also provides a CPP conjugate comprising a sequence set forth in SEQ ID NO: 2504, linked to an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 112.

[0143] The present disclosure provides a CPP conjugate comprising or consisting of a CPP consisting of a sequence RASARRASARRANARRANARRSGGR (SEQ ID NO: 2504), a GASGGASG (SEQ ID NO: 2521) linker and an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 112.

[0144] The present disclosure provides a CPP conjugate consisting of:

[0145] (i) a CPP consisting of a sequence set forth in SEQ ID NO: 2504

[0146] (ii) a linker consisting of a sequence set forth in SEQ ID NO: 2521; and

[0147] (iii)an antisense oligonucleotide consisting of a sequence set forth in SEQ ID NO: H2, wherein the C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide via the linker.

[0148] The present disclosure provides a CPP conjugate comprising a CPP consisting of a sequence set forth in SEQ ID NO: 2504, and an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 112, wherein the C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide via a linker. For example, the linker comprises or consists of a sequence set forth in SEQ ID NO: 2521. In one example, the amino acid sequence of the linker only includes D-amino acids. For example, the linker comprises or consists of the sequence glycyl-D-alanyl-D-seryl-glycyl-glycyl-D- alanyl-D-seryl-glycyl (SEQ ID NO: 2521). In one example, the C-terminus of the CPP is linked to the secondary amino at the 3' end of the antisense oligonucleotide via a linker. For example, the carboxylic acid C-terminus of the CPP is linked to the secondary amino at the 3' end of the antisense oligonucleotide via a linker.

[0149] The present disclosure further provides a CPP conjugate consisting of:

[0150] (i) a CPP consisting of a sequence set forth in SEQ ID NO: 2504, wherein the amino acid sequence of the CPP includes only D-amino acids;

[0151] (ii) a linker consisting of a sequence set forth in SEQ ID NO: 2521; and

[0152] (iii)an antisense oligonucleotide consisting of a sequence set forth in SEQ ID NO: H2, wherein the C-terminus of the CPP is linked to the 3' end of the antisense oligonucleotide via the linker.

[0153] The present disclosure further provides a CPP conjugate comprising a sequence set forth in SEQ ID NO: 2504, linked to an antisense oligonucleotide that binds to a targeted portion of the 3' UTR of an OPA1 gene transcript in a cell and increases transcript stability of an OP Al mRNA.

[0154] Agents for Increasing OP Al Protein Levels

[0155] OPA1 mitochondrial dynamin like GTPase gene (also known as OPA1, FLJ12460, KIAA0567, MGM1, NPG andNTG; referred to herein as OPA1) is composed of 30 coding exons distributed across more than 90 kb of genomic DNA. It is located on chromosome 3q29 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, or SEQ ID NO: 2489, and UniProt ID 060313, or SEQ ID NO: 2490.

[0156] 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 OP Al 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.

[0157] 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.

[0158] 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.

[0159] Also described herein, the ASOs according to any example bind to the 5' UTR or 3' UTR of OPA1 mRNA and increase expression of OPA1 protein.

[0160] Without being bound by theory or mode of action, the ASOs that bind to the 5' UTR are thought to increase expression of OP Al protein through steric inhibition of translation from upstream Open Reading Frames (uORF) start site and / or steric inhibition of secondary structure in the UTR and / or inhibiting the binding and / or activity of a decapping enzyme.

[0161] Also described herein, the ASOs according to any example bind to the 3' UTR or 3' UTR of OPA1 mRNA and increase expression of OPA1 protein. Without being bound by theory or mode of action, such ASOs may sterically inhibit binding of a miRNA to the 3' UTR.

[0162] Antisense Oligonucleotides (ASOs)

[0163] In some examples of the ASOs, 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.

[0164] In some examples, ASOs “specifically hybridize” to or are “specific” to a target nucleic acid or a targeted portion of the OPA1 mRNA 5' UTR. 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 Tmis the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.

[0165] 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. 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 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).

[0166] 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 5' UTR region of a mRNA or over one or more segments of intron 7 of the OP Al 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).

[0167] In some examples the nucleotide sequences of ASOs described herein are complementary to a targeted portion of OPA1 mRNA 5' UTR. For example, the ASOs are complementary to a targeted portion of the 5' UTR of an OPA1 mRNA corresponding to SEQ ID NO: 55. In some examples, the ASOs are complementary to a targeted portion of an OPA1 mRNA corresponding to SEQ ID NO:55, which encompasses the 5' UTR. In some examples the nucleotide sequence of the ASO is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the nucleotide sequence of the targeted portion of the OPA1 5' UTR over the length of the ASO.

[0168] In some examples, the nucleotide sequences of ASOs that bind to targeted portions of the 5' UTR of OPA1 mRNA comprise or consist of any one of SEQ ID NOs: 56-138.

[0169] In some examples the nucleotide sequences of ASOs described herein are complementary to a targeted portion of intron 7 of the OP Al 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: 2-54. 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- 54 or SEQ ID NOs: 2491-2499 or SEQ ID NOs: 2505-2517 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: 2491-2499 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: 2505-2517. In some examples the nucleotide sequence of the ASO is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the nucleotide sequence of the targeted portion of intron 7 of the OPA1 pre-mRNA over the length of the ASO.

[0170] In some examples the nucleotide sequences of ASOs described herein are complementary to a targeted portion of OPA1 mRNA 3' UTR. For example, the ASOs are complementary to a targeted portion of the 3' UTR of an OP Al mRNA corresponding to SEQ ID NO: 139. In some examples, the ASOs are complementary to a targeted portion of an OPA1 mRNA corresponding to SEQ ID NO: 139, which encompasses the 3' UTR. In some examples the nucleotide sequence of the ASO is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the nucleotide sequence of the targeted portion of the OPA1 3' UTR over the length of the ASO.

[0171] In some examples the nucleotide sequences of ASOs described herein are complementary to a targeted portion of intron 7 of the OP Al 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: 2505-2517. 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: 2505-517.

[0172] 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 an intron 7 of the OP Al 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).

[0173] 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,

[0174] 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,

[0175] 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.

[0176] In some examples, the ASO nucleotide sequence is from 8 to 50 nucleotides, 8 to

[0177] 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, 10 to 25 nucleotides, 10 to 20 nucleotides, 10 to 15 nucleotides, 11 to 50 nucleotides, 11 to 40 nucleotides, 11 to 35 nucleotides, 11 to 30 nucleotides, 11 to 25 nucleotides, 11 to 20 nucleotides, 11 to 15 nucleotides, 12 to 50 nucleotides, 12 to 40 nucleotides, 12 to 35 nucleotides, 12 to 30 nucleotides, 12 to 25 nucleotides, 12 to 20 nucleotides, 12 to 15 nucleotides, 13 to 50 nucleotides, 13 to 40 nucleotides, 13 to 35 nucleotides, 13 to 30 nucleotides, 13 to 25 nucleotides, 13 to 20 nucleotides, 14 to 50 nucleotides, 14 to 40 nucleotides, 14 to 35 nucleotides, 14 to 30 nucleotides, 14 to 25 nucleotides, 14 to 20 nucleotides, 15 to 50 nucleotides, 15 to 40 nucleotides, 15 to 35 nucleotides, 15 to 30 nucleotides, 15 to 25 nucleotides, 15 to 20 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 examples, the ASOs are 17 nucleotides in length. In some preferred examples, the nucleotide sequence of the ASO nucleotide is 25 nucleotides in length.

[0178] ASO Chemistry and Modifications The ASOs 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.

[0179] 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 hydroxymethoylcytosine.

[0180] 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 et 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.

[0181] In some examples, the stereochemistry at each of the phosphorus intemucleotide linkages of the ASO backbone is random. In other examples, the stereochemistry at each of the phosphorus intemucleotide 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%.

[0182] 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.

[0183] 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), 2'-O- methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F, N3'->P5' phosphoramidate,

[0184] 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O- guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. In some examples, the sugar moiety modification is selected from among 2'-(9-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 (LNA). 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 BNA 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-N- 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 phosphorothioate linkage, or each ribose sugar moiety comprises a 2'-(9-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).

[0185] 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 phosphorothioate backbone. In some examples, the ASO comprises a peptide nucleic acid (PNA).

[0186] In some examples, the ASO comprises a phosphorodiamidate morpholino (PMO).

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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 the OPA1 mRNA 5' UTR. In other examples, two or more ASOs that are complementary to different targeted portions of the OPA1 mRNA 5' UTR.

[0191] 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 OP Al pre-mRNA. 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 the OPA1 mRNA 3' UTR. In other examples, two or more ASOs that are complementary to different targeted portions of the OP Al mRNA 3' UTR.

[0192] The present disclosure provides an ASO described herein linked to a cellpenetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

[0193] The present disclosure provides an ASO described herein linked to a CPP comprising the sequence RASARRASARRANARRANARRSGGR (SEQ ID NO: 2504).

[0194] The present disclosure also provides an ASO described herein linked to a CPP, wherein the CPP consists of the sequence RASARRASARRANARRANARRSGGR (SEQ ID NO: 2504).

[0195] In some examples, the CPP is covalently linked to the ASO. In other examples, the CPP is non-covalently linked to the ASO.

[0196] The CPP of the present disclosure 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 CPP is linked to the 5' end of the ASO. In other examples, the CPP is linked to the 3' end of the ASO. For example, the C terminus of the CPP is linked to the 3' end of the ASO. In further examples, the CPP is linked to the 5' end and the 3' of the ASO.

[0197] 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.

[0198] In one example, the ASO is formulated with at least a pharmaceutically acceptable excipient, including a carrier, filler, preservative, adjuvant, solubilizer and / or diluent.

[0199] 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.

[0200] 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, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemi sulfate, 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.

[0201] Exemplary salts useful in a composition of the present disclosure include calcium chloride, magnesium chloride or sodium chloride.

[0202] In one example, a composition comprises a buffer. Exemplary buffers useful in a composition of the present disclosure include sodium phosphate.

[0203] 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 carboxymethylcellulose, 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).

[0204] 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.

[0205] 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 an ocular condition.

[0206] Methods o f Treating or Preventing Ocular Conditions

[0207] 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 an ASO of the present disclosure or 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.

[0208] In one example, the subject to be treated is suffering from an ocular condition, such as ADOA, glaucoma, geographic atrophy, acute ischemic optic neuritis or acute ischemic optic neuropathy. For example, the subject has been diagnosed as having or suffering from an ocular condition, such as ADOA, glaucoma, geographic atrophy, acute ischemic optic neuritis or acute ischemic optic neuropathy. In one example, the subject suffers from an ocular condition, such as ADOA, glaucoma, geographic atrophy, acute ischemic optic neuritis or acute ischemic optic neuropathy. 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, glaucoma, geographic atrophy, acute ischemic optic neuritis or acute ischemic optic neuropathy.

[0209] In one example, administration of an ASO or pharmaceutical composition as described herein slows progression of an ocular condition, such as ADOA, glaucoma, geographic atrophy, acute ischemic optic neuritis or acute ischemic optic neuropathy.

[0210] In one example, the subject is at risk of developing an ocular condition, such as ADOA, glaucoma, geographic atrophy, acute ischemic optic neuritis or acute ischemic optic neuropathy. Such subjects can be administered the ASOs or pharmaceutical composition as described here to prevent onset of an ocular condition, such as ADOA, glaucoma, geographic atrophy, acute ischemic optic neuritis or acute ischemic optic neuropathy. As used herein, the term “at risk” means that the subject has an increased chance of developing an ocular condition, compared to a normal individual. Subjects can be identified as at risk of developing an ocular condition 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 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.

[0211] Also provided herein is a method for increasing the OPA1 transcript or OPA1 protein in a cell, 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. Also provided herein is a method for increasing the level of OPA1 transcript or OPA1 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] As the skilled person will understand, the treatment methods disclosed herein include administration of the ASO or 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 pathological inflammation in 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.

[0216] Combination Therapies

[0217] The ASOs described herein or 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 an ocular condition, such as ADO A, glaucoma, geographic atrophy, acute ischemic optic neuritis or acute ischemic optic neuropathy. 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.

[0218] ASOs 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 ocular condition 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 ocular condition being treated and the condition of the patient.

[0219] 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.

[0220] 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.

[0221] 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, bioavailability, 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®)).

[0222] The present disclosure is not to be limited by the following non-limiting examples.

[0223] EXAMPLES

[0224] Example 1: ASO design to target exclusion of an OPA1 NMD exon 7x

[0225] ASOs with 24-25 nucleotides in length (Table 1, SEQ ID NOs: 2-9) were designed to target the intronic splice enhancer motifs (prediction using SpliceAid online tool) in intron 7 to mediate exclusion of exon 7x and generate productive OPA1 transcripts. The identified ASO sequences are synthesized as PMOs and / or 2'MOE chemistry) and nucleofected into HEK293 cells or ADOA patient fibroblasts carrying the OPA1 mutation (c.2708_2711delTTAG) using the NEON® electroporation system (ThermoFisher) at 25 pM and 50 pM and the nucleofected cells are cultured for 48 hr. Total RNA was extracted using the MagMAX™- 96 Total RNA Isolation kit and the level of OPA1 transcript is assessed by digital droplet PCR (Qiagen; probe catalogue number: dHsaCPE5043545). OPA1 transcript expression is normalized to GAPDH, PPL27 and SCL25A3 transcript levels (Qiagen; probe catalogue number: dHsaCPE5031596, dHsaCPE5036407, dHsaCPE5032926 respectively). PMOs that show induced OPA1 mRNA levels were further validated for the ability to increase OPA1 protein upregulation using a western blot assay. Further refinement of ASO sequences (Table 1; SEQ ID NOs: 10-31 and Table 2; SEQ ID NOs: 32-54) is performed to reduce or extend ASO length and micro-walk or engineered mismatch oligos and re-validated by ddPCR and protein assays.

[0226] Example 2 ASO design to target the 5' UTR of an OPA1 transcript

[0227] ASOs with 18-25 nucleotides in length (Table 3, SEQ ID NOs: 56-116) are designed to sterically inhibit a uORF or reduce the complexity of RNA secondary structure in the 5' UTR. The secondary structure of RNA is predicted using RNAfold online tool. The identified ASO sequences are synthesized as PMO and or 2'MOE chemistry) and nucleofected into HEK293 cells or ADOA patient fibroblasts carrying OPA1 mutation (c.2708_271 IdelTTAG) using the NEON® electroporation system (ThermoFisher) at 25 pM and 50 pM and the nucleofected cells were cultured for 48 hr. Total protein is 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, 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). Beta-actin serves as loading control and is detected using monoclonal mouse anti -beta actin antibody (Sigma- Aldrich, catalogue number A5441) followed by goat anti -mouse IgG H&L antibody (Abeam, catalogue number ab216776, IRDye® 680RD). ASO sequences are further refined by micro-walk or engineered mismatch oligos and / or extended up to 30 nucleotides (Table 3; SEQ ID NOs: 117-138). ASOs was subsequently re-validated using a western blot assay.

[0228] Example 3 ASO design to target the 3' UTR to increase OPA1 expression levels

[0229] An ASO sequence “micro-walk” of 25-mers (Table 4; SEQ ID NOs: 140-1312) or 17-mers (Table 5; SEQ ID NOs: 1313-2488) in 3 bp increments of distance was performed over the sequences of the 3' UTR of the ENST00000361510 transcript to mediate improvement in RNA stability. ASOs are screened to guide the ASO selection for OPA1 expression upregulation using ddPCR and western blot assays described in Examples 1 and 2.

[0230] Example 4: PMOs targeting intron 7 of OPAI

[0231] Antisense oligonucleotides with 26-30 nucleotides in length (SEQ ID NO: 2505- 2517) were designed to target the intronic splice enhancer motifs in intron 7 to mediate exclusion of exon 7x and generate productive OPAI 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 OPAI transcript was assessed using ddPCR and protein assays.

[0232] Example 5: Synthesis of peptide-oligonucleotide conjugate The peptide with SEQ ID NO: 2504 was synthesised using standard Fmoc solid phase peptide synthesis techniques. Upon cleavage from the supporting resin, the peptide was purified using reverse phase HPLC chromatography, and lyophilized to yield a fluffy white solid.

[0233] Conjugation to the phosphorodiamidate morpholino oligomer (PMO) cargo (OPA1 HlA(+10+32)lmml0C>T (SEQ ID NO: 112)) was achieved through standard amide conjugation conditions using HATU and DIEA. The carboxylic acid C-terminus of the peptide was conjugated to the secondary amine located at the PMO 3’ end. The PPMO conjugate was then purified through strong cation exchange chromatography and desalting to yield the PPMO conjugates used for subsequent in vitro and in vivo experiments.

[0234] Example 6: Evaluation of a peptide-PMO conjugate to target the 5' UTR of OPA1 in iPSC-RGCs derived from ADOA patients

[0235] A phosphorodiamidate morpholino oligomer was designed for steric inhibition of the 5' UTR of OPA1 mRNA (SEQ ID NO: 112) and conjugated with the CPP (SEQ ID NO: 2504) and utilized to assess OPA1 upregulation. iPSC-RGCs derived from three non-familial ADOA patients, each harbor distinct OPA1 mutation were used in the study. The cell culture conditions involved the systematic differentiation of induced pluripotent stem cells (iPSCs) sourced from ADOA patients into retinal ganglion cells (RGCs) over a minimum period of 35 days. On day 35 of RGC differentiation, iPSC-RGCs were seeded at 7x104cells / well in a 24-well tissue culture plate with 3 technical replicates for protein assessment. iPSC-RGCs were allowed to continue to mature for at least 10 days prior to PPMO treatment. The CPP -PMO conjugate was incubated with ADOA patient iPSC-RGCs carrying the OPA1 mutations (c.2708_271 IdelTTAG, c.985-lG>A and c.2608delA) for 5 days. At day 3 post-treatment media top up to 1 mL with 0.5 mL / well N2B27 media (DMEM / F12, Neurobasal media, GlutaMAX, Antibiotic / Antimycotic, N2 supplement and B27 supplement).

[0236] To assess of OPA1 protein, cells were harvested on day 5 post-treatment and equal amounts (10 pg) of each protein samples were denatured with 4X laemlli and TCEP for 5 minutes at 95 °C. Denatured protein was loaded onto 4-15% Criterion™ TGX™ Precast Midi Protein Gel (18 wells) in 1 x Tris / Glycine / SDS running buffer and run at 150 V for 60 min. Protein was transferred from the gel to nitrocellulose membrane by wet transfer in 25 mM Tris, 192 mM Glycine, 10% methanol at 25 V overnight (18 hours) at 4 °C. Following an overnight transfer, the membrane was stained for total protein using the REVERT® 700 total protein stain. Then, the membrane was blocked with 5% BSA / TBST for 1 hour. After blocking, the membrane was trimmed between 55 kDa to 200 kDa prior to incubation with rabbit anti-OPAl antibody (Cell Signaling, 67589S) primary antibody, clone D7C1A, diluted 1 :250 in 5% BSA / TBST for 16 hours at 4°C. Then, the membrane was washed 3 times in IX TBST for 5-10 mins and subsequently incubated with goat anti-rabbit (IRDye® 800CW preadsorbed) secondary antibody diluted 1 in 10,000 in 5% BSA in IX TBST for 1 hour at room temperature on a rocker. Finally, the membrane was washed 3 times in IX TBST for 5-10 mins. Protein bands were visualized using an Odyssey Imager and densitometry was performed using Image Studio Lite version 5.2 software (LI-COR Biosciences).

[0237] Western blot analysis was used to determine the upregulation of OPA1 protein in CPP-PMO-treated cells. The band intensity of OPA1 expression was normalised to total protein staining (assessed by ImageJ™). The untreated cells served as the baseline with a normalized value of 1. A non-targeting control (NTC) PMO [5'- GTCACGATCGAACAAGGTATCACGA-3'], conjugated with the same CPP, was employed as a negative control. Statistical analysis was conducted using Student’s Ltest. The CPP -PMO targeting the 5' UTR showed a significant upregulation of OPA1 protein in multiple ADOA patient iPSC-RGCs. Patient# 1 harboring OPA1 c.985-lG>C mutation (1.3-fold upregulation), Patient #2 harboring OPA1 c.2608delA mutation (1.4-fold upregulation) and Patient#3 harboring OPA1 c.2708 271 IdelTTAG mutation (1.3-fold upregulation).

[0238] Example 7: Evaluation of a peptide-PMO conjugate to target the NMD exon 7x of OPA1 to increase OAP1 levels in iPSC-RGCs derived from ADOA patients

[0239] A phosphorodiamidate morpholino oligomer was designed for steric inhibition of the intron 7 of the OPA1 mRNA (SEQ ID NO: 2512) and conjugated with the CPP (SEQ ID NO: 2504) to evaluate its potential for enhancing OPA1 mRNA levels. iPSC-RGCs derived from an ADOA patient carrying OPA1 c.2708 271 IdelTTAG mutation was studied. The cells were systematically differentiated from iPSCs obtained from the patient into retinal ganglion cells (RGCs) over a minimum period of 35 days. Upon reaching day 35 of RGC differentiation, iPSC-RGCs were seeded at a density of 7xl04cells / well in a 24-well tissue culture plate, with three technical replicates for protein assessment. Following seeding, iPSC-RGCs were allowed to mature for a minimum of 10 days before PPMO treatment. The CPP -PMO conjugate was then incubated with the ADOA patient iPSC-RGCs for 5 days. At day 3 post-treatment media top up to 1 mL with 0.5 mL / well N2B27 media (DMEM / F12, Neurobasal media, GlutaMAX, Antibiotic / Antimycotic, N2 supplement and B27 supplement). For the assessment of OPA1 mRNA, total RNA was extracted using the MagMAX™- 96 Total RNA Isolation kit and the level of OPA1 transcript is assessed by digital droplet PCR (Qiagen; probe catalogue number: dHsaCPE5043545). OPA1 transcript expression is normalized to GAPDH, PPL27 and SCL25A3 transcript levels (Qiagen; probe catalogue number: dHsaCPE5031596, dHsaCPE5036407, dHsaCPE5032926 respectively). The results presented in Figure 2 illustrate that the CPP- PMO conjugate induced up to 1.44-fold increase in OPA1 mRNA levels.

[0240] The ability of the CPP-PMO to enhance OPA1 protein levels was further validated in multiple patient lines. Patient iPSC-RGCs were incubated with the CPP-PMO for 5 days, and subsequently, OPA1 protein upregulation was assessed using a western blot assay. For the evaluation of OPA1 protein, cells were harvested on day 5 post-treatment and equal amounts (10 pg) of each protein samples were denatured with 4X laemlli and TCEP for 5 minutes at 95°C. Denatured protein was loaded onto 4-15% Criterion™ TGX™ Precast Midi Protein Gel (18 wells) in 1 x Tris / Glycine / SDS running buffer and run at 150 V for 60 min. Protein was transferred from the gel to nitrocellulose membrane by wet transfer in 25 mM Tris, 192 mM Glycine, 10% methanol at 25 V overnight (18 hours) at 4 °C. Following an overnight transfer, the membrane was stained for total protein using the REVERT® 700 total protein stain. Then, the membrane was blocked with 5% BSA / TBST for 1 hour. After blocking, the membrane was trimmed between 55 kDa to 200 kDa prior to incubation with rabbit anti-OPAl antibody (Cell Signaling, 67589S) primary antibody, clone D7C1A, diluted 1 :250 in 5% BSA / TBST for 16 hours at 4°C. Then, the membrane was washed 3 times in IX TBST for 5-10 mins and subsequently incubated with goat anti-rabbit (IRDye® 800CW preadsorbed) secondary antibody diluted 1 in 10,000 in 5% BSA in IX TBST for 1 hour at room temperature on a rocker. Finally, the membrane was washed 3 times in IX TBST for 5-10 mins. Protein bands were visualized using an Odyssey Imager and densitometry was performed using Image Studio Lite version 5.2 software (LI-COR Biosciences). The findings depicted in Figure 3 indicate a significant increase in OPA1 protein expression induced by the CPP- PMO, irrespective of the mutation. SEQUENCES

[0241] SEQ ID NO: 1 : OPA1 intron 7 (lowercase) and exon7x (uppercase) cDNA sequence (GRCh38 / hg38: chr3 193626203-193628616) gtgatggatggtttaagggggctaccgatacattcacactaatcagccatttctgccaagatcatgtcacctcaatctgttcatgg actccaaatacaagaaattaatttgacaaagtgaaaatataaaagatgcatcatataaatatgtaacttttctggagtgggtagtat aggtaaagccaaaagaaacaaattcaagcagaggaattttggtttctgaaaattaggttgtctgtagggtccctgtatttatactta gaacaaaattaggaatttctgtttatgtggtccagttattgagtcaccctaagtttgtaggcatcttacctacctacttgctccccaa gtttttatttctaaaatgaaaagcattgctgtagatgaccagtttacactaaagaataacatttatttatttgttttagctaaagtatatg gacagggaacattcatattcttgtagaagaaaattattttgacttttgggcaaaagcatgtagttcttatacactttgacaaactcatt gcgtacatttttcacattaatcaaagtcagcacaaataaattttcaccttggaccacggagggtttgaacactggaaatttgatata attctggttgctaaagaacaagttctaataaaagcttaagtgtataccaatatgtggctgttggtgcaatcagcaggtccgtaaaa atatgattttaatggttaggtaatcccacaacggagatcccaaagttcatgtttggaagagacttttgggtcaaagtgaaatcagt gtaatgaatttaaaattatactctgagatcttgaaatcagctaattatgttacatcttattagctcagaaaagttttgaagttatataca aatgctagtcaggaaaaaagattcagtcatgtaattcttgtacattctactatttaaatcaaccaatattatagattatgatttagtgc agtaattctgctggctaaccttatctcatttggtggtggttagtacttcagagtactcaccatagtttcatttatgttttcagcatcactt cctggtttttctcaattccatggctgtggaatcaattcatatgtatatttagcttcggtgagcaaaaacatagctagaaaaagaaaa gaagtgagtttcctacctggttaaattaaagtcgatgtgttaagccaaggaggacttcttttgaatggtactttaacaatccctgttc tgtatactgtgaatatatcatttaaatagcctaataaattggatgcttaggctgagccacctatactttagttttgttatggaaagaag ggagaggagcaagtatgttcttatatgttacttagaaataagaatgtagctgtagttacacattgttcttaagtttttttcgtaagaca acttgaaatgagtcccataggcctgctatttaacattctaagatatgacttaaggttaatgatgagcttttgaatctgacaattcaag agatatccataatgaatactgattcattttctacattgctgaaagctaatgttcattttaagcctactttagtagcctttatttgggctta gagatgttattcctctttctgatatttattgggttatctgtttaacccttttatatctccctttcccgatttgtaaattagagactggcaag actttttaccctgagtagagcaccaaacatggcttgtttctgcccacactgtagttaccttgaggggaagtaaatgggactttaaa agcaatttatgctcttttatagtgaaattatccctcttactatcccgaaagactgttaccttacaatatcctccactcctttccccctgt agttactatagagatgacttttcggttcttcactgccataatgatcaaaatcctaattcatgagatttttatcattccaggcatgtgag gtttacttgatgcataaaaccgcaagtactttttgttgttttttaattgttttttctctcttatcttcttgaaagtctaagtagatcatcatttt tgatgtcttattagtagcaactaataaattttccctgtatcttctcagcaaaagaactcaagcagagacagaagattagaactacc attggtagttttgcttcctatggatatgttcacatacatagaaatttttacaatgacctttttatatatgtatttcagaatttcagaatggc ctcaatgccttaataggaagaaatacttgaaatttttaaattagggcttggttttgtgaggagctagtaaaggtttttctctttcagC TTTAGCTTGTTTCTGCGGAGGATTCCGCTCTTTCTCCATCAGTTTCATAGCCC TGGAATTGTAGAAAAGCTCTGGTTTCAAGACCATTGATATCCATTTCTGTCA GG Table 1: List of antisense oligonucleotide sequences targeting removal of NMD exon 7x of an 0PA1 transcript

[0242] Table 2 : List of antisense digonudeotide micro-walked sequences with 17 nucleotides targeting intron 7 of an OPAl transcript.

[0243] 5 SEQ ID:55: cDNA sequence of the 5' UTR of an OPAl transcript (GRCh38 / hg38: chr3 193593064-193593380).

[0244] 10

[0245] Table 3: List of antisense oligonucleotide sequences targeting the 5’ UTR of an OP.4J transcript

[0246]

[0247] eDMA sequence of the 3' UTR located within exon 30 of an DPA I transcript (GRCh38 / hg38: cl»3 193694606- 193697811)

[0248] GTCCGTTCCCGACGCACTGTGCGCATGCGCTGGTCCTCCGCGGACCGTTCGTGCTGCCCGCCTAG.AAAGGGTGAAGAATCGTACTCATAATCAGCT

[0249] CTGGATACATCTGAAGAACAAAAACATCAACGTCrnTGTCCAGCCTCTTTn’CTrCTGCTGTrCCACCrTTCTAAACATACAATAAAGTCATGGGA

[0250] 5 TAAAAATAATCGATGTATGTTACGGGCGCTTTAACCATCAGCTGCCTCTCGAATGGAAGAACA(iTGGTAATGGATTAACATCCTATTTTGTTGTAC

[0251] TAAAGTGACAAATCGGAATAATATAATTGGTATGGC'CATTAGGTTCAGTCCTTGAAGATAAGAAACTTGTTCTCTGTTTGTTGTCTTATTTGTGGTG

[0252] GC A .CTCGTTTAATGGATTA ACTGAGGTTGCTCA ATGTTCAGTTTCTTTTCCAG A A ATAC A ATGCT AGGTGTTTTGA AATA A A ACTTATATAGCA ATT

[0253] GTTrAAAGTTATCAATTGTATATAAAATCACAGTAGCCTGCTAAATCATTGTATGTGTCTGTAGTATTCTATTCCCAGAAACTATTTGACCATGATA

[0254] ATTC'AGTTTATATTCACCACATGAAAGAAAAATCGGTAACAGAAGAACCCnTAAAACAGGTTAATrTGGATTGTAACG-TTCAGTGAAAGAAATTT

[0255] 10 CAACCCTTCATAGCC'AGCGAAGAAATTTGCCTTGGAAGCCAAGTCAGTACCAGCTTACCTATTTGATTCAGTTGCTGTTTTCTCACTCTCTATATCC

[0256] ATTTGAAATTGATTTATTTTAGATGTTGTATACTTACGTTAGGCTTTCTGTTAATAGTGGTTTTTCTCCTGTTGACAGAGCCACCGGATTATGACAC'A

[0257] GGATGAGGAAGATTAAGGATAATCAATrGACrAATTTCATTTAGAATATTATCAAACATTTCAACTAGGTATCAGAAAAAGGCnTCTTTCATAAG

[0258] ACTATTTTAAATAGAAATTATTTCAACAATTAAAGTAATGTTGACCAKrCCXTCTCAGCnGAATAAAGAAAAATTTAGTTCAATTTATTGC^AATTT

[0259] AATTACAATACTACCTTCACAACATTTTCATGTGTTTTAAATAAATATTTTTTAATTGGCTAAAGGACATTCAAGGAAAGAAATGCTTTCTTTACTT

[0260] 15 AAAATGTCrATCTCATTTGCTGCCTTTTCACTAAGCCTTTACTTTGTTAATAAAAGTGTCCATTGTGTGATGTTTTTGATTrrACACrTTTGCTAAATC

[0261] TTATTI7CTT(KUGTrGCTTTTTGGTAACA(KXX:CATTGCTACTCCCCATTTrATTGTTTTACATCAAT(K7ATCJC'TTCGTTGTGATCCCTCAAGA.TGT

[0262] AACACrrGGTATCKXCGGTTGAGGATATGAAAAAATACTTCCGAAACCAGGAATTCAATGTATGTTTGTTTTATACTGI'n’GATAAGA.AAAGTAGG

[0263] TCCAGCCTTAAGCAGCACAGATGCGCTGGTAGATGCATAGTCAGGAACTTTTTTTATTTCTTTTAGGTCTAGGGACAGGAGTGAATAGAAAGGGA

[0264] GGAGAGCTCTATTATGTTCTATACACAGATTAGGAGATGACCTTACTGGGTACACCCCTCTAACCAGTGCTTACAGGTTAATGCATGTTAATGAAT

[0265] 20 ATTTTTGCAGTTGTAAAGCATAACAATTACAACTACACATC'TATTTCTAAAGAATAAAACAGGACCATATTTATTTACTTGTGTCAACTATAGAAA

[0266]

[0267] 5 AAAA(^CAGAAATGATTCTAATTTAAACAAAAAGATA(^AAATATACAGAAGTTAAATTCGAACTAGCCACAGAATCATTT‘GTTTTTATGTCAGA ATrrGCAAAGAGTGGAGTGGACAAAGCTCTGTATGGAAGACrGAACAACTGTAAATAGATGATATCCAAACTTAATTTGGCTAGGACTTCAATrr TAAAAATCAGTGTACCTAGGCAGTGCACAGCACGAAATAAGTGGCCCTTGCAGCTTCCCCG'rrrAACCCACTGTGCTATAGTTGCGGGTGGAACA GTCAAC(JFnX^AGTAGTTTATGATATTGCCCTCTTTGTATTCCCATrrrC^

[0268] TGCAAATGAATGATATAAAAATAAGTAGGGAACATGGCAGAGAGTGGTGCITCCCAGCCTCACAATGTGGGAA'rrTGACATAGGATGAGAG'rCA

[0269] 10 GAGTATAGGTTTAAAAGATAAAATCTTTAGTrAATAATrrrGTATrrATTTATTCTAGATGTATGTATCTGAGGAAAGAAATCTGGTATrTrrGCTT 'rcCAATAAAGGGGATCAAAGTAATGarrmVICrCAG'nrrrCTAAGCI’GGTCrATG'rrATAGCrCfAGCAGTATGGAAATnrGC'rrrAAAATATGC TTACCTTTTGA ATG ATC ATGGCTATATGTTGTTG AGATATTTG A A AC1TACCTFGTITTC ACTTGTG CACTGTG AATG AA CXXXGilNVlKVGGnXl-A AAACCriTACATTACGTGTAGATA'nATTGCAACrrATAITTlTTCCTGAGCTTGATCAAAGGTGATTTGTGTAGATGAGTAATTAAAAAATATrTAA ATCACATTATAAlTGTArrA'ITGGAGAGCATCITrTAAATrTTTrTCTGTTrTAACGAGCXiAAAGAGAAACCTGTATACCTAGGGTCATTATTTGAC

[0270] 15 CXCA^'AGIATAACCAGAI'TCAl'GGrCTAACAAGCTCTCAGTG^'GGCTrrirrcrrGAAIGC'TTGAA'riTCACA'rGCCrrGCA'fqTCACAGI'IGrACTC CATGGTCAACCGGTGCTTrrTTlX^CATCGTGGTACTrGTCA^^

[0271] Table 4. List of antisense oligonucleotide sequences with 25 nucleotides in length that target the 3' UTR of an OPAI transcript.

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304] Table 5« List of antisense oligonucleotide micro-walked sequences with 17 nucleotides in that target the 3' UTR of an OPAl transcript.

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331] SEQ ID:2489: OP A I transcript cDNA sequence (NM 130837)

[0332] AGGCTCTTGCGGAAGTCCATGCGCCATTGGGAGGGCCTCGGCCGCGGCTCTGTGCCCTTGCTGCTGAGGGCCACTTCCTGGGTCATTCCTGGACCG

[0333] GGAGCCGGGCTGGGGCTCACACGGGGGCTCCCGCGTGGCCGTCTCGGCGCCTGCGTGACCTCCCCGCCGGCGGGATGTGGCGACTACGTCGGGCC

[0334] GCTGTGGCCTGTGAGGTCTGCCAGTCTTTAGTGAAACACAGCTCTGGAATAAAAGGAAGTTTACCACTACAAAAACTACATCTGGTTTCACGAAG

[0335] 5 CATTTATCATTCACATCATCCXACCTTAAAGC^TCAACGACCCCAATTAAGGACATCCTTTCAGCAGTTCTCTTCTCTGACAAACCTTCCrTTACGT

[0336] AAACTGAAATTCTCTCCAATTAAATATGGCTACCAGCCTCGCAGGAATTTTTGGCCAGCAAGATTAGCTACGAGACTCTTAAAACTTCGCTATCTC

[0337] ATACTAGGATCGGCrGTrGGGGGTGGCTACACAGCCAAAAAGACTTTTGATC’AGTGGAAAGATATGATACCGGACCTTAGTGAATATAAATGGAT

[0338] TGTGCCrGACATTGTGTGGGAAATTGATGAGTATATCGATTTTGAGAAAATTAGAAAAGa?CTTCCTAGTTCAGAAGACCTTGTAAAGTTAGCACC

[0339] AGACTTTGACAAGATTGTrGAAAGCCTTAGCTTATTGAAGGACTrTTTTACCTCAGGTCACAAAITGGTrAGTGAAGTCATAGGAGCTrCTGACCT

[0340] ACITCTGn'GTTAGGTrcrCCGGAAGAAACGGCG'm'AGAGCAACAGATCGTGGATCrGAAAG'rGACAAGCAITrr'AGAAAGGGTCrGCITGGrGA

[0341] 5 GCTCATTCTCTTACAACAACAAATTCAAGAGCATGAAGAGGAAGCGCGCAGAGtXGCTGGCCAATATAGCACGAGCTATGCCCAACAGAAGCGC

[0342] AAGGTGTCAGACAAAGAGAAAATTGACCAACTTCAGGAAGAACTTCTGCACACTCAGTTGAAGTATCAGAGAATCTTGGAACGATTAGAAAAGG

[0343] AGAACAAAGAATTGAGAAAATTAGTATrGCAGAAAGATGACAAAGGCATTCATCATAGAAAGCTTAAGAAATCTrTGATTGACATGTArrCTGAA

[0344] GTTCrrGATGTTCTCTCTGATTATGATGCCAGTrATAATACGCAAGATCATCTGCCACGGGn'GTFGTGGTTGGAGATCAGAGTGCTGGAAAGACT

[0345] AGTGTGTrGGAAATGATTGCCCAAGCTCGAATATTCCCAAGAGGATCTGGGGAGATGATGACACGTTCTCCAGTTAAGGTGACTCTGAGTGAAW

[0346] TCCTCACCATGTGGGCCl'ATTrAAAGATAGTTCTCGGGAGTrTGATCTTACCAAAGAAGAAGATCTTGCAGCATTAAGACATGAAATAGAACTfCG

[0347] AATGAGGAAAAATGTGAAAGAAGGCfGTACCGlTAGCCCrcAGACCATATCCfTAAATGTAAAAGGCCCrGGACrACAGAGGATGGTGCTTGlTG

[0348] ACTTACCAGGTGTGATTAATACTGTGACATCAGGCATGGCTCCTGACACAAAGGAAACTATTTTCAGTATCAGCAAAGCTrACATGC-AGAATCCTA

[0349] ATGCXrATCATACTGTGTATrCAAGATGGATCrGTGGATGCTGAACGCAGTATTGTTACAGACTTGGTCAGTCAAATGGACCCTCATGGAAGGAGA

[0350] ACCATATTCGTrrTGACCAAAGTAGACCTGGCAGAGAAAAATGTAGCCAGTCCAAGCAGGATTCAGCAGATAATTGAAGGAAAGCTCTTCCCAAT

[0351] 15 GAAAGCTrrAGGlTATm-GCfGTrGTAACAGGAAAAGGGAACAGCI'CTGAAAGCAITGAAGCrATAAGAGAATATGAAGAAGAG’ITmTCAGA

[0352] ATTCAAAGCTCCTAAAGACAAGCATGCTAAAGGCACACCAAGTGACTACAAGAAATTrAAGCCTTGCAGTATCAGACrGCTTTTGGAAAATGGTA

[0353] CGAGAGTCTGTrGAACAACAGGCTGATAGTTTCAAAGCAACACGTTTTAACCTTGAAACIGAATGGAAGAATAACTATCCTCGCCTGCGGGAACT

[0354] TGACCGGAATGAACTATTTGAAAAAGCTAAAAATGAAATCCTTGATGAAGTTATCAGTCTGAGCCAGGTTACACCAAAACATrGGGAGGAAATCC

[0355] TrCAACAATCrrrGrGGGAAAGAGTATCAACrCATGrGA'rrGAAAACATCTACC'FrCCAGCrGCGCAGACCATGAATTCAGGAACrnTAACACCA

[0356] 20 CAGTGGATATCAAGCTTAAACAGTGGACTGATAAACAACTTCX'TAATAAAGCAGTAGAGGTTGCTTGGGAGACCCTACAAGAAGAAT'nTCCCGC

[0357] TTTATGACAGAACCGAAAGGGAAAGAGCATGATGACATATTTGATAAACTTAAAGAGGCTGTTAAGGAAGAAAGTATTAAACGACACAAGTGGA

[0358] ATGACTTTGCGGAGGACAGCTTGAGGGTrArrCAACACAATCCrrTGGAAGACCGATCCATATCTGATA.AACAGCAATGGGATGCAGCTATFTATT

[0359] •ITATGGAAGAGGCICTGCAGGCrCG’I'CrCAAGGATACrGAAAA'rGCAA'rrGAA.AACATGGTGGGTCCAGACTGGAA.AAAGAGG'rGGTrATACTGG

[0360] AAGAATCGGACCCAAGAACAGTGTGTrCACAATGAAACCAAGAATGAATrGGAGAAGATGTrGAAATGTAArGAGGAGCACCCA.GCTTATCTTG

[0361] 25 CAAGTGATGAAATAACCACAGTCCGGAAGAACCrrGAATCCCGAGGAGTAGAAGTAGATCCAAGCTrGATTAAGGATACTrGGCATCAAGTTTAT

[0362] AGAAGACATTnrTAAAAACAGCrCrAAACCATTGTAACCTTTGTCGAAGAGGTTrTTATTACTAC^AAAGGCATrTTGTAGATTCTGAGTTGGAA TGCAATGATGTGGTCTTGTrrrGGCGTATACAGCGCATGCTTGCrATCACCGCAAATACTTTAAGGCAACAACTTACAAATACTGAAGTTAGGCGA TrAGAGAAAAATGTTAAAGAGGTATTGGAAGATTTTGCTG.AAGATGGTGAGAAGAAGATTAAA'TTGCTTACTGGTAAACGCGTTCAACTGGCGGA AGACCnrAAGAAAGTrAGAGAAATTCAAGAAAAzVClTGATGCTrrCATrGAAGCTCH'CATCAGGAGAAATAAATI'AAAATCGTACrCATAATCA

[0363] 5 GCrClVCATACATCTGAAGAACAAAAACATCAACGTCrnTGTCCAGCCrClTr7TCrrcrGCTGTTCCAG:CTTTCTAAACATACAATAAAGTCArG

[0364] GGATAAAAATAATCGATGTATGITACGGGCGCTTTAACCATCAGCn'GCGTCTCGAATGGAAGAACAGTGGTAATGGAITAACATCCTATTTTG'TTG TACTAAAGTGACAAATCGGAATAATATAATTGGTATGGCCATTAGGTTCAGTCCnGAAGATAAGAAACTTGTrCTCrGrrrGTTGrCTTA'rn'GTG GTGGCACTCGTTTAATGGATTAACTGAGGTrGCTCAATGTrCAGTTTCTTTT(XAGAAATACAATGCTAGGTOTTTTGAAATA / \AACTTATATAGCA ATTGTTTAAAGTTATCAATrGTATATAAAATCACAGTAGCCTGCTAAATCAnXJTATGTGTCTGTAGTATTCTATTCCCAGAAACTATTTGACCATG ATAATTCAGTrTATATTCACCACATOAAAGAAAAATGGGTAACAGAAGAACCCTTAAAACAGGTTAA'TTTGGATTGTAACGTTCAGTGAAAGAAA TrrCAACCCrrCATAGCCAGCGAAGAAA’nTGCClTGGAAGCCAAG^CAGrACCAGCrrACCTA'mGA'rrCAG'H’GCrG'mTCTCACTCTCTATA

[0365] TCCATTTCAAATTGATTTATrTrAGATGTTGTATACTrACGTTAGGCTTTXTGTTAATAGrGGTTTTT(TCCTGTTGACAGAGCCACCGGATTATGAC ACAGGATGAGGAAGATTAAGGATAATCAATrGACTAATn’CATrTAGAATATTATCAAACATTrCAACTAGGTATCAGAAAAAGGCTTTCTTTCAT AAGACrATTTTAAATAGAAATrATTTCAACAATrAAAGTAATGTTGACCATCCCCCTCTCAGCrGAATAAAGAAAAATTTAGTTCAATrTATrGCA

[0366] 15 ATnAATTACAATACrACCTrCACAACAnTrCATGI'GITrrAAATAAATATrniTAA'nGGCl’AAAGGACATrCAAGCAAAGAAATGCLTrClTr ACrTAAAATOTCTATCTCATTTGCTGCCTrrrcACTAAGCCTTTACmGTTAATAAAAGTGTCCATIGTOT

[0367] AATCTTATTTlTTTGGAG'rrGCTrrrTGGTAACAGCCCCAn,GCrACrCCCCATTTrArrGlTn'ACATCAAirrCAlTTC'n€GlTGTGATCCCTCAAG

[0368] ATGTAACACTTGGTATGCTCGGTTGAGGATATGAAAAAATACTTfXGAAACCAGGAATTCAATGTATGTrrGTTTTATACTGTn'GATAAGAAAAG TAGGTGCAGCCrrAAGCAGCACAGATGCGCrGGTAGATGCATAGTCAGGAACmTnTAmrnTrAGGTCrAGGGACAGGAGTGAATAGAAAG

[0369] 20 GG AGGAG AGCTCTATTATGTTCT ATAC A CAG AIT AGG AGATGACCTTACTGGGT ACACCCCTCTAA CCAGTGCTTAC AGGTTA ATGCATGTTA ATG AATATTTTTGCAGTTGTAAAGCATAACAATTACAACTACACATCTATTrCTAAAGAATAAAACAGGACCATATTTATTFACTTCTGTCAACTATAG AAAGAAAGACCTTCAGCTGTATTTCCACAGATrrCTCCCAAGGAAAAGGCTAATATTAGTCACTACTGTTATCACATCanTTGTATAAGTnTAA AAAGAGA7 GGA€KSGAGA7CTirAin''ICTT,rGAGGAGATCAGTATrGTAACGTATG'rGAArAGATGA'IAACAAriAATATrACrAAAAG'f'CCCACA

[0370] 25 ATrmCCrTGAAAGCrAGGTATTrATC.AACTGCAGATGTTATTGAAAGAAAATAAAATTCAGTCTCAAGAGTAAACCCTGTGTCTTGTGTCTGTA

[0371] GTTCAAAAGTCAGAAATGATTCTAATrTAAACAAAAAGATACTAAATATACAGAAGTTAAATTCGAACTAGCCACAGAATCATTTGTTTTTATGTC

[0372] AGAATTTGCAAAGAGTGGAGTGGACAAAGCTCTGTATGGAAGACTGAACAACTGTAAATAGATGATATCCAAACTrAATrrGGCTAGGACTrCAA

[0373] TrTTAAAAATCAGTGTACCTAGGCAGTGCACAGCACGAAATAAGTGGCCCTTGCAGCTTCCCCGTTTAACCCACTGTGCTATAGTTGCGGGTGGAA

[0374] CAGTCAACciTrci’AGTAGTn,ATGATATrGCCCivrnxjTAiirccArnivrA€AGrmTLx:cGCAGAcri,ciTTcrGCAAAirArrcAGCcrccA

[0375] 5 AATGCAAATGAATGATATAAAAATAAGTAGGGAACATGGCAGAGAGTGGTGCTTCCCAGCCTCACAATGTGGGAAnTGACATAGGATGAGAGT

[0376] CAGAGTATAGGTITAAAAGATAAAATCTTrAC / rrAATAATfrrGTATITA'ITTATTCTAGATGTATGTATCTGAGGAAAGAAATCTGGTA'mTrGC

[0377] TrrCCAATAAAGGGGATCAAAGTAATGGTrrrrCTCTCAGTTCTCTAAGCTGGTCTATGTTATAGCTCTAGCAGTATGGAAATGTGCTTTAAAATAT

[0378] GCTTACCTnrGAATGATCATGG&ATATGTrGTTGAGATATTTGAAACTTACCrTGr^

[0379] TAAAACCnGACA,n\ACGTGTAGATATrATrGCAACTTATATlTrGCCTGAGCTrGATCAAAGGTClTrGTGTAGATGAGTAAlTAAAAAATATTrA

[0380] 10 AATCACATTATAATrCTATrATTGGAGAGCATCTrTTAAATrrrTTrCTGTmAAa}AGGGAAAGAGAAACCTGTATACCTAGGGTCATTATTTGA

[0381] {XCCATAGrATAACCAGA'HGATGGTCTAACAAGCTC'rCAGTG'rGGCmTCrCTGAATGCri’GAAmrACATGCCrrGCA’ITrCACAGlTGl’ACf

[0382] CCATGGTCAACCGGTGCTTrTTTTCACATCGTGGTACrTGTCAAAACAITITGTrATTriCCITGGTAAAATATATAAAAAAGGTTrTCTAATfTCA

[0383] SEQ ID:2490: OPAI protein sequence (UniProt lD 060313)

[0384] MWRLRRAAVACXVCQSLVKHSSGIKGSLPLQKLHLVSRSrYHSHHPTLKLQRPC^RTSFQQFSSLTNLPLRKLKFSPlKYGYQPRRNFWPARLATRLLKL RYLILGSAVGGGYTAKKTFDQWKDMIPDLSEYXWTVPDIVWEIDEYTDFEKIRKALPSSEDLVKLAPDFDKIVESLSLLKDFFTSGSPEETAFRATDRGSES DKHFRKVWKEK1DQLQEEIzLHTQIzKYQRILERLEKENKEl.RKI,VLQK.DDKGIHHRKLKKSLIDMYSEVI„DVIzSDYDASYNTQDHIzPRVVVVGDQSAG

[0385] 5 KTSVIzEMlAQARIFPRGS(^MMIRSPVKVTIzSEGPHHVAIzFKDSSREFDLTK^EDLAALRHEIFJzRMRKNVKEGCTVSPETlSIzNVKGPGizQRMVLzVI)LP GVT?Tr\T’SGMAPDTKETiFSISKAYMQFIPNAnLClQDGSVDAERSIVTDLVSQMDPHGRRTI.FVLTKVDLAEKNVA^»SRiQQnEGKLFPMKALGYFAVy?TGKGNSSESIEAIREYEEEFFQNSKLLKTSMLKAHQVnRNLSLAVSDCFWTCAIVRESVEQQADSFKATRF^ETEWKNNYPRLRELDRNELFEKAKNEJL DE\aSLSQVTPKHWEEIL(XM.WERVSTHVIEN[YLPAAQ™NSGlFNTTVDIKLKQWTDKQLPNXAVEVAWTLQEEFSRFNfrEPKGKEHDDiroKLKE A\^EESTKRHKWDFAEDSLzRVIQHNAlzEDRSTSDKQQWAATYFMEEALQARLKDlENAIEWVGPDWKKRWIzYWKNRTQEQCVHNETKNELEKM

[0386] 10 LKO>IEEHPAYlJ\SDElTTWK?^ESRGVEVI)PSLIKDTWHQVYRRHFLKTALNHa^LCRRGFYYYQRHFVDSELEC?©WLFWRIQRMLAn'ANTLRQQ LTNTEVRRLjSCNVKEVLEDFAEDGEKKTKLLTGRRVQLAEDLKKVRElQEKLDAFIEALHQEK

[0387]

[0388] Table ? : PMO refinement to target the 5TJTR of an OPA l transcript

[0389] 5 SEQ ID NO: 2504: CPP Sequence (N to C terminus) RASARRASARRAN ARRAN ARRSGGR

[0390] Table 8: 1 ,ist of antisense oligonucleotide sequences targeting intron 7 of 0PA1 transcript

Claims

CLAIMS1. A cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

2. A cell-penetrating peptide (CPP), wherein the CPP consists of a sequence set forth in SEQ ID NO: 2504.

3. The CPP of claim 1 or 2, wherein the CPP is linked to an antisense oligonucleotide that modulates mRNA productive transcript, stability and / or translation of OPA1 gene transcript or part thereof.

4. A cell-penetrating peptide (CPP) conjugate comprising a sequence set forth in SEQ ID NO: 2504 linked to an antisense oligonucleotide that modulates mRNA productive transcript, stability and / or translation of OPA1 gene transcript or part thereof.

5. An antisense oligonucleotide that modulates mRNA productive transcript, stability and / or translation of OPA1 gene transcript or part thereof, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

6. The CPP of claim 3, or the CPP conjugate of claim 4, or the antisense oligonucleotide of claim 5, wherein the antisense oligonucleotide increases the level of OPA1 mRNA or the amount of functional OPA1 protein in a cell and / or a tissue of the subject.

7. The CPP of claims 3 or 6, or the CPP conjugate of claims 4 or 6, or the antisense oligonucleotide of claims 5 or 6, wherein the amount of functional OPA1 protein in the cell and / or the tissue is increased by about 1.1 to about 10-fold.

8. The CPP of any one of claims 3, 6 or 7, or the CPP conjugate of any one of claims 4, 6 or 7, or the antisense oligonucleotide of any one of claims 5 to 7, wherein the tissue is selected from the group consisting of the retina, retinal pigment epithelium and combinations thereof.

9. The CPP of any one of claims 3 or 6 to 8, or the CPP conjugate of any one of claims 4 or 6 to 8, or the antisense oligonucleotide of any one of claims 5 to 8, wherein the antisense oligonucleotide binds to a targeted portion of: i. an OPA1 gene pre-mRNA in a cell to promote exclusion of a nonsense-mediated RNA decay-inducing (NMD) exon during splicing of the OPA1 pre-mRNA to increase the level of OP Al mRNA transcripts encoding full length, functional OPA1; ii. the 5' untranslated region (UTR) of an OPA1 gene transcript in a cell to increase translation efficiency of an OP Al mRNA; iii. the 5' UTR of an OPA1 gene transcript in a cell to increase transcript stability; and / or iv. the 3' UTR of an OPA1 gene transcript in a cell to increase transcript stability.

10. The CPP, the CPP conjugate or antisense oligonucleotide of claim 9, wherein: i. the antisense oligonucleotide binds to intron 7 of an OPA1 gene pre-mRNA in a cell and increases the level of OP Al gene transcripts encoding full length, functional OPA1 by exclusion of NMD exon 7x; or ii. the antisense oligonucleotide binds to intron 7 of an OPA1 transcript and reduces expression of OPA1 gene transcript lacking exon 7x but does not substantially affect the relative expression levels of OPA1 gene transcripts comprising exon 7 or lacking exon 7.

11. The CPP of any one of claims 3 or 6 to 10, or the CPP conjugate of any one of claims 4 to 6 to 10, or the antisense oligonucleotide of any one of claims 5 to 10, wherein the antisense oligonucleotide binds within a targeted portion of the OPA1 pre-mRNA nucleotide sequence corresponding to SEQ ID NOs: 1, 55, 139.

12. The CPP of any one of claims 3 or 6 to 11, or the CPP conjugate of any one of claims 4 to 6 to 11, or the antisense oligonucleotide of any one of claims 5 to 11, wherein the antisense oligonucleotide comprises one or more mismatches relative to the sequence set forth in SEQ ID NO: 1.

13. The CPP, the CPP conjugate or the antisense oligonucleotide of claim 12, which comprises two mismatches relative to the sequence set forth in SEQ ID NO: 1.

14. The CPP, the CPP conjugate or the antisense oligonucleotide of claims 12 or 13, 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.

15. The CPP of any one of claims 3 or 6 to 14, or the CPP conjugate of any one of claims 4 to 6 to 14, or the antisense oligonucleotide of any one of claims 5 to 14, wherein the nucleotide sequence of the antisense oligonucleotide is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the nucleotide sequence of the targeted portion over the length of the antisense oligonucleotide.

16. An antisense oligonucleotide comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 2-54 or SEQ ID NOs: 2491-2677 or SEQ ID NOs: 2505-2517 or a sequence having at least about 50% identity thereto, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

17. An antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 2512, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

18. An antisense oligonucleotide that binds to a targeted portion of the intron 7x of an OPA1 gene transcript in a cell and increases the level of OPA1 gene transcripts encoding full length, functional OPA1 by exclusion of NMD exon 7x, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

19. The CPP of any one of claims 3 or 6 to 11, or the CPP conjugate of any one of claims 4 to 6 to 11, or the antisense oligonucleotide of any one of claims 5 to 11 or 18, wherein the antisense oligonucleotide comprises or consists of any one of SEQ ID NOs: 2-54 or SEQ ID NOs: 2491-2499 or SEQ ID NOs: 2505-2517.

20. An antisense oligonucleotide that binds to a targeted portion of the 5' UTR of an OP Al gene transcript in a cell and increases transcript stability of an OP Al mRNA, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP)comprising a sequence set forth in SEQ ID NO: 2504 and wherein the targeted portion is within the nucleotide sequence corresponding to SEQ ID NO: 55.

21. The CPP of any one of claims 3 or 6 to 9, or the CPP conjugate of any one of claims 4 to 6 to 9, or the antisense oligonucleotide of any one of claims 5 to 9 or 20, wherein the antisense oligonucleotide comprises or consists of any one of SEQ ID NOs: 56-138 or SEQ ID NOs: 2500-2503.

22. An antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO: 112, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

23. An antisense oligonucleotide that binds to a targeted portion of the 3' UTR of an OP Al gene transcript in a cell and increases transcript stability of an OP Al mRNA, wherein the antisense oligonucleotide is linked to a cell-penetrating peptide (CPP) comprising a sequence set forth in SEQ ID NO: 2504.

24. The CPP of any one of claims 3 or 6 to 9, or the CPP conjugate of any one of claims 4 to 6 to 9, or the antisense oligonucleotide of any one of claims 5 to 9 or 23, wherein the antisense oligonucleotide binds within a targeted portion of the 3' UTR of OPA1 mRNA, wherein the targeted portion is within the nucleotide sequence corresponding to SEQ ID NO: 139.

25. The CPP, or the CPP conjugate or the antisense oligonucleotide of claim 24, wherein the antisense oligonucleotide comprises or consists of any one of SEQ ID NOs: 140-2488.

26. The CPP of any one of claims 3 or 6 to 15, or 19, or 21 or 24, or 25, or the CPP conjugate of any one of claims 4 to 6 to 15, or 19, or 21 or 24, or 25, or the antisense oligonucleotide of any one of claims 5 to 25, wherein the antisense oligonucleotide comprises a backbone modification.

27. The CPP, the CPP conjugate or the antisense oligonucleotide of claim 26, wherein the antisense oligonucleotide comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

28. The CPP of any one of claims 3 or 6 to 15, or 19, or 21 or 24, or 25 to 27, or the CPP conjugate of any one of claims 4 to 6 to 15, or 19, or 21 or 24, or 25 to 27, or the antisense oligonucleotide of any one of claims 5 to 27, wherein the antisense oligonucleotide comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-(9-methyl, a 2' -Fluoro, or a 2'-(9-methoxyethyl moiety.

29. The CPP of any one of claims 3 or 6 to 15, or 19, or 21 or 24, or 25 to 28, or the CPP conjugate of any one of claims 4 to 6 to 15, or 19, or 21 or 24, or 25 to 28, or the antisense oligonucleotide of any one of claims 5 to 28, wherein the antisense oligonucleotide comprises at least one modified sugar moiety.

30. The CPP of any one of claims 3 or 6 to 15, or 19, or 21 or 24, or 25 to 29, or the CPP conjugate of any one of claims 4 to 6 to 15, or 19, or 21 or 24, or 25 to 29, or the antisense oligonucleotide of any one of claims 5 to 29, wherein each sugar moiety in the antisense oligonucleotide is a modified sugar moiety.

31. The CPP of any one of claims 3 or 6 to 15, or 19, or 21 or 24, or 25 to 30, or the CPP conjugate of any one of claims 4 to 6 to 15, or 19, or 21 or 24, or 25 to 30, or the antisense oligonucleotide of any one of claims 5 to 30, wherein the antisense oligonucleotide comprises a 2'-(9-methoxy ethyl moiety.

32. The CPP, or the CPP conjugate, or the antisense oligonucleotide of claim 31, wherein each nucleotide of the antisense oligonucleotide comprises a 2'-(9-methoxy ethyl moiety.

33. The CPP of any one of claims 3 or 6 to 15, or 19, or 21 or 24, or 25 to 32, or the CPP conjugate of any one of claims 4 to 6 to 15, or 19, or 21 or 24, or 25 to 32, or the antisense oligonucleotide of any one of claims 5 to 32, wherein the nucleotide sequence of the antisense oligonucleotide 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.

34. The CPP, or the CPP conjugate, or the antisense oligonucleotide of claim 33, wherein the nucleotide sequence of the antisense oligonucleotide consists of 20 to 30 nucleotides.

35. The CPP, or the CPP conjugate or the antisense oligonucleotide of claim 34, wherein the antisense oligonucleotide comprises one or more phosphorodiamidate morpholino moieties.

36. The CPP of any one of claims 3 or 6 to 15, or 19, or 21 or 24, or 25 to 35, or the CPP conjugate of any one of claims 4 to 6 to 15, or 19, or 21 or 24, or 25 to 35, or the antisense oligonucleotide of any one of claims 5 to 35, wherein the antisense oligonucleotide is covalently or non-covalently linked to the CPP.

37. The CPP, or the CPP conjugate, or the antisense oligonucleotide of claim 36, wherein the CPP is linked to the 5' end of the antisense oligonucleotide or is linked to the 3' end of the antisense oligonucleotide.

38. The CPP of any one of claims 3 or 6 to 15, or 19, or 21 or 24, or 25 to 37, or the CPP conjugate of any one of claims 4 to 6 to 15, or 19, or 21 or 24, or 25 to 37, or the antisense oligonucleotide of any one of claims 5 to 37, wherein the antisense oligonucleotide is complexed with a delivery nanocarrier.

39. The CPP, or the CPP conjugate, or the antisense oligonucleotide of claim 38, wherein the delivery nanocarrier is selected from the group consisting of: lipoplexes, liposomes, exosomes, inorganic nanoparticles, and DNA nanostructures.

40. The CPP, or the CPP conjugate, or the antisense oligonucleotide of claim 39, wherein the delivery nanocarrier comprises a lipid nanoparticle (LNP) encapsulating the antisense oligonucleotide.

41. The CPP of any one of claims 3 or 6 to 15, or 19, or 21 or 24, or 25 to 40, or the CPP conjugate of any one of claims 4 to 6 to 15, or 19, or 21 or 24, or 25 to 40, or the antisense oligonucleotide of any one of claims 5 to 40, wherein the antisense oligonucleotide 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.

42. A pharmaceutical composition comprising the CPP of any one of claims 3 or 6 to 15, or 19, or 21 or 24, or 25 to 41, or the CPP conjugate of any one of claims 4 to 6 to15, or 19, or 21 or 24, or 25 to 41, or the antisense oligonucleotide of any one of claims 5 to 41 and a pharmaceutically acceptable excipient.

43. A method of treating , preventing and / or delaying progression of a condition associated with 0PA1 expression in a subject, the method comprising administering theCPP of any one of claims 3 or 6 to 15, or 19, or 21 or 24, or 25 to 41 , or the CPP conjugate of any one of claims 4 to 6 to 15, or 19, or 21 or 24, or 25 to 41, or the antisense oligonucleotide of any one of claims 5 to 41 or the pharmaceutical composition of claim 38 to the subject.

44. The method of claim 43, wherein the condition is glaucoma, autosomal dominant optic atrophy (ADOA), geographic atrophy, acute ischemic optic neuritis or acute ischemic optic neuropathy.