Treatment methods for optic atrophy

Abstract

The present invention relates to isolated or purified antisense oligonucleotides that bind to intron 7 of the OPA1 gene pre-mRNA. The present invention also relates to methods for manipulating the translation of OPA1 gene transcripts and to the use of antisense oligonucleotides to treat, prevent or alleviate the effects caused by mutations in the gene OPA1.

Description

[Technical Field]

[0001] Related application data This application claims priority from Australian Patent Application No. 2022 / 900163, filed 31 January 2022, entitled "Method of Treatment for Optic Atrophy", the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to the use of antisense oligonucleotides to treat, prevent or alleviate the effects caused by mutations in the gene OPA1. [Background technology]

[0004] Autosomal dominant optic atrophy (ADOA) is the most common hereditary optic neuropathy worldwide, with an estimated disease prevalence ranging from 1 in 100 million to 1 in 50,000. OPA1 is the primary causative gene in most families with ADOA.

[0005] ADOA typically presents with progressive visual impairment in early childhood, with selective retinal ganglion cell loss as its defining pathological feature. The mean age of onset of visual impairment is 7 years, with a range of 1 to 16 years. Eighty percent of affected individuals are symptomatic before the age of 10 years. In up to 20% of cases, ADOA is associated with other clinical signs, most commonly sensorineural hearing loss, ataxia, myopathy, and progressive external ophthalmoplegia. The importance of these syndromic ADOA variants is underscored by the identification of cytochrome c oxidase (COX)-deficient muscle fibers and multiple mtDNA deletions in skeletal muscle biopsies from these patients, suggesting a role for OPA1 in mitochondrial DNA maintenance.

[0006] OPA1 consists of 30 exons spanning 100 kb of genomic DNA on the long arm of chromosome 3 (3q28-q29). The protein product is a 960-amino acid polypeptide that colocalizes 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 mitochondrial network stability. Over 400 different OPA1 mutations have been reported to cause optic nerve degeneration and visual loss, ranging from isolated "Dominant Optic Atrophy" (DOA; OMIM #165500), including several biallelic cases with Behr syndrome (OMIM #210000), to a more severe multisystem syndrome termed "DOAplus" (OMIM #125250).

[0007] OPA1 is a ubiquitously expressed mitochondrial GTPase essential for mitochondrial function. In humans, OPA1 generates at least eight isoforms by differential splicing of exons 4, 4b, and 5b, or exons 4, 5, and 7, as shown in Figure 1A. The OPA1 precursor protein is targeted by its mitochondrial targeting sequence (MTS) and recruited to mitochondria. In mitochondria, the OPA1 precursor protein is cleaved into either a long form (l form) anchored to the inner mitochondrial membrane or a short soluble form (s form).

[0008] The coding sequence of the entire OPA1 gene exceeds the packaging capacity of AAV vectors, which have a size limit of less than 5 kb. Furthermore, certain forms of CRISPR / Cas9 gene correction require different products for each unique OPA1 mutation, and splicing-switching strategies using antisense oligomers targeting the OPA1 pre-mRNA only target the region of the mutation (e.g., within an exon, as with antisense oligomer drugs such as eteplirsen) and may not provide a functional protein product. Additionally, both gene replacement and gene editing approaches require subretinal injection of viral vectors to achieve sufficient transfection. This procedure carries the risk of retinal trauma.

[0009] There is a need to provide new treatments or preventative measures for ADOA, or at least methods that complement previously known treatments. The present invention seeks to provide improved or alternative methods for treating, preventing, or mitigating the effects of ADOA.

[0010] The foregoing discussion of the background art is intended only to facilitate an understanding of the present invention. This discussion is not an admission or acknowledgement that any of the material referred to is or was part of the common general knowledge at the priority date of the application. Summary of the Invention

[0011] The present invention provides compositions comprising antisense oligonucleotides that bind to a target portion of the OPA1 gene pre-mRNA in a cell, promote the exclusion of nonsense-mediated RNA decay-induced (NMD) exons during splicing of the OPA1 pre-mRNA, and increase the level of OPA1 mRNA transcripts encoding the OPA1 protein.

[0012] The present invention provides a means for increasing the level of functional OPA1 protein expression. Preferably, the increase in functional OPA1 is in patients with ADOA, more preferably in ADOA associated with OPA1 haploinsufficiency.

[0013] Generally, one aspect of the present invention provides an isolated or purified antisense oligonucleotide that binds to intron 7 of the OPA1 gene pre-mRNA in a cell and promotes the exclusion of nonsense-mediated RNA decay (NMD) exon 7x from intron 7 during splicing of the OPA1 pre-mRNA. Preferably, the isolated or purified antisense oligonucleotide increases translation of functional OPA1 protein by excluding the NMD exon. Preferably, the isolated or purified antisense oligonucleotide is for inducing increased production of functional OPA1 protein or a portion thereof.

[0014] In one aspect of the invention, there are provided antisense oligonucleotides of 10 to 50 nucleotides that contain a targeting sequence complementary to a region near or within intron 7 or a portion thereof. In another aspect of the invention, there are provided antisense oligonucleotides of 10 to 50 nucleotides that contain a targeting sequence complementary to a region near or within an exon or a portion thereof of the OPA1 gene transcript.

[0015] According to one aspect of the present invention, there is provided an isolated or purified antisense oligonucleotide designed to increase translation of functional OPA1 protein by excluding nonsense-mediated RNA decay (NMD) exon 7x. Preferably, the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide.

[0016] Preferably, the antisense oligonucleotides are selected from the group comprising the sequences shown in Table 1 and Table 2, and combinations or cocktails thereof.

[0017] More specifically, the antisense oligonucleotide may be selected from the group including any one or more of SEQ ID NOs: 1 to 31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3, and 4, and combinations or cocktails thereof.

[0018] In one example, the antisense oligonucleotide is selected from the group comprising any one or more of SEQ ID NOs: 1-31.

[0019] In one example, the antisense oligonucleotide is selected from the group comprising any one or more of SEQ ID NOs: 1, 2, 3, 4 and 7.

[0020] In one example, the antisense oligonucleotide is selected from the group comprising any one or more of SEQ ID NOs: 1, 3 and 4.

[0021] Preferably, the antisense oligonucleotides are selected from the group comprising the sequences shown in Tables 1, 2 and 4, and combinations or cocktails thereof.

[0022] More specifically, the antisense oligonucleotide may be selected from the group including any one or more of SEQ ID NOs: 1 to 31 and 33 to 54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7 and 33 to 44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40 and 44, and combinations or cocktails thereof.

[0023] In one example, the antisense oligonucleotide is selected from the group comprising any one or more of SEQ ID NOs: 1-31 and 33-54.

[0024] In one example, the antisense oligonucleotide is selected from the group comprising any one or more of SEQ ID NOs: 33-54.

[0025] In one example, the antisense oligonucleotide is selected from the group comprising any one or more of SEQ ID NOs: 1, 2, 3, 4, 7, and 33-44.

[0026] In one example, the antisense oligonucleotide is selected from the group comprising any one or more of SEQ ID NOs: 33-44.

[0027] In one example, the antisense oligonucleotide is selected from the group comprising any one or more of SEQ ID NOs: 1, 3, 4, 37, 40 and 44.

[0028] In one example, the antisense oligonucleotide is selected from the group comprising any one or more of SEQ ID NOs: 37, 40, and 44. For example, the antisense oligonucleotide is set forth in SEQ ID NO: 1. In another example, the antisense oligonucleotide is set forth in SEQ ID NO: 3. In a further example, the antisense oligonucleotide is set forth in SEQ ID NO: 4. For example, the antisense oligonucleotide is set forth in SEQ ID NO: 37. In another example, the antisense oligonucleotide is set forth in SEQ ID NO: 40. In a further example, the antisense oligonucleotide is set forth in SEQ ID NO: 44.

[0029] Also provided is a method for manipulating translation of an OPA1 gene transcript, the method comprising: Methods are provided that include the steps of: a) providing one or more of the antisense oligonucleotides described herein and allowing the oligonucleotide to bind to a target nucleic acid site.

[0030] Also provided is a pharmaceutical, prophylactic, or therapeutic composition for treating, preventing, or ameliorating the effects of a disease associated with OPA1 expression in a patient, the composition comprising: a) one or more antisense oligonucleotides described herein; and b) one or more pharmaceutically acceptable carriers and / or diluents.

[0031] Preferably, the disease associated with OPA1 expression is associated with a decrease in the level of functional OPA1 protein expression. Preferably, the decrease in functional OPA1 is in patients with ADOA, more preferably in ADOA associated with OPA1 haploinsufficiency.

[0032] Also provided are methods for treating, preventing, or alleviating the effects of a disease associated with OPA1 expression, comprising: Methods are provided that include the step of: a) administering to a patient an effective amount of one or more antisense oligonucleotides described herein or a pharmaceutical composition comprising one or more antisense oligonucleotides.

[0033] Also provided is the use of a purified and isolated antisense oligonucleotide described herein for the manufacture of a medicament for treating, preventing, or ameliorating the effects of a disease associated with OPA1 expression.

[0034] Also provided are kits for treating, preventing, or ameliorating the effects of a disease associated with OPA1 expression in a patient, the kits including at least the antisense oligonucleotides and combinations or cocktails thereof described herein packaged in a suitable container, together with instructions for their use.

[0035] Preferably, the disease associated with OPA1 expression in the patient is autosomal dominant optic atrophy (ADOA). The subject with a disease associated with OPA1 expression can be a mammal, including a human.

[0036] Further aspects of the present invention will now be described with reference to the accompanying non-limiting examples and drawings. [Brief explanation of the drawings]

[0037] [Figure 1]Schematic diagram of antisense oligonucleotides that target intron 7 of OPA1 and remove exon 7x. (A) Illustration of intron 7 and exon 7x in OPA1, SEQ ID NO: 32. (B) Antisense oligonucleotides were designed to target the upstream region of OPA1 exon 7x (intron 7). PTC, premature stop codon. [Figure 2] Screening of PMOs (25 μM and 50 μM) in ADOA patient fibroblasts is shown. Patient fibroblasts were transfected for 48 hours with PMOs targeting the deletion of OPA1 exon 7x, as indicated. OPA1 transcript expression was assessed by digital droplet PCR (ddPCR) and normalized to GAPDH, RPL27, and SCL25A3 transcript levels. OPA1 expression in untreated cells was set to 1. [Figure 3] Screening of PMOs (50 μM and 100 μM) in ADOA patient fibroblasts. (A) Western blot gel image showing expression of the long and short OPA1 isoforms in patient fibroblasts transfected with PMOs targeting intron 7 of the OPA1 transcript at 48 hours. (B) Band intensity of OPA1 expression was normalized to beta-actin (evaluated by ImageJ™). OPA1 expression in untreated cells was set to 1. [Figure 4] Screening of cell-penetrating peptide-conjugated PMOs (PPMOs) (5, 10, and 20 μM) in ADOA patient fibroblasts. ADOA patient fibroblasts were transfected for 5 days with PPMOs targeting intron 7 of the OPA1 transcript, as indicated. OPA1 transcript expression was assessed by ddPCR and normalized to HPRT1. OPA1 expression in untreated cells was set to 1. [Figure 5]Schematic diagram of the refinement of antisense oligonucleotides to improve OPA1 upregulation. (A) Illustration of OPA1 exons and the location of exon 7x present in the transcript. (B) Binding region of the parent PMO on the OPA1 transcript upstream of exon 7x. Exon 7x is not drawn to scale. (C) Binding region of the daughter sequence by microwalking, nucleotide base substitution, and extension to improve PMO efficacy. [Figure 6] Screening of Microwalk PMO (25 μM and 50 μM) in ADOA patient fibroblasts is shown. Patient fibroblasts were transfected in triplicate for 48 h with PMOs targeting the deletion of OPA1 exon 7x, as indicated. Experiments were performed with one to four biological replicates, as indicated by the number of data points in the bar graph. OPA1 transcript expression was assessed by ddPCR and normalized to HPRT1 transcript levels. OPA1 expression in untreated cells was set to 1. [Figure 7] Figure 1 shows the efficacy of PMO OPA1_Ex7xA(-134-105)2mmA>G (25 μM and 50 μM) for inducing OPA1 protein upregulation in ADOA patient fibroblasts. OPA1 protein expression was assessed using a WB assay, and the band intensity of OPA1 expression was normalized to HPRT-1 (evaluated by ImageJ™). OPA1 expression in untreated cells was set to 1. [Figure 8]The balance of major OPA1 isoforms in transcripts with and without exon 7 after PMO treatment is shown. Evaluation of total OPA1 expression and OPA1 isoforms with (top) and without (bottom) exon 7 in PMO-treated and untreated fibroblasts derived from ADOA patients. Total OPA1 transcript expression was assessed using ddPCR and normalized to HPRT1 transcript levels. OPA1 expression in untreated cells was set to 1. OPA1 isoform expression was analyzed by RT-PCR following automated capillary electrophoresis separation using a LabChip GX analyzer to quantify band intensities of OPA1 isoforms. [Figure 9] Quantitative analysis of the major OPA1 isoform after PMO treatment is shown. The PMO OPA1_Ex7xA(-134-105)2mmA>G (25 μM and 50 μM) was incubated in patient-derived fibroblasts, and samples were collected for quantification of total and OPA1 isoform upregulation using a ddPCR assay. Primers targeting exons 22-23 and exons 6-8 were used to determine the levels of total OPA1 mRNA and the OPA1 splice isoform without exon 7, respectively. Both total OPA1 mRNA and the major OPA1 isoform were normalized to the expression level of HPRT1. OPA1 expression in untreated cells was set to 1. Statistical significance of data from three technical replicates was tested using two-way ANOVA. *p<0.05, **p<0.01. [Figure 10]Figure 1 shows PPMO-mediated upregulation of total OPA1 mRNA in iPSC-RGCs derived from ADOA patients. Peptide-conjugated PMOs were incubated with iPSC-RGCs at concentrations of 5, 10, and 20 μM for 120 hours, and RNA was harvested and analyzed for total OPA1 mRNA levels. OPA1 transcript expression was assessed by ddPCR and normalized to HPRT1 levels. OPA1 expression in untreated cells was set to 1. Two-way ANOVA was used to test statistical significance of data from three technical replicates. **p<0.01, ****p<0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0038] Detailed Description of the Invention general Throughout this specification, unless expressly stated otherwise or unless the context requires otherwise, a reference to a single step, composition of matter, group of steps, or group of compositions of matter should be construed to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps, or group 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, a reference to "a" includes two or more and not just the singular, a reference to "an" includes two or more and not just the singular, a reference to "the" includes two or more and not just the singular, etc.

[0039] Each example of the present disclosure described herein should be applied mutatis mutandis to each and every other example unless expressly stated otherwise.

[0040] Those skilled in the art will understand that the disclosure herein is susceptible to variations and modifications other than those specifically described. The present disclosure is to be understood to include all such variations and modifications. The present disclosure also includes all of the steps, features, compositions, and compounds referred to or shown herein, individually or collectively, and any and all combinations, or any two or more of the steps or features described above.

[0041] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure as described herein.

[0042] This disclosure will be carried out without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid-phase peptide synthesis, and immunology, as described and explained throughout the literature, including Perbal 1984, Sambrook et al., 2001, Brown (editor) 1991, Glover and Hames (editors) 1995 and 1996, Ausubel et al. (including all updates until present), Coligan et al. (editors) (including all updates until present), Maniatis et al. 1982, Gait (editor) 1984, Hames and Higgins (editors) 1984, Freshney (editor) 1986, and the like.

[0043] 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 interpreted as providing explicit support for both meanings or either meaning.

[0044] The term "about," unless indicated to the contrary, refers to + / - 20%, more preferably + / - 10%, of the specified value. For the avoidance of doubt, the term "about" preceding a specified value should be interpreted as including the exact specified value itself (e.g., "about 10" includes exactly 10).

[0045] 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.

[0046] The terms "antisense oligonucleotide," "antisense oligomer," or "ASO," as used herein, encompass oligonucleotides and any other oligomeric molecules containing nucleobases capable of hybridizing to a complementary sequence on a target RNA transcript, including, but not limited to, those that do not contain sugar moieties, such as in the case of peptide nucleic acids (PNAs). Preferably, the ASO is an ASO that is resistant to nuclease cleavage or degradation.

[0047] As used herein, the phrases "bind to a target moiety" or "bind within a target moiety" with respect to an ASO or antisense RNA (AR) refer to specific hybridization between the ASO or AR nucleotide sequence and a target nucleotide sequence that is complementary within the scope set forth herein. In some instances, specific hybridization occurs when hybridization occurs under high stringency conditions under ex vivo conditions. "High stringency conditions" means that the ASO or AR hybridizes to the target sequence under such ex vivo conditions in an amount detectably stronger than nonspecific hybridization. Thus, high stringency conditions are conditions that distinguish polynucleotides with exact complementary sequences, or those containing only a few scattered mismatches, from random sequences that happen to have a few small regions (e.g., 1-5 bases) that match the probe. Such small regions of complementarity are more easily resolved than full-length complements of 12-17 or more bases, and moderate stringency hybridization makes them easily distinguishable. In one example, high stringency conditions include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70° C. One of skill in the art will understand that under in vivo conditions, the specificity of hybridization between an ASO or AR and its target sequence is defined in terms of the level of complementarity between the ASO or AR and the target sequence to which it hybridizes in a cell.

[0048] The term "peptide" is intended to include compounds composed of amino acid residues linked by amide bonds. Peptides can be natural or non-natural, ribosomal-encoded, or synthetic. Typically, peptides consist of 2 to 200 amino acids. For example, peptides can have lengths ranging from 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 the above ranges. Peptides can 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.

[0049] As referred to herein, peptides include "inverso" peptides, in which all L-amino acids are replaced with the corresponding D-amino acids, and "retro-inverso" peptides, in which the amino acid sequence is reversed and all L-amino acids are replaced with D-amino acids.

[0050] Peptides can contain both L- and / or D-amino acids. For example, both L- and D-forms can be used for different amino acids within the same peptide sequence. In some instances, amino acids within a peptide sequence are L-forms, such as natural amino acids. In some instances, amino acids within a peptide sequence are a combination of L- and D-forms. Furthermore, peptides can contain unconventional, but naturally occurring, amino acids, including, but not limited to, hydroxyproline (Hyp), beta-alanine, citrulline (Cit), ornithine (Orn), norleucine (Nle), 3-nitrotyrosine, nitroarginine, and pyroglutamic acid (Pyr). Peptides can also incorporate unnatural amino acids, including, but not limited to, homoamino acids, N-methyl amino acids, alpha-methyl amino acids, beta (homo)amino acids, gamma amino acids, and N-substituted glycines. Peptides can be linear or cyclic.

[0051] The term "protein" should be interpreted to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains covalently or non-covalently bonded to one another (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently bonded using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0052] The percentage amino acid sequence identity for a given amino acid sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Amino acid sequence identity can be determined using the EMBOSS Pairwise Alignment Algorithms tool available from The European Bioinformatics Institute (EMBL-EBI), part of the European Molecular Biology Laboratory. This tool is available on the website at www.ebi.ac.uk / Tools / emboss / align / . This tool uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). Default settings are used, including Gap Open: 10.0 and Gap Extend 0.5. The default matrix "Blosum62" is used for the amino acid sequence and the default matrix. Percent (%) or percentage "nucleic acid sequence identity" for a nucleotide sequence disclosed herein is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST or ALIGN. Those skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the entire length of the sequences being compared.

[0053] The term "cell-penetrating peptide" (CPP) refers to a peptide that can cross a cell membrane. In one example, a CPP can translocate across a mammalian cell membrane and enter the cell. In another example, a CPP can target a conjugate to a desired subcellular compartment. Thus, a CPP can direct or facilitate penetration of a molecule of interest across a phospholipid, mitochondrial, endosomal, lysosomal, vesicle, or nuclear membrane. A CPP can translocate across a membrane with its amino acid sequence intact or, alternatively, partially degraded.

[0054] A CPP can direct a molecule of interest, such as an ASO disclosed herein, from the extracellular space through the plasma membrane to the cytoplasm or a desired subcellular compartment. Alternatively, or in addition, a CPP can direct a molecule of interest across the blood-brain, mucosal, blood-retinal, skin, gastrointestinal, and / or pulmonary barriers.

[0055] The term "peptide ligand" or "receptor-binding domain" refers to a peptide capable of binding to a membrane surface receptor to enable translocation of the peptide across the cell membrane. In one example, the peptide ligand may enable translocation across the cell membrane via the natural endocytosis of the target receptor. In another example, the peptide ligand may utilize a complementary mechanism of translocation across the cell membrane, including utilizing a conjugated CPP. In one example, the peptide ligand may translocate across a mammalian cell membrane and enter the cell. In another example, the peptide ligand may target the conjugate to a desired subcellular compartment. Thus, the peptide ligand may direct or promote cellular uptake of a molecule of interest through phospholipids, mitochondria, endosomes, lysosomes, vesicles, or nuclear membranes. The peptide ligand may translocate across the membrane with its amino acid sequence intact or, alternatively, partially degraded.

[0056] Peptide ligands, via binding to target receptors, can direct molecules of interest, such as the ASOs disclosed herein, from the extracellular space through the plasma membrane to the cytoplasm or desired subcellular compartment. Alternatively, or in addition, peptide ligands, via binding to target receptors, can direct molecules of interest across relevant biological barriers, such as the blood-brain, mucosal, blood-retinal, skin, gastrointestinal, and / or pulmonary barriers.

[0057] Compositions for increasing OPA1 mRNA and / or protein levels ADOA is characterized by degeneration of retinal ganglion cells, and at least 75% of cases are caused by mutations in the OPA1 gene. OPA1 is ubiquitously expressed and is most abundant in the retina. The OPA1 protein is a mitochondrial GTPase that plays an important role in regulating mitochondrial structure, function, and apoptosis.

[0058] The unique distribution of mitochondria in retinal ganglion cells (RGCs) points to the critical function of this network and may underlie the susceptibility of these cells to OPA1 loss (potentially also caused by exposure of these cells to photooxidative stress). The majority of OPA1 mutations (approximately 27% missense, 27% splice variant, 23.5% frameshift, 16.5% nonsense, and 6% deletion or duplication) cause transcript degradation by mRNA decay, supporting the hypothesis that haploinsufficiency is a major mechanism underlying ADOA pathogenesis. OPA1 mutations that cause haploinsufficiency result in impaired mitochondrial function, including defective complex I-driven ATP synthesis. Because ATP is the primary energy currency within cells, insufficient ATP ultimately leads to cellular dysfunction and death.

[0059] OPA1 haploinsufficiency accounts for a significant proportion of ADOA cases, and OPA1 haploinsufficiency causing ADOA is amenable to treatment by upregulating wild-type OPA1.

[0060] The present invention provides a means for increasing the level of functional OPA1 protein expression. Preferably, the increase in functional OPA1 is in patients with ADOA, more preferably in ADOA associated with OPA1 haploinsufficiency.

[0061] The term "nonsense-mediated RNA decay (NMD) exon" or "NMD exon" refers to an exon or pseudoexon present in a region within an intron that, when included in a mature RNA transcript, can activate the NMD pathway. The NMD pathway is triggered when the exon or pseudoexon contains a premature termination codon (PTC) or induces a frameshift in the transcript. In constitutive splicing events, introns containing NMD exons are typically spliced ​​out, but the intron or portions thereof may be retained during alternative or aberrant splicing events. Mature mRNA transcripts containing such NMD exons may be unproductive due to a reading frameshift that induces the NMD pathway. Inclusion of an NMD exon in a mature OPA1 RNA transcript can downregulate overall OPA1 mRNA and protein expression.

[0062] Introns are removed by a large RNA-protein complex called the spliceosome, which orchestrates complex interactions between the primary transcript, small nuclear RNAs (snRNAs), and numerous proteins. The spliceosome assembles on each intron in an ordered manner, beginning with recognition of the 5' splice site (5'ss) by U1 snRNA or the 3' splice site (3'ss) by the U2 pathway. This involves binding of the U2 accessory factor (U2AF) to the 3'ss region, facilitating U2 binding to the branch point sequence (BPS). U2AF is a stable heterodimer composed of a U2AF2-encoded 65 kD subunit (U2AF65), which binds to the polypyrimidine tract (PPT), and a U2AF1-encoded 35 kD subunit (U2AF35), which interacts with a highly conserved AG dinucleotide at the 3'ss, stabilizing U2AF65 binding. In addition to the BPS / PPT units and 3'ss / 5'ss, accurate splicing requires auxiliary sequences or structures known as intronic or exonic splicing enhancers or silencers that activate or repress splice site recognition. These elements enable true splice sites to be recognized among the vast number of cryptic or pseudosites in the genomes of higher eukaryotes, which have the same sequence but outnumber the true sites by an order of magnitude.

[0063] Recently, it has been recognized that missplicing of many mammalian genes results in one or more NMD transcripts. Therefore, for such genes, the steady-state levels of productive RNA transcripts and functional proteins for that gene are reduced in proportion to the abundance of NMD transcripts. Despite this "inefficiency," in a genetic background of two functional (wild-type) alleles, the levels of productive RNA transcription and translation result in sufficient levels of functional protein for a given gene. However, in the case of certain monogenic diseases, loss of one functional allele results in haploinsufficiency and the associated disease. Therefore, a useful strategy for treating such diseases seeks to increase the levels of the associated protein expressed from the remaining healthy allele in affected target tissues. Recently, it has been shown that for a given gene of interest, pre-mRNA can be targeted with antisense oligonucleotides, which can reduce the level of missplicing of that pre-mRNA and increase the number of productive transcripts (Lim et al., 2020, Nature Communications, 11:3501).

[0064] Strategies using ASOs to exclude mutation-induced retained introns by PTCs have been used to treat β-thalassemia (Seirakowska et al.; Proc Natl Acad Sci USA 1996;93(23):12840-12844), cystic fibrosis (Friedman et al.; Nucleic Acids, Protein Synthesis, and Molecular Genetics 1999;274(51):36193-36199), and ataxia-telangiectasia (Du et al.; Proc Natl Acad Sci USA 2007 Apr 3;104(14):6007-6012). Further use of intron-containing PTCs as gene expression regulation mechanisms has been reviewed in Lewis et al (Proc Natl Acad Sci USA 2003;100(1):189-92), Mendell et al Science 2002;298(5592):419-22), Ni et al (Genes Dev 2007;21(6):708-718), Wong et al (Cell 2013;154:583-595), and Aartsma-Rus and Ommen (RNA 2007;13(10):1609-1624).

[0065] The terms "antisense oligonucleotide," "antisense oligonucleotide," "oligonucleotide," and "antisense compound" may be used interchangeably to refer to an oligonucleotide.

[0066] Without wishing to be bound by theory, it is believed that antisense sequences may sterically hinder access of the spliceosome to the target splice site sequence by binding at or near the splicing motif.

[0067] The OPA1 gene contains an intron with a premature termination codon (PTC) in intron 7 (located between exons 7 and 8). In all subjects, a proportion of OPA1 RNA transcripts derived from the wild-type OPA1 gene retain the segment of intron 7 containing this PTC; this retained intron segment is referred to as exon 7x in the transcribed RNA. RNA transcripts containing exon 7x (the retained intron segment containing the PTC) are subject to nonsense-mediated RNA decay. Thus, in all subjects, a proportion of OPA1 RNA is translated into the mature wild-type protein, while a portion of OPA1 RNA is almost immediately degraded by RNase due to the presence of the PTC. This immediately degraded OPA1 RNA does not affect vision in normal subjects, as combined production of functional protein from the two available healthy alleles is sufficient for normal, healthy function of the eye. Recently, OPA1 exon 7 has also been referred to as exon 5b, and intron 7x has been referred to as intron 5x. Thus, a reference herein to exon 7 is also a reference to exon 5, and a reference herein to intron 7x is also a reference to intron 5x.

[0068] However, if a subject has a mutation in one allele of the OPA1 gene, they rely on the production of normal functional protein from the remaining, non-mutated OPA1 gene alone, and the amount of functional OPA1 protein produced by the single wild-type gene is insufficient for the needs of the eye.

[0069] The present invention seeks to address this insufficiency by ensuring that an increased amount of OPA1 transcript is produced due to the lack of exon 7x in the wild-type OPA1 gene. This means that the increased amount of RNA transcribed from the wild-type OPA1 gene can be translated into a functional protein because the additional RNA is "rescued" from conversion to an RNA form subject to nonsense-mediated RNA decay (NMD) by the presence of the PTC in exon 7x. This additional functional protein can then compensate for the inability of the mutant allele to produce a functional OPA1 protein.

[0070] Antisense oligonucleotides (ASOs) and antisense RNAs (Ars) The present invention seeks to provide one or more antisense oligonucleotides (ASOs) that reduce or prevent the inclusion of the portion of intron 7 that becomes the NMD-prone exon 7x in mRNA transcripts.

[0071] Antisense oligonucleotides were designed to bind to target nucleic acid sites in OPA1 pre-mRNA transcripts. Binding to representative OPA1 transcripts is shown in Figures 1 and 5 and pertains to all OPA1 transcripts.

[0072] Generally, according to one aspect of the present invention, there is provided an isolated or purified antisense oligonucleotide that targets intron 7 or a portion thereof of the OPA1 gene transcript. Preferably, the isolated or purified antisense oligonucleotide prevents the aberrant retention of part or all of intron 7 during pre-mRNA splicing, thus avoiding the production of OPA1 mRNA containing an NMD-prone PTC that contains an aberrantly retained exon 7x derived from intron 7, thereby increasing translation of functional OPA1 protein. Preferably, the isolated or purified antisense oligonucleotide is for inducing increased production of the OPA1 gene transcript or a portion thereof.

[0073] The antisense oligonucleotide may be selected from Table 1 and Table 2. Preferably, the antisense oligonucleotide is selected from the list comprising SEQ ID NOs: 1 to 31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3, and 4.

[0074] The antisense oligonucleotide may be selected from Tables 1, 2 and 4. Preferably, the antisense oligonucleotide is selected from the list comprising SEQ ID NOs: 1 to 31 and 33 to 54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7 and 33 to 44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40 and 44.

[0075] For example, one embodiment of the present invention provides antisense oligonucleotides of 10 to 50 nucleotides that contain a targeting sequence complementary to a region near or within intron 7 or a portion thereof of the OPA1 gene transcript. Another embodiment of the present invention provides antisense oligonucleotides of 10 to 50 nucleotides that contain a targeting sequence complementary to a region near or within an exon or a portion thereof of the OPA1 gene transcript.

[0076] 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 is 26 nucleotides. In another example, the nucleotide sequence of the ASO is 27 nucleotides. In another example, the nucleotide sequence of the ASO is 28 nucleotides. In another example, the nucleotide sequence of the ASO is 29 nucleotides. In another example, the nucleotide sequence of the ASO is 30 nucleotides.

[0077] Generally, one aspect of the present invention provides isolated or purified antisense oligonucleotides designed to hybridize to a region within intron 7 or a portion thereof of the OPA1 gene transcript and prevent retention of all or part of intron 7. The antisense oligonucleotides (ASOs) are designed to target intron 7 with the goal of preventing retention of all or part of intron 7, including exon 7x, by blocking an intronic splice enhancer or other splicing motif. This disruption preferably regulates expression of functional OPA1 protein by increasing the level of correctly spliced ​​wild-type OPA1 gene transcript and, therefore, OPA1 protein. Increased OPA1 protein production can be useful in treating diseases associated with OPA1 expression, particularly ADOA.

[0078] In general, according to another aspect of the invention, there are provided isolated or purified antisense oligonucleotides designed to increase translation of functional OPA1 protein through binding to intron 7.

[0079] For example, one embodiment of the present invention provides antisense oligonucleotides of 10 to 50 nucleotides that contain a targeting sequence complementary to a region near or within intron 7 or a portion thereof of the OPA1 gene transcript. Another embodiment of the present invention provides antisense oligonucleotides of 10 to 50 nucleotides that contain a targeting sequence complementary to a region near or within exon 7 or a portion thereof of the OPA1 gene transcript.

[0080] The antisense oligonucleotide may be selected from Table 1 and Table 2. Preferably, the antisense oligonucleotide is selected from the list comprising SEQ ID NOs: 1 to 31, more preferably SEQ ID NOs: 1, 2, 3 and 4, even more preferably SEQ ID NOs: 1, 3 and 4.

[0081] The antisense oligonucleotide may be selected from Tables 1, 2 and 4. Preferably, the antisense oligonucleotide is selected from the list comprising SEQ ID NOs: 1 to 31 and 33 to 54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7 and 33 to 44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40 and 44.

[0082] Preferably, the antisense oligonucleotide of the present invention comprises: It hybridizes to intron 7 of the OPA1 gene transcript and prevents the aberrant retention of part or all of intron 7 during pre-mRNA splicing, thus avoiding the production of OPA1 mRNA containing NMD-prone PTCs containing the aberrantly retained exon 7x derived from intron 7, thereby increasing the translation of functional OPA1 protein.

[0083] In the context of this document, "aberrant splicing," "aberrantly spliced," and "aberrant retention" refer to alternative OPA1 gene transcripts that retain all or part of their intron 7. Intron retention may function as a regulatory mechanism to fine-tune the expression of various genes in specific cell types. It is unclear whether retention of all or part of OPA1 intron 7 serves this purpose.

[0084] Preferably, the antisense oligonucleotide of the present invention inhibits the following in cells of a patient with an OPA1 mutation: ●Repairs mitochondrial structure, Increase mitochondrial ATP levels, and / or • Acts to reduce apoptosis.

[0085] Preferably, the antisense oligonucleotide of the present invention inhibits the following in cells of a patient with an OPA1 mutation: ●Repairs mitochondrial structure, Increases oxygen consumption rate, Increase mitochondrial ATP levels, and / or • Acts to reduce apoptosis.

[0086] The antisense oligonucleotide of the present invention can comprise the sequence that can hybridize to such sequence under stringent hybridization conditions, the sequence that is complementary to it, the sequence that contains modified bases, the modified backbone, and its functional truncation or extension that has or regulates the splicing activity in gene transcription product.In certain embodiments, antisense oligonucleotide can be 100% complementary to target sequence, or can comprise mismatches, for example, to accommodate variants, as long as the heteroduplex formed between oligonucleotide and target sequence is stable enough to withstand the action of cellular nuclease and other degradation modes that may occur in vivo. Thus, a particular oligonucleotide may have about or at least about 70% sequence complementarity between the oligonucleotide and the target sequence, e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity.

[0087] 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 target portion over the length of the ASO.

[0088] The present invention also extends to combinations of two or more antisense oligonucleotides capable of binding to a selected target to induce increased translation of functional OPA1 protein, including constructs containing two or more antisense oligonucleotides, which constructs can be used in antisense oligonucleotide-based therapy.

[0089] According to a still further aspect, the present invention extends to cDNA or cloned copies of the antisense oligonucleotide sequences of the invention, and to vectors containing the antisense oligonucleotide sequences of the invention. The present invention further extends to cells containing such sequences and / or vectors.

[0090] "Isolated" refers to material that is substantially or essentially free from components that normally accompany it in its natural state. For example, as used herein, "isolated polynucleotide" or "isolated oligonucleotide" can refer to a polynucleotide that has been purified or removed from sequences that flank it in its naturally occurring state, e.g., a DNA fragment that has been removed from sequences adjacent to the fragment in a genome. The term "isolate" in reference to a cell refers to the purification of a cell (e.g., a fibroblast, lymphoblast) from a source subject (e.g., a subject with a polynucleotide repeat disorder). In the context of RNA, mRNA, or protein, "isolate" refers to the recovery of RNA, mRNA, or protein from a source, e.g., a cell.

[0091] Antisense oligonucleotides can be said to be "directed" or "targeted" to the target sequence to which they hybridize. In certain embodiments, the target sequence includes a region near or within intron 7. The target sequence is preferably within intron 7, more preferably near the 5' end of intron 7 and / or near the junction of exon 7 and intron 7. Antisense oligonucleotides can be selected from Tables 1 and 2. Preferably, antisense oligonucleotides are selected from the list comprising SEQ ID NOS: 1-31, more preferably SEQ ID NOS: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOS: 1, 3, and 4. Antisense oligonucleotides can be selected from Tables 1, 2, and 4. Preferably, antisense oligonucleotides are selected from the list comprising SEQ ID NOS: 1-31 and 33-54, more preferably SEQ ID NOS: 1, 2, 3, 4, 7, and 33-44, even more preferably SEQ ID NOS: 1, 3, 4, 37, 40, and 44.

[0092] In certain embodiments, the antisense oligonucleotide has sufficient sequence complementarity to the target RNA (i.e., the RNA whose translation is regulated) to effectively block a region of the target RNA (e.g., mRNA), such as binding to intron 7 in an exemplary embodiment.

[0093] An antisense oligonucleotide that has sufficient sequence complementarity to a target RNA sequence to bind to a portion of the target RNA means that the antisense oligonucleotide has sufficient sequence to alter the three-dimensional structure of the target RNA.

[0094] The selected antisense oligonucleotide can be made shorter, e.g., about 12 bases, or longer, e.g., about 50 bases, containing a small number of mismatches, so long as the sequence is sufficiently complementary to result in mRNA structure upon hybridization to the target sequence, and optionally forms a duplex with the RNA having a Tm of 45°C or higher.

[0095] Preferably, the antisense oligonucleotide is selected from the group comprising the sequences shown in Table 1 and Table 2. Preferably, the antisense oligonucleotide is selected from the group comprising the sequences of SEQ ID NOs: 1 to 31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3, and 4.

[0096] Preferably, the antisense oligonucleotides are selected from the group comprising the sequences shown in Tables 1, 2 and 4. Preferably, the antisense oligonucleotides are selected from the group comprising the sequences of SEQ ID NOs: 1 to 31 and 33 to 54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7 and 33 to 44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40 and 44.

[0097] In certain embodiments, the degree of complementarity between the target sequence and the antisense oligonucleotide is sufficient to form a stable duplex. The region of complementarity between the antisense oligonucleotide and the target RNA sequence can be as short as 8-11 bases, but can also be 12-15 bases or longer, for example, 10-50 bases, 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases, or 15-20 bases, including all integers between these ranges. An antisense oligonucleotide of about 16-17 bases is generally long enough to have a unique complementary sequence. In certain embodiments, as discussed herein, a minimum length of complementary bases may be required to achieve the required binding Tm.

[0098] In certain embodiments, oligonucleotides of about 50 bases in length may be suitable, with at least a minimum number of bases, for example, 10-12 bases, being complementary to the target sequence.However, in general, enhanced or active uptake in cells is optimized with an oligonucleotide length of less than about 30 bases.For phosphorodiamidate morpholino oligonucleotide (PMO) antisense oligonucleotides, the optimal balance of binding stability and uptake generally occurs with a length of 17-25 bases. Included are antisense oligonucleotides (e.g., CPP-PMO, PPMO, PMO, PMO-X, PNA, LNA, 2'-Ome, 2'-MOE, 2'-F oligonucleotides, thiomorpholino and other hybrid oligonucleotide chemistries) consisting of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 bases. PMO-phosphorodiamidate morpholino oligonucleotide, CPP-cell penetrating peptide, PPMO-peptide conjugated phosphorodiamidate morpholino oligonucleotide, PNA-peptide nucleic acid, LNA-locked nucleic acid, 2'-Ome-2'-O-methyl modified oligonucleotide, 2'-MOE-2'-O-methoxyethyl oligonucleotide, 2'-F-2'-fluoro

[0099] In certain embodiments, antisense oligonucleotides may be 100% complementary to the target sequence, or may contain mismatches, for example, to accommodate variants, so long as the heteroduplex formed between the oligonucleotide and the target sequence is sufficiently stable to withstand the action of cellular nucleases and other degradation modes that may occur in vivo. Thus, a particular oligonucleotide may have about or at least about 60% sequence complementarity between the oligonucleotide and the target sequence, e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity.

[0100] When mismatches exist, they are typically less unstable towards the end regions of the hybrid double strand than those in the middle.The number of mismatches that can be tolerated will depend on the length of oligonucleotide, the percentage of G:C nucleic acid base pairs in the double strand, and the position of the mismatch in the double strand, according to the well-understood principle of double strand stability.Although such antisense oligonucleotides are not necessarily 100% complementary to target sequences, they can effectively bind stably and specifically to target sequences, so as to improve the efficiency of mRNA translation.

[0101] The stability of the duplex formed between an antisense oligonucleotide and a target sequence is a function of the binding Tm and the susceptibility of the duplex to cellular enzymatic cleavage. The Tm of an oligonucleotide with respect to its complementary sequence RNA can be measured by conventional methods, such as those described by Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or Miyada CG and Wallace RB, 1987, Oligonucleotide Hybridization Techniques, Methods Enzymol. Vol. 154, pp. 94-107. In certain embodiments, the antisense oligonucleotide may have a binding Tm with respect to its complementary sequence RNA above body temperature, preferably above about 45°C or 50°C. Tms in the range of 60-80°C or higher are also included.

[0102] Additional examples of variants include antisense oligonucleotides having about or at least about 60% sequence identity, e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, over the entire length of any of the sequences provided in Table 1 and Table 2, or any of SEQ ID NOs: 1-31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3 and 4.

[0103] In one example, the antisense oligonucleotides of the present disclosure have about or at least about 75% sequence identity, e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, over the entire length of any of the sequences provided in Table 1 and Table 2, or any of SEQ ID NOs: 1-31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3 and 4. For example, antisense oligonucleotides of the present disclosure have about or at least about 80% sequence identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, over the entire length of any of the sequences provided in Table 1 and Table 2, or any of SEQ ID NOs: 1-31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3 and 4. In another example, the antisense oligonucleotides of the present disclosure have about or at least about 85% sequence identity, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, over the entire length of any of the sequences provided in Table 1 and Table 2, or any of SEQ ID NOs: 1-31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3 and 4. In further examples, the antisense oligonucleotides of the present disclosure have about or at least about 90% sequence identity, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, over the entire length of any of the sequences provided in Table 1 and Table 2, or any of SEQ ID NOs: 1-31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3 and 4.In one example, the antisense oligonucleotides of the present disclosure have about or at least about 95% sequence identity, e.g., 95%, 96%, 97%, 98%, 99% or 100% sequence identity, over the entire length of any of the sequences provided in Table 1 and Table 2, or any of SEQ ID NOs: 1-31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3 and 4.

[0104] Additional examples of variants include those that share about or at least about 60% sequence identity, e.g., 60%, 61%, 62%, 63%, 64%, 65%, or more preferably, 66% or more of the sequences provided in Tables 1, 2, and 4, or any of SEQ ID NOs: 1-31 and 33-54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7, and 33-44, and even more preferably SEQ ID NOs: 1, 3, 4, 37, 40, and 44. , 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0105] In one example, the antisense oligonucleotides of the present disclosure have about or at least about 75% sequence identity, e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, over the entire length of any of the sequences provided in Tables 1, 2 and 4, or any of SEQ ID NOs: 1-31 and 33-54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7 and 33-44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40 and 44. For example, antisense oligonucleotides of the present disclosure have about or at least about 80% sequence identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, over the entire length of any of the sequences provided in Tables 1, 2 and 4, or any of SEQ ID NOs: 1-31 and 33-54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7 and 33-44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40 and 44. In another example, the antisense oligonucleotides of the present disclosure have about or at least about 85% sequence identity, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, over the entire length of any of the sequences provided in Tables 1, 2 and 4, or any of SEQ ID NOs: 1-31 and 33-54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7 and 33-44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40 and 44. In further examples, the antisense oligonucleotides of the present disclosure have about or at least about 90% sequence identity, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, over the entire length of any of the sequences provided in Tables 1, 2 and 4, or any of SEQ ID NOs: 1-31 and 33-54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7 and 33-44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40 and 44.In one example, the antisense oligonucleotides of the present disclosure have about or at least about 95% sequence identity, e.g., 95%, 96%, 97%, 98%, 99% or 100% sequence identity, over the entire length of any of the sequences provided in Tables 1, 2 and 4, or any of SEQ ID NOs: 1-31 and 33-54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7 and 33-44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40 and 44.

[0106] More specifically, antisense oligonucleotides are provided that can bind to selected target sites and increase nonsense-mediated RNA decay (NMD) exon exclusion in OPA1 gene transcripts or portions thereof. The antisense oligonucleotides are preferably selected from those provided in Tables 1 and 2, or SEQ ID NOs: 1-31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, and even more preferably SEQ ID NOs: 1, 3, and 4.

[0107] More specifically, antisense oligonucleotides are provided that can bind to selected target sites and increase nonsense-mediated RNA decay (NMD) exon exclusion in OPA1 gene transcripts or portions thereof. The antisense oligonucleotides are preferably selected from those provided in Tables 1, 2, and 4, or SEQ ID NOS: 1-31 and 33-54, more preferably SEQ ID NOS: 1, 2, 3, 4, 7, and 33-44, and even more preferably SEQ ID NOS: 1, 3, 4, 37, 40, and 44.

[0108] The modification of mRNA translation preferably increases the production of functional OPA1 protein by excluding nonsense-mediated RNA decay (NMD) exons. The modification of mRNA translation of the present invention using antisense oligonucleotides can promote the exclusion of nonsense-mediated RNA decay (NMD) exon 7x from intron 7 during splicing of the OPA1 pre-mRNA.

[0109] The antisense oligonucleotides of the present invention can be a combination of two or more antisense oligonucleotides capable of binding to a selected target to induce nonsense-mediated RNA decay (NMD) exon exclusion. The combination can be a cocktail of two or more antisense oligonucleotides and / or a construct comprising two or more antisense oligonucleotides linked together.

[0110] In the present invention, antisense oligonucleotides are also known as antisense oligonucleotides, ASOs, Aos, AONs, antisense phosphorodiamidate morpholino oligomers, PMOs, peptide-conjugated PMOs and PPMOs, and these terms are used interchangeably.

[0111] Preferably, the antisense oligonucleotide is selected from the group comprising the sequences shown in Table 1 and Table 2. Preferably, the antisense oligonucleotide is selected from the group comprising the sequences of SEQ ID NOs: 1 to 31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3, and 4.

[0112] Preferably, the antisense oligonucleotides are selected from the group comprising the sequences shown in Tables 1, 2 and 4. Preferably, the antisense oligonucleotides are selected from the group comprising the sequences of SEQ ID NOs: 1 to 31 and 33 to 54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7 and 33 to 44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40 and 44. [Table 1] [Table 2] [Table 3] [Table 4] SEQ ID NO: 55. Exemplary amino acid sequence of a CPP for PMO conjugation: RRSRTARAGRPGRNSSRPSAPRGASGGASG

[0113] Also provided is a method for excluding an NMD exon in an OPA1 gene transcript, the method comprising the steps of: a) providing one or more of the antisense oligonucleotides described herein and allowing the oligonucleotide to bind to a target nucleic acid site.

[0114] According to yet another aspect of the present invention, there is provided a target nucleic acid sequence for exclusion of the NMD exon in OPA1, comprising the DNA equivalent of a nucleic acid sequence selected from the group consisting of Table 1 and Table 2, or SEQ ID NOs: 1-31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3, and 4.

[0115] According to yet another aspect of the present invention, there is provided a target nucleic acid sequence for exclusion of an NMD exon in OPA1, comprising the DNA equivalent of a nucleic acid sequence selected from the group consisting of Tables 1, 2 and 4, or SEQ ID NOs: 1-31 and 33-54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7 and 33-44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40 and 44.

[0116] Designing an antisense oligonucleotide to sterically block splicesome access to the target splice site sequence by and / or adjacent to the antisense oligonucleotide may not necessarily result in changes in the splicing of the target gene transcript. Furthermore, the present inventors have discovered that the size or length of the antisense oligonucleotide itself is not necessarily a major consideration when designing an antisense oligonucleotide. For some targets, antisense oligonucleotides as short as 17 bases can induce some changes in splicing, and in certain cases, can do so more efficiently than other longer (e.g., 25 base) oligonucleotides directed to the same regulatory element.

[0117] We also found that there do not appear to be any canonical motifs that can be blocked or masked by antisense oligonucleotides to redirect splicing. While tool-assisted analysis of RNA can predict likely functional motifs within RNA sequences, we found that antisense oligonucleotides must be designed and their individual effectiveness empirically evaluated.

[0118] More specifically, the antisense oligonucleotides may be selected from those shown in Tables 1 and 2. The sequences are preferably selected from the group consisting of any one or more of SEQ ID NOS: 1-31, more preferably SEQ ID NOS: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOS: 1, 3, and 4, and combinations or cocktails thereof. More specifically, the antisense oligonucleotides may be selected from those shown in Tables 1, 2, and 4. The sequences are preferably selected from the group consisting of any one or more of SEQ ID NOS: 1-31 and 33-54, more preferably SEQ ID NOS: 1, 2, 3, 4, 7, and 33-44, even more preferably SEQ ID NOS: 1, 3, 4, 37, 40, and 44, and combinations or cocktails thereof. This includes sequences capable of hybridizing to such sequences under stringent hybridization conditions, sequences complementary thereto, sequences containing modified bases, modified backbones, and functional truncations or extensions thereof that retain or modulate intron retention activity in OPA1 gene transcripts.

[0119] An oligonucleotide and a DNA, cDNA, or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity or pairing so that stable and specific binding occurs between the oligonucleotide and the DNA, cDNA, or RNA target. It is understood in the art that the sequence of an antisense oligonucleotide does not need to be 100% complementary to the sequence of its target sequence to be specifically hybridizable. In certain examples, the nucleotide sequence of the ASO 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 target portion of the RNA transcript over the length of the ASO nucleotide sequence. For example, an ASO in which 18 out of 20 nucleotides of the ASO sequence are complementary to the target region and will therefore specifically hybridize would represent 90% complementarity. In such an example, the remaining non-complementary nucleotides of the ASO can be clustered or interspersed with complementary nucleotides and need not be contiguous. The complementarity of an ASO sequence to a target nucleotide sequence (expressed as "percent complementarity" to the target sequence or "percent identity" to its reverse complement sequence) can be routinely determined using algorithms known in the art, as exemplified by the BLAST program (Basic Local Alignment Search Tool) and PowerBLAST program (Altschul, et al., 1990, J. Mol. Biol., 215:403-410; Zhang et al., 1997, Genome Res., 7:649-656).An antisense oligonucleotide is capable of specific hybridization when binding of the compound to a target DNA or RNA molecule interferes with the function of the target DNA or RNA product, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense oligonucleotide to non-target sequences under conditions where specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic treatments, or conditions under which the assay is performed in the case of in vitro assays.

[0120] Selective hybridization can occur under low, medium, or high stringency conditions, but is preferably under high stringency. Those skilled in the art will recognize that hybridization stringency is influenced by conditions such as salt concentration, temperature, and organic solvents, in addition to the base composition, length of complementary strands, and number of nucleotide base mismatches between hybridizing nucleic acids. Stringent temperature conditions generally include temperatures greater than 30°C, typically greater than 37°C, preferably greater than 45°C, and preferably at least 50°C, typically 60°C to 80°C or higher. Stringent salt conditions are usually less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is far more important than the measurement of any single parameter. An example of stringent hybridization conditions is 65°C and 0.1x SSC (1x SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). Thus, antisense oligonucleotides of the present invention may include oligonucleotides that selectively hybridize to the sequences provided in Table 1 and Table 2, or SEQ ID NOs: 1 to 31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3, and 4. Antisense oligonucleotides of the present invention may also include oligonucleotides that selectively hybridize to the sequences provided in Table 1, 2, and 4, or SEQ ID NOs: 1 to 31 and 33 to 54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7 and 33 to 44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40, and 44.

[0121] Typically, selective hybridization will occur when there is at least about 55% identity, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%, 95%, 98% or 99% identity with the nucleotide of the antisense oligonucleotide over a stretch of at least about 14 nucleotides. The length of identity comparison can be over a longer stretch as described, and in certain embodiments, often over a stretch of at least about 9 nucleotides, usually at least about 12 nucleotides, more usually at least about 20 nucleotides, often at least about 21, 22, 23 or 24 nucleotides, at least about 25, 26, 27 or 28 nucleotides, at least about 29, 30, 31 or 32 nucleotides, at least about 36 or more nucleotides.

[0122] Therefore, the antisense oligonucleotide sequence of the present invention preferably has at least 75%, more preferably at least 85%, more preferably at least 86, 87, 88, 89 or 90% homology with the sequence shown in the sequence listing herein. More preferably, there is at least 91, 92, 93, 94 or 95%, more preferably at least 96, 97, 98% or 99% identity. Generally, the shorter the length of the antisense oligonucleotide, the higher the identity required to achieve selective hybridization. Consequently, when the antisense oligonucleotide of the present invention consists of less than about 30 nucleotides, the identity percentage is preferably greater than 75%, preferably greater than 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95%, 96, 97, 98% or 99% compared to the antisense oligonucleotide shown in the sequence listing herein. Nucleotide identity comparisons may be performed using sequence comparison programs such as the GCG Wisconsin Bestfit program or GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). In this manner, sequences of similar or substantially different lengths to those cited herein can be compared by inserting gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0123] The antisense oligonucleotides of the present invention may have regions of reduced identity and regions of complete identity with the target sequence. The oligonucleotide need not have complete identity over its entire length. For example, the oligonucleotide may have a continuous stretch of at least 4 or 5 bases identical to the target sequence, preferably a continuous stretch of at least 6 or 7 bases identical to the target sequence, and more preferably a continuous stretch of at least 8 or 9 bases identical to the target sequence. The oligonucleotide may have a stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 bases identical to the target sequence. The remaining stretches of the oligonucleotide sequence may be intermittently identical to the target sequence; for example, the remaining sequence may have an identical base followed by a non-identical base followed by an identical base. Alternatively (or similarly), the oligonucleotide sequence may have several stretches of identical sequence (e.g., 3, 4, 5, or 6 bases) interspersed with stretches of less than complete identity. Such sequence mismatches preferably result in little or no loss of splice regulatory activity, and even more preferably, such sequence mismatches will result in increased activity to reduce intron retention.

[0124] The term "modulate" or "modulates" optionally includes "increasing" or "decreasing" one or more quantifiable parameters by a defined and / or statistically significant amount. The terms "increase" or "increasing," "enhance" or "enhancing," or "stimulate" or "stimulating," or "enhance" or "enhancing" generally refer to the ability of one or more antisense oligonucleotides or compositions to produce or cause a greater physiological response (i.e., downstream effect) in a cell or subject compared to the response caused by either not containing the antisense oligonucleotide or a control compound. The term "decreasing" or "decreasing" generally refers to the ability of one or more antisense oligonucleotides or compositions to produce or cause a decreased physiological response (i.e., downstream effect) in a cell or subject compared to the response caused by either not containing the antisense oligonucleotide or a control compound.

[0125] The relevant physiological or cellular response (in vivo or in vitro) will be apparent to one of skill in the art and can include an increase in the expression of functional OPA1 protein in a cell, tissue, or subject in need thereof. An "increased" or "enhanced" amount can be a statistically significant amount and can include an increase of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more times (e.g., 500, 1000-fold) (e.g., including all integers and decimal points therebetween and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8) over the amount produced in the absence of the antisense oligonucleotide (in the absence of drug) or when a control compound is used.

[0126] The terms "reduce" or "inhibit" may generally relate to the ability of one or more antisense oligonucleotides or compositions to "reduce" a relevant physiological or cellular response, such as a symptom of a disease described herein, as measured according to routine techniques in the diagnostic arts. The relevant physiological or cellular response (in vivo or in vitro) will be apparent to one of ordinary skill in the art and may include a reduction in the symptoms or pathology of a disease such as ADOA.

[0127] A "reduction" in response may be statistically significant compared to the response produced by the antisense oligonucleotide or a control composition and may include 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all integers therebetween.

[0128] The length of an antisense oligonucleotide may vary as long as it is capable of selectively binding to the intended site within an mRNA molecule. The length of such a sequence can be determined according to the selection procedures described herein. Generally, antisense oligonucleotides are about 10 nucleotides in length, with a maximum length of about 50 nucleotides. However, it is understood that any length within this range can be used in the present method. Preferably, the length of an antisense oligonucleotide is 10-40, 10-35, 15-30, or 20-30 nucleotides, and most preferably about 25-30 nucleotides. For example, the oligonucleotide may be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0129] In some examples, the ASO nucleotide sequence is 8-50 nucleotides, 8-40 nucleotides, 8-35 nucleotides, 8-30 nucleotides, 8-25 nucleotides, 8-20 nucleotides, 8-15 nucleotides, 9-50 nucleotides, 9-40 nucleotides, 9-35 nucleotides, 9-30 nucleotides, 9-25 nucleotides, 9-20 nucleotides, 9-15 nucleotides, 10-50 nucleotides, 10-40 nucleotides, 10-35 nucleotides, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, 10-15 nucleotides, 11-50 nucleotides, 11-40 nucleotides, 11-35 nucleotides, 11-30 nucleotides, 11-25 nucleotides, 11-20 nucleotides, 11-15 nucleotides, 12-50 nucleotides, 12-40 nucleotides, 12-35 nucleotides, 12-30 nucleotides, 12-25 nucleotides, 12-20 nucleotides, 12-15 nucleotides, 13-50 nucleotides, 13-40 nucleotides, 13-35 nucleotides, 13-30 nucleotides, 13-25 nucleotides, 13-20 nucleotides, 14-50 nucleotides, 14-40 nucleotides, 14-35 nucleotides, 14-30 nucleotides, 14-25 nucleotides, 14-20 nucleotides The ASO may be 15-50 nucleotides, 15-40 nucleotides, 15-35 nucleotides, 15-30 nucleotides, 15-25 nucleotides, 15-20 nucleotides, 20-50 nucleotides, 20-40 nucleotides, 20-35 nucleotides, 20-30 nucleotides, 20-25 nucleotides, 25-50 nucleotides, 25-40 nucleotides, 25-35 nucleotides, or 25-30 nucleotides in length. In some examples, the ASO is 17 nucleotides in length. In some preferred examples, the nucleotide sequence of the ASO nucleotide is 25 nucleotides in length.

[0130] As used herein, "antisense oligonucleotide" refers to a linear sequence of nucleotides or nucleotide analogs, which allows the nucleobases to hybridize to a target sequence in RNA via Watson-Crick base pairing, forming an oligonucleotide:RNA heteroduplex within the target sequence. The cyclic subunits can be based on ribose or another pentose sugar, or in certain embodiments, morpholino groups (see the description of morpholino oligonucleotides below). Also contemplated are peptide nucleic acids (PNAs) or other artificial entities that allow for polynucleotide-like structures, locked nucleic acids (LNAs), 2'-O-methyl oligonucleotides, and other antisense agents known in the art.

[0131] Non-naturally occurring antisense oligonucleotides, or "oligonucleotide analogs," include antisense oligonucleotides or oligonucleotides having (i) modified backbone structures, e.g., backbones other than the standard phosphodiester linkages found in naturally occurring oligonucleotides and polynucleotides, and / or (ii) modified sugar moieties, e.g., morpholino moieties rather than ribose or deoxyribose moieties. Oligonucleotide analogs support bases that can hydrogen bond to standard polynucleotide bases by Watson-Crick base pairing, and the backbone of the analog presents the bases in a manner that allows such hydrogen bonding in a sequence-specific manner between the oligonucleotide analog molecule and the bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). Preferred analogs are those with a substantially uncharged phosphorus-containing backbone.

[0132] One method for producing antisense oligonucleotides is methylation of the 2' hydroxyribose position, and incorporation of a phosphorothioate backbone produces molecules that superficially resemble RNA but are much more resistant to nuclease degradation, although those skilled in the art will recognize other forms of suitable backbones that may be used for the purposes of the present invention.

[0133] To avoid degradation of pre-mRNA and / or mRNA during duplex formation with the antisense oligonucleotide, the antisense oligonucleotide used in the present method can be adapted to minimize or prevent cleavage by endogenous RNase H. This property is highly desirable, since treatment of RNA with unmethylated oligonucleotides, either in cells or in crude extracts containing RNase H, causes degradation of the pre-mRNA:antisense and / or mRNA:antisense oligonucleotide duplex. Any form of modified antisense oligonucleotide that can bypass or avoid inducing such degradation may be used in the present method. Nuclease resistance can be achieved by modifying the antisense oligonucleotide of the present invention to include a partially unsaturated aliphatic hydrocarbon chain and one or more polar or charged groups, including carboxylic acid groups, ester groups, and alcohol groups.

[0134] Antisense oligonucleotides that do not activate RNase H can be prepared according to known techniques (see, for example, U.S. Patent No. 5,149,797). Such antisense oligonucleotides may be deoxyribonucleotide or ribonucleotide sequences, simply containing any structural modifications that sterically hinder or prevent RNase H from binding to a double-stranded molecule containing the oligonucleotide as one of its members, without substantially disrupting or disrupting duplex formation. Because the portion of the oligonucleotide involved in duplex formation is substantially different from the portion involved in RNase H binding to it, numerous antisense oligonucleotides that do not activate RNase H are available. For example, such antisense oligonucleotides can be oligonucleotides in which at least one or all of the internucleotide bridge phosphate residues are modified phosphates, such as methylphosphonate, methylphosphorothioate, phosphoromorpholidate, phosphoropiperazidate boranophosphate, amide bond, and phosphoramidate. For example, all other internucleotide bridge phosphate residues may be modified as described. In another non-limiting example, such an antisense oligonucleotide is a molecule in which at least one or all nucleotides contain a 2' lower alkyl moiety (e.g., C1-C4, straight or branched, saturated or unsaturated alkyl, e.g., methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). All other nucleotides may be modified, e.g., as described.

[0135] An example of an antisense oligonucleotide that is not cleaved by cellular RNase H when duplexed with RNA is a 2'-O-methyl derivative. Such 2'-O-methyl oligoribonucleotides are stable in cellular environments and animal tissues, and their duplexes with RNA have higher Tm values ​​than their ribo- or deoxyribo- counterparts. Alternatively, the nuclease-resistant antisense oligonucleotides of the present invention may have at least one of the last 3'-terminal nucleotides fluorinated. Further alternatively, the nuclease-resistant antisense oligonucleotides of the present invention have phosphorothioate linkages between at least two of the last 3'-terminal nucleotide bases, and preferably have phosphorothioate linkages between the last four 3'-terminal nucleotide bases.

[0136] Increased translational regulation can also be achieved with alternative oligonucleotide chemistries. For example, antisense oligonucleotides may be selected from the list including phosphoramidate or phosphorodiamidate morpholino oligonucleotides (PMO); PMO-X; PPMO; peptide nucleic acids (PNAs); locked nucleic acids (LNAs) and derivatives including alpha-L-LNA, 2'-amino LNA, 4'-methyl LNA, and 4'O-methyl LNA; ethylene-bridged nucleic acids (ENA) and their derivatives; phosphorothioate oligonucleotides; tricyclo-DNA oligonucleotides (tcDNA); tricyclophorothioate oligonucleotides; 2'-O-methyl modified oligonucleotides (2'-O-Me); 2'-O-methoxyethyl (2'-MOE); 2'-fluoro (2'-F), 2'-fluoroarabino (FANA); non-locked nucleic acids (UNA); hexitol nucleic acids (HNA); cyclohexyl nucleic acids (CeNA); 2'-amino (2'-NH2); 2'-O-ethyleneamine, or any combination of the above, either as a mixture or as a gapmer. To further improve delivery efficacy, the above-mentioned modified nucleotides are often conjugated to sugar or nucleobase moieties using fatty acids, lipids, cholesterol, amino acids, carbohydrates, polysaccharides, nanoparticles, etc. These conjugated nucleotide derivatives can also be used to construct exon-skipping antisense oligonucleotides. Antisense oligonucleotide-induced translational regulation of human OPA1 gene transcripts generally uses oligoribonucleotides, PNAs, 2'-O-Me, or MOE-modified bases on a phosphorothioate backbone. 2'-O-Me ASOs are used in oligo design, but these compounds, despite their efficient in vitro uptake when delivered as cationic lipoplexes, are susceptible to nuclease degradation and are not considered ideal for in vivo or clinical use. When using alternative chemistries to generate the antisense oligonucleotides of the present invention, thymine (T) in the sequences provided herein can be replaced with uracil (U).

[0137] While the antisense oligonucleotides described above are preferred forms of antisense oligonucleotides of the present invention, the present invention includes other oligonucleotide antisense molecules, including but not limited to oligonucleotide mimetics as described below.

[0138] The preferred specific example of antisense oligonucleotide useful in the present invention includes the oligonucleotide that contains modified backbone or non-natural internucleoside bond.As defined herein, the oligonucleotide with modified backbone includes those that retain phosphorus atom in backbone and those that do not have phosphorus atom in backbone.For the purpose of this specification and as sometimes referred to in the art, the modified oligonucleotide that does not have phosphorus atom in internucleoside backbone can also be considered as antisense oligonucleotide.

[0139] In other preferred oligonucleotide mimetics, both the sugar and internucleoside linkages (i.e., the backbone) of the nucleotide units are replaced with novel groups, while maintaining the base units for hybridization with appropriate nucleic acid target compounds. One such oligonucleotide compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone.

[0140] Another preferred chemistry is phosphorodiamidate morpholino oligonucleotide (PMO) compounds, which are not degraded by any known nucleases or proteases, are uncharged, and do not activate RNase H activity when bound to an RNA strand.

[0141] Modified oligonucleotides may also contain one or more substituted sugar moieties. Oligonucleotides may also contain modified or substituted nucleobases (often simply referred to in the art as "bases"). Certain nucleobases are particularly useful for increasing the binding affinity of the oligonucleotide compounds of the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C, more specifically when combined with 2'-O-methoxyethyl sugar modifications.

[0142] Another modification of the oligonucleotides of the invention involves chemically linking one or more moieties or conjugates to the oligonucleotide that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-S-tritylthiol, thiocholesterol, fatty chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, myristyl, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0143] Cell-penetrating peptides have been added to phosphorodiamidate morpholino oligonucleotides to enhance cellular uptake and nuclear localization. Different peptide tags have been shown to affect the efficiency of uptake and target tissue specificity, as shown by Jearawiriyapaisarn et al. (2008), Mol. Ther. 16 9, 1624-1629.

[0144] Not all positions in a given compound need to be uniformly modified, and in fact, more than one of the above modifications can be incorporated into a single compound, or into a single nucleoside in an oligonucleotide.The present invention also includes the antisense oligonucleotide of chimeric compound.In the context of the present invention, " chimeric " antisense oligonucleotide or " chimera " is an antisense oligonucleotide, particularly an oligonucleotide that contains two or more chemically different regions, which are composed of at least one monomer unit, i.e., in the case of an oligonucleotide compound, nucleotide.These oligonucleotides typically contain at least one region that oligonucleotide is modified to give oligonucleotide or antisense oligonucleotide an additional region for increasing resistance to nuclease degradation, increasing cellular uptake, and increasing binding affinity to target nucleic acid.

[0145] The activity of antisense oligonucleotides and their variants can be assayed according to conventional techniques in the art. The expression level of the investigated RNA and protein can be evaluated by any of a variety of well-known methods for detecting the expression of transcribed nucleic acids or proteins. Non-limiting examples of such methods include RT-PCR of spliced ​​forms of RNA followed by size separation of PCR products, nucleic acid hybridization methods, such as Northern blots and / or nucleic acid arrays, nucleic acid amplification methods, immunological methods for detecting proteins, protein purification methods, and protein function or activity assays. Protein expression levels can be evaluated by Western blots and / or ELISA assays from cells, tissues, or organisms, and by evaluating downstream functional or physiological effects.

[0146] When two or more transcripts of different sizes are present, the resulting proteins can be assessed by any of a wide variety of well-known methods for detecting the expression of related proteins, including, but not limited to, immunological methods for detecting proteins, protein purification methods, mass spectrometry, and protein function or activity assays.

[0147] The present invention provides antisense oligonucleotide-induced pre-mRNA sequence modulation of OPA1 gene transcripts, a delivery system directed to clinically relevant oligonucleotide chemistry and exon structure, and therapeutic levels of functional OPA1 protein. Functional OPA1 translation, and therefore a clinically relevant increase in the amount of OPA1 protein from OPA1 gene transcription, is 1) In vitro oligonucleotide purification using a fibroblast cell line with experimental evaluation of (i) target motif, (ii) antisense oligonucleotide length and oligonucleotide cocktail development, (iii) chemistry selection, and (iv) addition of cell-penetrating peptides (CPPs) to enhance oligonucleotide delivery; and 2) This will be achieved by detailed evaluation of novel approaches to regulate OPA1 splicing through interactions with the OPA1 pre-mRNA transcript and to exclude exon 7X from the OPA mRNA transcript.

[0148] Thus, it is demonstrated herein that splicing of OPA1 exon 7X can be modulated with specific antisense oligonucleotides, thereby obtaining a functionally significant increase in the amount of OPA1 wild-type mRNA transcript and OPA1 protein, thereby reducing the severe pathology associated with ADOA caused by OPA1 haploinsufficiency.

[0149] The antisense oligonucleotide used according to the present invention can be easily carried out by the well-known technique of solid phase synthesis.The equipment for this synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, California).One method for synthesizing oligonucleotide on modified solid support is described in U.S. Patent No. 4,458,066.

[0150] Any other means for such synthesis known in the art may also be used in addition or alternatively. It is well known to use similar techniques to prepare oligonucleotides, such as phosphorothioates and alkylated derivatives. In one such embodiment, diethyl-phosphoramidites are used as starting materials and can be synthesized as described in Beaucage, et al., (1981) Tetrahedron Letters, 22:1859-1862.

[0151] The antisense oligonucleotides of the present invention are synthesized in vitro and do not include antisense compositions of biological origin or genetic vector constructs designed to direct the in vivo synthesis of antisense oligonucleotides. The molecules of the present invention may also be mixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds, such as, for example, liposomes, receptor-targeting molecules, etc.

[0152] Antisense oligonucleotides can be formulated for oral, topical, parenteral, or other administration, particularly for topical and injectable ocular administration. Formulations can be formulated to aid uptake, distribution, and / or absorption at the site of administration or activity. Preferably, the antisense oligonucleotides of the present invention are formulated to be delivered locally to the eye or by intraocular injection or implantation, so that the effect on OPA1 production is spatially limited and not systemic.

[0153] ASO chemistry and modification The ASOs used in the compositions described herein may contain naturally occurring nucleotides, nucleotide analogs, 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 modified backbones. In some examples, all nucleotides in the ASO are modified nucleotides. Chemical modifications of ASOs or ASO components compatible with the compositions and methods described herein are known in the art, as disclosed, for example, 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.

[0154] One or more nucleotides of the ASO can be any naturally occurring unmodified nucleobase, such as adenine, guanine, cytosine, thymine, uracil, and inosine, or any synthetic or modified nucleobase sufficiently similar to an unmodified nucleobase so as to be 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-hydroxymethylcytosine.

[0155] ASOs contain a "backbone" structure, which refers to the connections between the nucleotides / monomers of the ASO. In naturally occurring oligonucleotides, the backbone contains 3'-5' phosphodiester bonds connecting the 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, 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, for example, Roberts et al. (supra) and Agrawal (2021), Biomedicines, 9:503. In some examples, the backbone structure of the ASO does not contain phosphorus-based linkages, but rather contains peptide linkages, for example, peptide nucleic acids (PNAs), or linking groups including carbamates, amides, and linear and cyclic hydrocarbon groups.

[0156] In some examples, the stereochemistry at each phosphorus internucleotide linkage of the ASO backbone is random. In other examples, the stereochemistry at each phosphorus internucleotide linkage of the ASO backbone is controlled and not random. For example, U.S. Patent No. 9,605,019 describes methods for independently selecting the handedness of chirality at each phosphorus atom in an oligonucleotide. In some examples, ASOs used in the compositions and methods provided herein, including but not limited to the ASO sequences disclosed herein as SEQ ID NOS: 1-31 and 33-54, are ASOs with non-random phosphodiester internucleotide linkages. In some examples, the compositions or compositions used in the methods disclosed herein comprise pure diastereomeric ASOs. In other examples, the composition comprises an ASO having a 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%.

[0157] In some examples, the ASO has a non-random mixture of Rp and Sp configurations at its phosphorus internucleotide linkages. In some examples, the ASO used in the compositions and methods disclosed herein contains 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 being Sp, or about 100% Rp.

[0158] In some examples, the ASOs described herein contain a sugar moiety comprising ribose or deoxyribose, or a modified sugar moiety or sugar analog containing a morpholine ring. Suitable examples of modified sugar moieties include, but are not limited to, 2'-O-modified, 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F, N3'-P5' phosphoramidate, 2' dimethylaminooxyethoxy, 2' dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and bicyclic-modified sugars. In some examples, the sugar moiety modification is selected from 2'-O-Me, 2'F, and 2'MOE. In other examples, the sugar moiety modification is an extra crosslink, such as in locked nucleic acids (LNAs). In some examples, the sugar analog contains a morpholine ring, such as in phosphorodiamidate morpholinos (PMOs). In some examples, the sugar moiety comprises a ribofuranyl or 2' deoxyribofuranyl modification. In some examples, the sugar moiety comprises a 2'4'-constrained, 2'-O-methyloxyethyl (cMOE) modification. In some examples, the sugar moiety comprises a cE'2"4'-constrained, 2'-O-ethyl BNA modification. In other examples, the sugar moiety comprises a tricycloDNA (tcDNA) modification. In some examples, the sugar moiety comprises an ethylene nucleic acid (ENA) modification. In some examples, the sugar moiety comprises a 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.

[0159] In some instances, each constituent nucleotide of an ASO is modified in the same manner, e.g., all linkages in the backbone of the ASO contain phosphorothioate linkages or each ribose sugar moiety contains a 2'-O-methyl modification. In other instances, ASOs are used that contain a combination of different modifications, e.g., a combination of phosphorodiamidate linkages and sugar moieties that contain morpholine rings (morpholinos).

[0160] In some examples, the ASO comprises one or more backbone modifications. In some examples, the ASO comprises one or more sugar moiety modifications. 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).

[0161] In some preferred examples, the ASO comprises a phosphorodiamidate morpholino (PMO).

[0162] Those skilled in the art will understand that ASOs can be modified to achieve desired properties or activities or to reduce undesirable properties or activities of the ASO. In some instances, ASOs are modified to alter one or more properties. For example, such modifications can increase binding affinity to a target sequence on a pre-mRNA transcript, reduce binding to any non-target sequences, reduce degradation by cellular nucleases (e.g., RNase H), improve uptake of the ASO into cells and / or specific subcellular compartments, alter the pharmacokinetics or pharmacodynamics of the ASO, and / or modulate the half-life of the ASO in vivo.

[0163] In some instances, the ASO contains one or more 2'-O-(2-methoxyethyl) (MOE) phosphorothioate modified nucleotides, which have been shown to significantly enhance the resistance of the ASO to nuclease degradation and increase bioavailability.

[0164] Methods for the synthesis and chemical modification of ASOs, as well as the synthesis of ASO conjugates, are well known in the art, and such ASOs are commercially available.

[0165] In some examples, the compositions (e.g., pharmaceutical compositions) provided herein include two or more ASOs that have different chemistries but are complementary to the same target portion of the OPA1 pre-mRNA. In other examples, the two or more ASOs are complementary to different target portions of the OPA1 pre-mRNA.

[0166] In some examples, the compositions disclosed herein comprise an ASO linked to a functional moiety. In some examples, the functional moiety is a delivery moiety, a targeting moiety, a detection moiety, a stabilizing moiety, or a therapeutic moiety. In some examples, the functional moiety comprises a delivery moiety or a targeting moiety. In some examples, the functional moiety comprises a stabilizing moiety. In some preferred examples, the functional moiety is a delivery moiety.

[0167] Suitable delivery moieties include, but are not limited to, lipids, polyethers, peptides, carbohydrates, glycans, receptor binding domains (RBDs), and antibodies.

[0168] In some examples, the delivery moiety comprises a cell-penetrating peptide (CPP). Suitable examples of CPPs are described, for example, in PCT / AU2020 / 051397. In some examples, the amino acid sequence of the CPP comprises or consists of RRSRTARAGRPGRNSSRPSAPRGASGGASG (SEQ ID NO: 55). In one example, the CPP comprises the sequence RRSRTARAGRPGRNSSRPSAPRGASGGASG (SEQ ID NO: 55), optionally where any amino acid other than glycine is a D-amino acid. In other examples, the delivery moiety comprises an RBD.

[0169] In other examples, the delivery moiety comprises a carbohydrate. In some examples, the carbohydrate delivery moiety is selected from among N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), and mannose. In one example, the carbohydrate delivery moiety is GalNAc.

[0170] In other examples, the delivery moiety comprises a lipid. Examples of suitable lipids as delivery moieties include, but are not limited to, cholesterol moieties, cholesteryl moieties, and aliphatic lipids. In some examples, the delivery moiety comprises a fatty acid or lipid moiety. In some embodiments, the fatty acid chain length is about C8 to C20. Examples of suitable fatty acid moieties and their conjugation to oligonucleotides can be found, for example, in International Patent Publication No. 2019 / 232255 and Prakash et al., (2019).

[0171] In a further example, the delivery moiety includes an antibody, for example, as described in Dugal-Tessier et al., (2021), J Clin Med., 10(4):838.

[0172] Suitable examples of stabilizing moieties include, but are not limited to, polyethylene glycol (PEG), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), and poly(2-oxazoline) (POx).

[0173] In some instances, when an ASO is linked to a functional moiety, the functional moiety is covalently bound to the ASO, while in other instances, the functional moiety is non-covalently bound to the ASO.

[0174] The functional moiety can be linked to one or more of the nucleotides in the ASO at any of several positions on the sugar, base, or phosphate group, using, for example, a linker, as understood in the art and described in the literature. The linker can include a bivalent or trivalent branched linker. In some examples, the functional moiety is linked to the 5' end of the ASO. In other examples, the functional moiety is linked to the 3' end of the ASO.

[0175] In some examples, a composition comprising any of the ASOs disclosed herein also comprises a delivery nanocarrier complexed with the ASO. In some examples, the delivery nanocarrier is selected from among lipoplexes, liposomes, exosomes, inorganic nanoparticles, and DNA nanostructures. In other examples, the delivery nanocarrier comprises a lipid nanoparticle (LNP) encapsulating the ASO. In some examples, the LNP comprises a neurotransmitter-derived lipidoid (NT-lipidoid) to facilitate delivery across the blood-brain barrier, as described in Ma et al. (2020). Various delivery ASO-nanocarrier complex formats are known in the art, for example, as reviewed in Roberts et al. (supra).

[0176] Pharmaceutical Compositions Also provided herein are pharmaceutical compositions comprising any of the aforementioned ASOs, non-viral expression vectors, modified messenger RNA (mmRNA), and viral expression vectors disclosed herein, formulated with at least a pharmaceutically acceptable excipient, including a carrier, filler, preservative, adjuvant, solubilizer, and / or diluent.

[0177] For use in the methods disclosed herein, pharmaceutical compositions containing any of the ASO or expression vector compositions described 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 or expression vector disclosed herein.

[0178] 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 non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or 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, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, Salts include 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, and the like. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Additional pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates.

[0179] In some examples, the pharmaceutical compositions are formulated into any of several possible administration routes or dosage forms, including, but not limited to, intravenous administration, intrathecal administration, tablets, capsules, gel capsules, liquid syrups, and softgels. 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. Suspensions may also contain stabilizers. In some examples, the pharmaceutical formulations disclosed herein are provided in forms including, but not limited to, solutions, emulsions, microemulsions, foams, or liposome-containing formulations (e.g., cationic or non-cationic liposomes).

[0180] In some examples, pharmaceutical formulations containing any of the ASOs or expression vectors described herein may include one or more penetration enhancers, carriers, excipients, or other active or inactive ingredients, as appropriate and known to those skilled in the art. In some examples, when a pharmaceutical composition includes liposomes, such liposomes may also include sterically stabilized liposomes, for example, liposomes containing one or more specialized lipids. These specialized lipids result in liposomes with extended circulation lifetimes. In some examples, sterically stabilized liposomes include one or more glycolipids or are derivatized with one or more hydrophilic polymers, such as PEG moieties. In some examples, a surfactant is included in the pharmaceutical formulation.

[0181] In some examples, the pharmaceutical composition also includes a penetration enhancer to enhance delivery of the ASO or non-viral expression vector, e.g., to aid diffusion across cell membranes and / or to enhance the permeability of lipophilic drugs. In some examples, the penetration enhancer includes a surfactant, a fatty acid, a bile salt, or a chelating agent.

[0182] In some instances, when administration is via a systemic route, e.g., intravenous, the method also includes a step of facilitating movement of any of the ASOs or vectors described herein through the blood-brain barrier (BBB) ​​to the CNS, particularly the brain. In some instances, the BBB is transiently disrupted by administration of one or more antibodies that disrupt the binding of Netrin-1 to Unc5B, e.g., as described in Boye et al. (2022).

[0183] In some examples, the pharmaceutical composition comprises an ASO or non-viral vector at a dose ranging from about 0.01 mg / kg to 20 mg / kg, e.g., 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 8 mg / kg, 10 mg / kg, 15 mg / kg, or another dose ranging from about 0.01 mg / kg to 20 mg / kg. In some examples, when the ASOs disclosed herein are administered directly to the CNS or brain, for example, by intraventricular administration, the total dose is in the range of about 50 mg to about 500 mg, e.g., 60 mg, 70 mg, 80 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 220 mg, 250 mg, 270 mg, 290 mg, 300 mg, 350 mg, 400 mg, 450 mg, or another dose of about 50 mg to about 500 mg. This dose range is approximately 1200 cm 3 Assuming an average human brain volume of 1000, this is approximately 0.050 mg / cm of brain volume. 3 ~approximately 0.42 mg / cm of brain volume 3 Corresponds to.

[0184] In some examples, the pharmaceutical composition comprises multiple ASOs or AR expression vectors. In some examples, the pharmaceutical composition comprises, in addition to the ASO or AR expression vector, another drug or therapeutic agent suitable for treating a subject suffering from a condition associated with OPA1 haploinsufficiency.

[0185] Treatment method According to yet another aspect of the present invention, there is provided one or more antisense oligonucleotides described herein for use in antisense oligonucleotide-based therapy.Preferably, the therapy is for a disease associated with OPA1 expression.More preferably, the therapy for a disease associated with OPA1 expression is a therapy for ADOA.Preferably, the disease associated with OPA1 expression is associated with a decrease in the level of functional OPA1 protein expression.Preferably, the decrease in functional OPA1 is in patients with ADOA, more preferably in ADOA associated with OPA1 haploinsufficiency.

[0186] More specifically, the antisense oligonucleotides may be selected from the group consisting of any one or more of Table 1 and Table 2, or SEQ ID NOs: 1-31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3, and 4, and combinations or cocktails thereof. More specifically, the antisense oligonucleotides may be selected from the group consisting of any one or more of Table 1, Table 2, and Table 4, or SEQ ID NOs: 1-31 and 33-54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7, and 33-44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40, and 44, and combinations or cocktails thereof. This includes sequences capable of hybridizing to such sequences under stringent hybridization conditions, sequences complementary thereto, sequences containing modified bases, modified backbones, and functional truncations or extensions thereof that possess or modulate mRNA translation in the OPA1 gene transcript.

[0187] The present invention also extends to combinations of two or more antisense oligonucleotides capable of binding to a selected target to induce splicing modulation in the OPA1 gene transcript, which may be a cocktail of two or more antisense oligonucleotides, or constructs comprising two or more antisense oligonucleotides linked together for use in antisense oligonucleotide-based therapy.

[0188] Accordingly, a method for treating, preventing, or ameliorating the effects of a disease associated with OPA1 expression, comprising: Methods are provided that include the step of: a) administering to a patient an effective amount of one or more antisense oligonucleotides described herein or a pharmaceutical composition comprising one or more antisense oligonucleotides.

[0189] Preferably, the disease associated with OPA1 expression in the patient is ADOA.

[0190] Accordingly, the present invention provides a method for treating, preventing, or ameliorating the effects of ADOA, comprising: a) administering to a patient an effective amount of one or more antisense oligonucleotides described herein or a pharmaceutical composition comprising one or more antisense oligonucleotides.

[0191] Also provided herein are methods for increasing OPA1 protein in cells, the method comprising contacting the cells with a composition or pharmaceutical composition disclosed herein, thereby increasing the amount of OPA1 protein in the cells. Also provided herein are methods for increasing the level of OPA1 protein in cells ex vivo or in tissues in vivo, the method comprising contacting the cells with an ASO or pharmaceutical composition disclosed herein, thereby increasing the amount of OPA1 protein in the cells.

[0192] Preferably, this therapy is used to increase the level of functional OPA1 protein through a splicing modulation strategy, preferably by reducing the amount of OPA1 RNA containing the abnormally retained exon 7x and increasing the amount of OPA1 RNA that does not contain exon 7x.

[0193] In some examples, administration to a subject or contacting a cell with any of the ASOs or pharmaceutical compositions disclosed herein increases the level of OPA1 protein by about 1.1 to about 10 times, e.g., 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, or more, compared to the level in the tissue before administration or contact. The increase is 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. In one example, administration to a subject or contacting a cell with any of the ASOs or pharmaceutical compositions disclosed herein increases OPA1 protein levels by about 1.1 to about 2.5-fold compared to levels in the tissue before administration or contact.

[0194] Preferably, administration of the antisense oligonucleotide results in expression of OPA1 protein that is 1.1 to 2.5 times higher than the expression of OPA1 protein in subjects with a syndromic OPA1 mutation.

[0195] From the disclosure herein, it will be apparent to those skilled in the art that antisense oligonucleotides of the present disclosure that reduce the expression of OPA1 gene transcripts lacking exon 7x do so substantially without altering the proportions of other transcripts of the OPA1 gene. For example, antisense oligonucleotides of the present disclosure reduce the expression of OPA1 gene transcripts lacking exon 7 without substantially affecting the relative expression levels of transcripts that include exon 7 or lack exon 7. In one example, antisense oligonucleotides of the present disclosure reduce the expression of OPA1 gene transcripts lacking exon 7x without reducing the expression levels of transcripts lacking exon 7. The data herein suggest that antisense oligonucleotides of the present disclosure reduce the expression of OPA1 gene transcripts lacking exon 7x without altering the normal physiological ratio of OPA1 gene transcripts that include exon 7 or lack exon 7. Such antisense oligonucleotides are of therapeutic interest because transcripts lacking exon 7 are thought to increase the cell's susceptibility to apoptosis, and as a result, oligonucleotides that increase the relative levels of transcripts lacking exon 7 are thought to have little therapeutic benefit.

[0196] In one example, an antisense oligonucleotide of the present disclosure that reduces the expression of an OPA1 gene transcript lacking exon 7x does not substantially affect the relative expression level of an OPA1 gene transcript that includes or lacks exon 7. In one example, an antisense oligonucleotide of the present disclosure that reduces the expression of an OPA1 gene transcript lacking exon 7x, when administered to a cell, does not affect the relative expression level of an OPA1 gene transcript that includes or lacks exon 7, compared to the level or ratio of the transcript in a cell to which the antisense oligonucleotide is not administered. For example, an antisense oligonucleotide of the present disclosure, when administered to a cell, reduces the expression of an OPA1 gene transcript that includes or lacks exon 7x without altering the normal physiological ratio of transcripts that include or lack exon 7, compared to the level or ratio of the transcript in a cell to which the antisense oligonucleotide is not administered. The normal physiological ratio of transcripts that include or lack exon 7 will be apparent to one of skill in the art and / or are disclosed herein. For example, the normal physiological ratio of OPA1 gene transcripts containing or lacking exon 7 is approximately 1:4 (i.e., approximately 20% of OPA1 gene transcripts contain exon 7 and approximately 80% of OPA1 gene transcripts lack exon 7). Transcript levels and / or ratios are determined using methods known in the art, such as qPCR.

[0197] In one example, the relative expression level of OPA1 gene transcripts that include or lack exon 7 is a ratio of approximately 20% OPA1 gene transcripts that include exon 7 and approximately 80% OPA1 gene transcripts that lack exon 7. For example, the relative expression level of OPA1 gene transcripts that include or lack exon 7 is a ratio of 15-25% OPA1 gene transcripts that include exon 7 and 75-85% OPA1 gene transcripts that lack exon 7.

[0198] In one example, the relative expression levels of OPA1 gene transcripts that include or lack exon 7 are a ratio of OPA1 gene transcripts that include exon 7 to OPA1 gene transcripts that lack exon 7 of approximately 1:4.

[0199] Increasing functional OPA1 will preferably result in a reduction in the amount, duration, or severity of symptoms of an OPA1-associated disorder, such as ADOA.

[0200] As used herein, "treatment" of a subject (e.g., a mammal such as a human) or cell is any type of intervention used in an attempt to alter the natural course of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and can be performed either prophylactically or after the initiation of a pathological event or after contact with a pathogenic agent. Also included is "prophylactic" treatment, which can be directed to reducing the rate of progression of the disease being treated, delaying the onset of the disease, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily indicate complete eradication, cure, or prevention of the disease or its associated symptoms.

[0201] The subject having a disease associated with OPA1 expression can be a mammal, including a human.

[0202] The antisense oligonucleotides of the present invention may also be used in conjunction with alternative therapies, such as drug therapy.

[0203] Thus, the present invention provides a method for treating, preventing, or alleviating the effects of a disease associated with OPA1 expression, wherein an antisense oligonucleotide of the present invention is administered sequentially or simultaneously with another alternative therapy associated with treating, preventing, or alleviating the effects of a disease associated with OPA1 expression, preferably, the disease is ADOA.

[0204] delivery The antisense oligonucleotides of the present invention can also be used as prophylactic or therapeutic agents, which can be utilized for the purpose of treating disease. Thus, in one embodiment, the present invention provides an antisense oligonucleotide that binds to a selected target within the OPA1 pre-mRNA to efficiently and consistently induce correctly spliced ​​OPA1 mRNA as described herein, in a therapeutically effective amount, mixed with a pharmaceutically acceptable carrier, diluent, or excipient.

[0205] Also provided is a pharmaceutical, prophylactic, or therapeutic composition for treating, preventing, or ameliorating the effects of a disease associated with OPA1 expression in a patient, the composition comprising: a) one or more antisense oligonucleotides described herein; and b) one or more pharmaceutically acceptable carriers and / or diluents.

[0206] Preferably, the antisense oligonucleotides of the present invention are delivered via a localized ocular route to avoid systemic effects.Administration routes include, but are not limited to, intravitreal, intracavitary, subconjunctival, subtenon, retrobulbar, juxtascleral, or topical (drops, eyewash, cream, etc.).Delivery methods include injection with a syringe and drug delivery device, for example, an implanted vitreous delivery device (i.e., VITRASERT®).

[0207] More preferably, the antisense oligonucleotide is administered by intravitreal injection at a dose of 0.005 to 5 mg / eye, 0.005 to 1 mg / eye, 0.005 to 0.5 mg / eye, 0.01 to 5 mg / eye, 0.02 to 1 mg / eye, 0.01 to 0.5 mg / eye, 0.01 to 0.1 mg / eye, 0.01 to 0.5 mg / eye, 2 to 20 mg / eye, 0.5 to 20 mg / eye, or more preferably 5 to 20 mg / eye. The antisense oligonucleotide may be administered by intravitreal injection at, for example, about 0.01 mg / eye, 0.02 mg / eye, 0.03 mg / eye, 0.04 mg / eye, 0.05 mg / eye, 0.06 mg / eye, 0.07 mg / eye, 0.08 mg / eye, 0.09 mg / eye, 0.1 mg / eye, 0.2 mg / eye, 0.3 mg / eye, 0.4 mg / eye, 0.5 mg / eye, 1 mg / eye, or 2 mg / eye. Preferably, the antisense oligonucleotide is administered by intravitreal injection at about 0.01 to 0.5 mg / eye.

[0208] Antisense oligonucleotide can be administered for a short period, for example, every day for 2 weeks or less, at regular intervals.However, in many cases, oligonucleotide is administered intermittently for a longer period.Administration can be followed by the administration of antibiotics or other therapeutic treatments, or can be administered simultaneously.The treatment regimen can be adjusted as indicated (dosage, frequency, route, etc.) based on the results of immunoassay, other biochemical tests and physiological tests of the subject under treatment.

[0209] Dosing may depend on the severity and responsiveness of the disease state being treated, with the course of treatment lasting from several days to several months, or until a cure or a reduction in the disease state is achieved. Alternatively, dosing can be titrated to the rate of disease progression. A baseline progression is established. The rate of progression is then monitored after the first administration is discontinued to confirm a decrease in the rate. Preferably, there is no progression after administration. Preferably, re-administration is only necessary if the rate of progression remains unchanged. Successful treatment preferably results in no further progression of the disease or some further recovery of vision. The optimal dosing schedule can be calculated from measurements of drug accumulation in the patient's body. Those skilled in the art can easily determine the optimal dosage, administration method, and repetition rate.

[0210] Optimal dosages may vary depending on the relative potency of individual oligonucleotides and can generally be estimated based on EC50 values ​​found to be effective in in vitro and in vivo animal models.

[0211] Generally, the dosage is 0.005-5 mg / eye administered via intravitreal injection, and may be administered daily, weekly, monthly, or once or more per year, or even once every 2-20 years. The repetition rate of dosing depends on the rate of disease progression. Those skilled in the art can easily estimate the repetition rate of dosing based on the measured residence time and drug concentration in body fluids or tissues. After successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent recurrence of the disease state, and the oligonucleotide is administered at a maintenance dose, and the dose may be 0.005-5 mg / eye administered via intravitreal injection, and may be administered daily, weekly, monthly, or once or more per year, or even once every 2-20 years.

[0212] Effective in vivo treatment regimens using the antisense oligonucleotides of the present invention can vary according to the duration, dose, frequency and route of administration, and the condition of the subject being treated (i.e., prophylactic administration versus administration in response to local or systemic infection).Therefore, such in vivo therapy often requires monitoring by tests appropriate for the specific type of disorder being treated, and corresponding adjustments in dosage or treatment regimen to achieve optimal therapeutic results.

[0213] Treatment can be monitored, for example, by common indicators of disease known in the art. The effectiveness of the in vivo administered antisense oligonucleotide of the present invention can be determined from biological samples (tissue, blood, urine, etc.) collected from a subject before, during, and after administration of the antisense oligonucleotide. Assays of such samples include: (1) monitoring the presence or absence of heteroduplex formation between target and non-target sequences using procedures known to those skilled in the art, for example, electrophoretic gel mobility assay; and (2) monitoring the amount of mutant mRNA relative to reference normal mRNA or protein, as determined by standard techniques such as RT-PCR, Northern blotting, ELISA, or Western blotting.

[0214] Nuclear oligonucleotide delivery is a major challenge for antisense oligonucleotides. Different cell-penetrating peptides (CPPs) localize PMOs to varying degrees in different conditions and cell lines, and novel CPPs have been evaluated by the present inventors for their ability to deliver PMOs to target cells. The terms CPP or "cellular uptake-enhancing peptide moiety" are used interchangeably and refer to cationic cell-penetrating peptides, also known as "transport peptides," "carrier peptides," or "peptide transduction domains." As demonstrated herein, the peptides have the ability to induce cell penetration within about or at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells in a given cell culture population, and upon systemic administration and / or macromolecule translocation, enable macromolecule translocation within multiple tissues in vivo when administered intraocularly. CPPs are well known in the art and are disclosed, for example, in U.S. Application No. 2010 / 0016215, the entire contents of which are incorporated by reference.

[0215] Thus, the present invention provides an antisense oligonucleotide of the present invention in combination with a cell-penetrating peptide for the preparation of a therapeutic pharmaceutical composition.

[0216] excipients The antisense oligonucleotides of the present invention are preferably delivered in a pharmaceutically acceptable composition. The composition may contain about 1 nM to 1,000 nM of each desired antisense oligonucleotide of the present invention. Preferably, the composition may contain about 1 nM to 500 nM, 10 nM to 500 nM, 50 nM to 750 nM, 100 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 40 nM, 1 nM to 30 nM, 1 nM to 20 nM, and most preferably 1 nM to 10 nM of each antisense oligonucleotide of the present invention.

[0217] The composition may contain about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM or 1000 nM of each desired antisense oligonucleotide of the invention.

[0218] The present invention further provides one or more antisense oligonucleotides adapted to assist in the prophylactic or therapeutic treatment, prevention or alleviation of symptoms of a disease, such as a disease or pathology associated with OPA1 expression, in a form suitable for delivery to a patient.

[0219] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and typically do not produce allergic or similar adverse reactions, such as stomach upset, when administered to a patient. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, 22nd Ed., Pharmaceutical Press, PA (2013).

[0220] In a more specific embodiment of the present invention, pharmaceutical compositions are provided comprising a therapeutically effective amount of one or more antisense oligonucleotides of the present invention in combination with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions may contain diluents of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength, as well as additives such as detergents and solubilizers (e.g., Tween 80, Polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol), and bulking substances (e.g., lactose, mannitol). Materials may be incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, or into liposomes. Hyaluronic acid may also be used. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the proteins and derivatives of the present invention. See, e.g., Remington: The Science and Practice of Pharmacy, 22nd Ed., Pharmaceutical Press, PA (2013). The composition may be prepared in liquid form, or may be a dry powder, such as a lyophilized form.

[0221] It will be understood that the pharmaceutical compositions provided according to the present invention can be administered by any means known in the art.The pharmaceutical compositions for administration can be administered by injection, orally, topically, or via pulmonary or nasal routes.For example, antisense oligonucleotides can be delivered by local, intravenous, intraarterial, intraperitoneal, intramuscular, or subcutaneous administration routes.The appropriate route can be determined by those skilled in the art to be appropriate for the condition of the subject being treated.Preferably, antisense oligonucleotides are delivered locally to the eye or by intraocular injection or intraocular implantation, so that the effect on OPA1 production is spatially limited and not systemic.

[0222] Formulations for topical administration include those in which the oligonucleotides of the present disclosure are mixed with topical delivery agents such as hydrogels, lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). For topical or other administration, the oligonucleotides of the present disclosure can be encapsulated in liposomes or complexed thereto, particularly cationic liposomes. Alternatively, the oligonucleotides can be complexed to lipids, particularly cationic lipids. Fatty acids and esters, their pharmaceutically acceptable salts, and their uses are further described in U.S. Patent No. 6,287,860, filed May 20, 1999, and / or U.S. Patent Application No. 09 / 315,298.

[0223] In certain embodiments, the antisense oligonucleotides of the present disclosure can be delivered by topical or transdermal methods, including delivery to the ocular surface (e.g., via incorporating the antisense oligonucleotide into an emulsion, optionally packaging such antisense oligonucleotide in a liposome). Such topical or transdermal and emulsion / liposome-mediated delivery methods are described in the art for the delivery of antisense oligonucleotides, for example, in U.S. Patent No. 6,965,025. Preferably, the topical delivery is to the eye.

[0224] Also, antisense oligonucleotides described herein can be delivered via implantable device.The design of such device is a recognized process in the art, for example, using the synthetic implant design described in U.S. Patent No. 6,969,400.Preferably, implant can be implanted into eye for sustained delivery of antisense oligonucleotides.

[0225] Compositions and formulations for ocular administration, including ocular injection, topical ocular delivery, and ocular implants, may comprise sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0226] Delivery of therapeutically useful amounts of antisense oligonucleotides can be achieved by previously published methods. For example, delivery of antisense oligonucleotides can be carried out via a composition comprising a mixture of antisense oligonucleotides and an effective amount of block copolymers. An example of this method is described in U.S. Patent Application No. 2004 / 0248833. Other methods for delivery of antisense oligonucleotides to the nucleus are described in Mann CJ et al. (2001) Proc. Natl. Acad. Science, 98(1)42-47 and Gebski et al. (2003) Human Molecular Genetics, 12(15):1801-1811. Methods for introducing nucleic acid molecules into cells either as naked DNA or via expression vectors complexed to lipid carriers are described in U.S. Patent No. 6,806,084.

[0227] Antisense oligonucleotides can be introduced into cells using art-recognized techniques (e.g., transfection, electroporation, fusion, liposomes, colloidal polymer particles, and viral and non-viral vectors, as well as other means known in the art). The delivery method selected will depend at least on the cells to be treated and the location of the cells, and will be clear to those skilled in the art. For example, localization can be achieved by liposomes with specific markers on the surface to guide the liposomes, direct injection into tissues containing target cells, specific receptor-mediated uptake, etc.

[0228] It may be desirable to deliver antisense oligonucleotides in colloidal dispersion systems.Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes or liposome preparations).These colloidal dispersion systems can be used to prepare therapeutic pharmaceutical compositions.

[0229] Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. These preparations may have net cationic, anionic, or neutral charge characteristics, which may be useful for in vitro, in vivo, and ex vivo delivery methods. It has been shown that large unilamellar vesicles can encapsulate a significant percentage of aqueous buffer containing large macromolecules. RNA and DNA can be encapsulated in the aqueous interior and delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci. 6:77, 1981).

[0230] For liposomes to be effective gene transfer vehicles, the following characteristics must be present: (1) highly efficient encapsulation of the desired antisense oligonucleotide without compromising its biological activity, (2) preferential and substantial binding to target cells compared to non-target cells, (3) highly efficient delivery of the aqueous contents of the vesicles to the target cytoplasm, and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988). The composition of liposomes is usually a combination of phospholipids (especially high-phase transition temperature phospholipids), usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Cationic liposomes are positively charged liposomes that are thought to interact with negatively charged DNA molecules to form stable complexes. pH-sensitive or negatively charged liposomes are thought to entrap DNA or other oligonucleotides rather than complexing with them. Both cationic and non-cationic liposomes have been used to deliver DNA or other oligonucleotides into cells.

[0231] Liposomes also include "sterically stabilized" liposomes, which term, as used herein, refers to liposomes containing one or more specialized lipids that, when incorporated into the liposome, result in enhanced circulation life compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the lipid moiety forming the liposome vesicle contains one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Liposomes and their uses are further described in U.S. Patent No. 6,287,860.

[0232] As is known in the art, antisense oligonucleotides may be delivered using methods including, for example, liposome-mediated uptake, lipid conjugates, polylysine-mediated uptake, nanoparticle-mediated uptake, and receptor-mediated endocytosis, as well as additional non-endocytic delivery modalities such as microinjection, permeabilization (e.g., streptolysin-O permeabilization, anionic peptide permeabilization), electroporation, and various non-invasive, non-endocytic delivery methods known in the art (see Dokka and Rojanasakul, Advanced Drug Delivery Reviews 44, 35-49, which is incorporated by reference in its entirety).

[0233] Antisense oligonucleotides can be combined with other pharmaceutically acceptable carriers or diluents to produce pharmaceutical compositions.Suitable carriers and diluents include isotonic saline solution, such as phosphate buffered saline.Compositions can be formulated for topical, parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral or transdermal administration.

[0234] The routes of administration described are intended as a guide only, as one skilled in the art can readily determine the optimum route of administration and any dosage for any particular animal and condition.

[0235] Several approaches have been attempted to introduce functional new genetic material into cells, both in vitro and in vivo (Friedmann (1989) Science, 244:1275-1280). These approaches include incorporation of genes expressed within modified retroviruses (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51(18) supra:5074S-5079S), incorporation into non-retroviral vectors (Rosenfeld, et al. (1992) Cell, 68:143-155; Rosenfeld, et al. (1991) Science, 252:431-434), or delivery of transgenes linked to heterologous promoter-enhancer elements via liposomes (Friedmann (1989) supra; Brigham, et al. (1989) Am. J. Med. Sci., 298:278-281; ​​Nabel, et al. (1990) Science, 249:1285-1288; Hazinski, et al. al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209, and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855), linked to ligand-specific cation-based transport systems (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624), or using naked DNA, expression vectors (Nabel et al. (1990) supra; Wolff et al. (1990) Science, 247:1465-1468). Direct injection of transgenes into tissues produces only localized expression (Rosenfeld (1992) supra; Rosenfeld et al. (1991) supra; Brigham et al. (1989) supra; Nabel (1990) supra; and Hazinski et al. (1991) supra).The Brigham et al. group (Am. J. Med. Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) reported lung-only in vivo transfection of mice after either intravenous or intratracheal administration of DNA-liposome complexes. Examples of review articles on human gene therapy procedures are Anderson, Science (1992) 256:808-813; Barteau et al. (2008), Curr Gene Ther;8(5):313-23; Mueller et al. (2008). Clin Rev Allergy Immunol;35(3):164-78; Li et al. (2006) Gene Ther.,13(18):1313-9; Simoes et al. (2005) Expert Opin Drug Deliv;2(2):237-54.

[0236] The antisense oligonucleotides of the present invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compounds that can yield (directly or indirectly) biologically active metabolites or residues thereof when administered to an animal, including a human. Thus, by way of example, the present disclosure also envisions prodrugs and pharmaceutically acceptable salts of the compounds of the present invention, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents.

[0237] The term " pharmaceutically acceptable salt " refers to the physiologically and pharmaceutically acceptable salt of the compound of the present invention, i.e., the salt that maintains the desired biological activity of the parent compound and does not impart undesired toxicological effects to it.For oligonucleotide, preferred examples of pharmaceutically acceptable salts include, but are not limited to, (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, (b) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc., (c) salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, etc., and (d) salts formed with elemental anions such as chlorine, bromine, and iron. The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be via topical (including ocular and mucosal, and rectal delivery), pulmonary (e.g., by inhalation or infusion of powder or aerosol, including nebulizer, intratracheal, intranasal, epithelial, and transdermal), oral, or parenteral routes. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intraocular, or intramuscular injection or infusion, or intracranial, for example, intrathecal or intraventricular, administration. Oligonucleotides with at least one 2'-O-methoxyethyl modification are considered particularly useful for intraocular administration. Preferably, antisense oligonucleotides are delivered via the intraocular route.

[0238] The pharmaceutical formulations of the present invention, which can be conveniently provided in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing the active ingredient into association with pharmaceutical carriers or excipients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0239] use According to another aspect of the present invention, there is provided the use of one or more antisense oligonucleotides described herein in the manufacture of a medicament for the modulation or control of a disease associated with OPA1 protein expression.

[0240] The present invention also provides the use of a purified and isolated antisense oligonucleotide described herein for the manufacture of a medicament for the treatment of a disease associated with OPA1 protein expression.

[0241] Also provided is the use of a purified and isolated antisense oligonucleotide described herein for the manufacture of a medicament for treating, preventing, or ameliorating the effects of a disease associated with OPA1 expression.

[0242] Preferably, the OPA1-related disease is ADOA.Preferably, the disease associated with OPA1 protein expression is associated with a decrease in the level of functional OPA1 protein.Preferably, the decrease in the level of functional OPA1 protein is in patients with ADOA, more preferably in ADOA associated with OPA1 haploinsufficiency.

[0243] According to a still further aspect, the present invention extends to cDNA or cloned copies of the antisense oligonucleotide sequences of the invention, and to vectors containing the antisense oligonucleotide sequences of the invention. The present invention further extends to cells containing such sequences and / or vectors.

[0244] kit Also provided are kits for treating, preventing, or alleviating the effects of a disease associated with OPA1 protein expression in a patient, the kit comprising at least the antisense oligonucleotides and combinations or cocktails thereof described herein packaged in a suitable container, together with instructions for use thereof. Preferably, the disease associated with OPA1 protein expression is ADOA.

[0245] In a preferred embodiment, the kit contains at least one antisense oligonucleotide as described herein or as shown in Tables 1 and 2. Preferably, the antisense oligonucleotide is selected from the list comprising SEQ ID NOs: 1-31, more preferably SEQ ID NOs: 1, 2, 3, 4, and 7, even more preferably SEQ ID NOs: 1, 3, and 4.

[0246] In a preferred embodiment, the kit contains at least one antisense oligonucleotide as described herein or shown in Tables 1, 2 and 4. Preferably, the antisense oligonucleotide is selected from the list comprising SEQ ID NOs: 1-31 and 33-54, more preferably SEQ ID NOs: 1, 2, 3, 4, 7 and 33-44, even more preferably SEQ ID NOs: 1, 3, 4, 37, 40 and 44.

[0247] In one embodiment, the kit contains a cocktail of antisense oligonucleotides described herein.

[0248] The kit may also contain peripheral reagents such as buffers, stabilizers, and the like.

[0249] When the components of the kit are provided in one or more liquid solutions, the liquid solution can be an aqueous solution, for example, a sterile aqueous solution. For in vivo use, the expression construct can be formulated into a pharmaceutically acceptable injectable composition. In this case, the container means itself can be an inhalant, syringe, pipette, eyedropper, or other similar device from which the formulation can be applied to the affected area of ​​an animal (such as the lungs), injected into an animal, or applied to and mixed with other components of the kit.

[0250] In one embodiment, the kit of the present invention includes a composition comprising a therapeutically effective amount of an antisense oligonucleotide capable of binding to a selected target on the OPA1 gene transcript and modifying translation of the OPA1 gene transcript or a portion thereof. In an alternative embodiment, the formulation is pre-measured, pre-mixed, and / or pre-packaged. Preferably, the intraocular solution is sterile.

[0251] The kits of the present invention may also include instructions designed to facilitate user compliance. As used herein, instructions refer to any label, insert, etc., and may be placed on one or more surfaces of the packaging material, or the instructions may be provided on a separate sheet, or any combination thereof. For example, in one embodiment, the kits of the present invention include instructions for administering the formulations of the present invention. In one embodiment, the instructions indicate that the formulations of the present invention are suitable for treating ADOA. Such instructions may also include instructions for dosing, as well as instructions for administration via topical delivery to the eye or via intraocular injection.

[0252] The antisense oligonucleotide and suitable excipients can be packaged separately, allowing the practitioner or user to formulate the components into a pharmaceutically acceptable composition as needed. Alternatively, the antisense oligonucleotide and suitable excipients can be packaged together, thereby requiring minimal formulation by the practitioner or user. In any case, the packaging must maintain the chemical, physical, and aesthetic integrity of the active ingredient.

[0253] general Those skilled in the art will understand that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions, and compounds referred to or shown in this specification, individually or collectively, and any and all combinations, or any two or more of the steps or features described above.

[0254] Each document, reference, patent application, or patent cited herein is expressly incorporated herein by reference in its entirety, meaning that it should be read and considered by the reader as part of this specification. It is solely for the sake of brevity that documents, references, patent applications, or patents cited herein are not repeated here.

[0255] Any manufacturer's instructions, descriptions, product specifications, and product sheets for any product described in this specification or any document incorporated herein by reference are hereby incorporated by reference and may be used in the practice of this invention.

[0256] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are for illustrative purposes only. Functionally equivalent products, formulations, and methods are clearly within the scope of the invention as described herein.

[0257] The inventions described herein may include one or more ranges of values ​​(e.g., size, displacement, field strength, etc.). A range of values ​​will be understood to include all values ​​within that range, inclusive of the value defining the range, and adjacent values ​​that produce the same or substantially the same result as the value immediately adjacent to that value defining the boundary to that range. Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Thus, "about 80%" can also mean "about 80%" and "80%." At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0258] Throughout this specification, unless the context requires otherwise, it will be understood that the term "comprise," or variations such as "comprises" or "comprising," imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. Also, in this disclosure, and particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," "comprising," and the like, can have the meaning ascribed to them in U.S. patent law, e.g., they can mean "includes," "included," "including," etc., and terms such as "consisting essentially of" and "consists essentially of" can have the meaning ascribed to them in U.S. patent law, e.g., they allow for elements not expressly recited, but exclude elements found in the prior art or that affect a basic or novel characteristic of the invention.

[0259] Other definitions of selected terms used herein can be found in the detailed description of the present invention and can be applied throughout.Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.The term "active agent" can refer to one active agent or can include two or more active agents.

[0260] The sequence identity numbers ("SEQ ID NOs") containing the nucleotide and amino acid sequence information contained herein are collected at the end of the specification and have been prepared using the program Patentln Version 3.0. Each nucleotide or amino acid sequence is identified in the sequence listing by a numeric indicator <210> followed by a sequence identifier (e.g., <210> 1. <210> 2, etc. The length of each nucleotide or amino acid sequence, the type of sequence, and the source organism are each identified by a numeric indicator field. <211> , <212> and <213> Nucleotide and amino acid sequences referred to herein are represented by the information provided in the numeric indicator fields. <400> followed by a sequence identifier (e.g., <400> 1. <400> 2, etc.).

[0261] To distinguish between different antisense oligonucleotides, an antisense oligonucleotide nomenclature system has been proposed and published (see Mann et al., (2002) J Gen Med 4,644-654). This nomenclature system has become particularly relevant when several slightly different antisense oligonucleotides are tested, all directed against the same target region, as shown below. H # A / D(x:y) The first letter indicates the species (e.g., H: human, M: mouse). "#" indicates the target exon number "A / D" indicates the acceptor or donor splice site at the beginning and end of the exon, respectively. (xy) represents the annealing coordinate, with "-" or "+" indicating the intron or exon sequence, respectively. As an example, A(-6+18) would indicate the last six bases of the intron preceding the target exon and the first 18 bases of the target exon. These coordinates are preceded by an "A" because the nearest splice site is the acceptor. Illustrating annealing coordinates at the donor splice site could be D(+2-18), where the last two exon bases and the first 18 intron bases correspond to the annealing site of the antisense oligonucleotide. The complete exon annealing coordinate is represented by A(+65+85), which is the site between (inclusive of) the 65th and 85th nucleotides from the start of that exon.

[0262] The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these methods in no way limit the true scope of the invention, but rather are presented for illustrative purposes. [Example]

[0263] Example 1 Identification of OPA1 target sequence and ASO design The OPA1 intron 7 and exon regions are shown in Figure 1A. Antisense oligonucleotides (ASOs) 24–25 nucleotides long (Table 1, Figure 1B) and 17 nucleotides long (Table 2) were designed to target the intronic splice enhancer within the intron, mediate exon 7x exclusion, and generate productive OPA1 transcripts. Further refinement of ASO sequences demonstrating protein upregulation was performed by microwalk or engineered mismatch oligos and revalidated by ddPCR and protein assays using the protocols described in Example 2 below.

[0264] Example 2 Screening of phosphorodiamidate morpholino oligonucleotides (PMOs) targeting intron 7 of OPA1 in autosomal dominant optic atrophy (ADOA) patient cells The 24- and 25-nucleotide ASO sequences identified above were synthesized as PMOs. Antisense PMOs targeting intron 7 of OPA1 (as described in Example 1) were transferred into ADOA patient fibroblasts carrying the OPA1 mutation (c.2708_2711delTTAG) using the NEON® electroporation system (ThermoFisher), and the transferred cells were cultured for 5 days. Total RNA was extracted using a MagMAX™-96 kit. OPA1 transcript levels were assessed using a total RNA isolation kit and digital droplet PCR (Qiagen, probe catalog number: dHsaCPE5043545). OPA1 transcript expression was normalized to GAPDH, RPL27, and SCL25A3 transcript levels (Qiagen, probe catalog numbers: dHsaCPE5031596, dHsaCPE5036407, and dHsaCPE5032926, respectively). The three PMOs upregulated OPA1 transcript expression 48 hours after treatment compared to untreated controls. The PMO hOPA1_Ex7xA(-100-76)1mmA-G showed the highest OPA1 transcript upregulation of up to 3.5-fold at 48 hours compared to untreated patient fibroblasts (n = 1 biological replicate) (Figure 2).

[0265] In addition to OPA1 transcript assessment, total protein was harvested from transfected cells (48 h) using CytoBuster Protein Extraction Reagent (Merck Millipore) according to the manufacturer's instructions and assessed by Western blot assay using rabbit anti-OPA1 monoclonal antibody (Cell Signaling, catalog no. 67589) at a dilution of 1:250 in 5% BSA in TBST buffer, followed by goat anti-rabbit IgG H&L antibody (Abcam, catalog no. ab216773, IRDye® 800CW). Beta-actin served as a loading control and was detected using monoclonal mouse anti-beta-actin antibody (Sigma-Aldrich, catalog no. A5441) followed by goat anti-mouse IgG H&L antibody (Abcam, catalog no. ab216776, IRDye® 680RD). The expression levels of OPA1 protein were compared between non-ASO-transfected cells (UT) and OPA1 NMD ASO-transfected cells. The PMO hOPA1_Ex7xA(-99-76) showed up to a 5-fold upregulation of OPA1 expression at 48 hours compared to untreated patient fibroblasts (Figure 3).

[0266] Example 3 Screening of cell-permeable peptide-conjugated phosphorodiamidate morpholino oligonucleotides (PPMOs) targeting intron 7 of OPA1 in autosomal dominant optic atrophy (ADOA) patient cells The selected OPA1 PMOs were conjugated to cell-penetrating peptides (PPMOs). The PPMOs were incubated with ADOA patient fibroblasts carrying the OPA1 mutation (c.985-1G>A), and the transfected cells were cultured for 5 days. Total RNA was extracted using a MagMAX™-96. Total RNA isolation kits were used, and OPA1 transcript levels were assessed by digital droplet PCR (Qiagen, probe catalog number: dHsaCPE5043545). OPA1 transcript expression was normalized to HPRT1 (Qiagen, probe catalog number: dHsaCPE5192872). The majority of PMOs upregulated OPA1 transcript expression 5 hours after treatment compared to untreated controls. The PMO hOPA1_Ex7xA(-100-76)1mmA-G showed upregulation of the highest OPA1 transcript levels by up to 2.2-fold at 5 h compared to untreated patient fibroblasts (n = 1 biological replicate) (Fig. 4).

[0267] Example 4 Refinement of PMO sequence targeting intron 7 of OPA1 in ADOA patient cells The PMO was redesigned to microwalk around the parental sequences identified in exons Examples 2 and 3. Figure 4 illustrates the binding sites of the refined ASOs (ASOs listed in Table 4) on the OPA1 target moiety in relation to the parental sequences in Table 1. Mismatched base substations and oligonucleotide extensions were used to improve the efficacy of OPA1 upregulation.

[0268] Example 5 Screening of PMO refined sequences in ADOA patient-derived fibroblasts The refined PMOs were transfected into ADOA patient fibroblasts carrying the OPA1 mutation (c.985-1G>A) using the NEON™ electroporation system (ThermoFisher) and incubated for 48 hours. Total RNA was extracted using a MagMAX™-96. Total RNA isolation kit and OPA1 transcript levels were assessed by digital droplet PCR (Qiagen, probe catalog number: dHsaCPE5043545). OPA1 transcript expression was normalized to HPRT1 levels (Qiagen, probe catalog number: dHsaCPE5192872). The majority of refined PMOs upregulated OPA1 transcript expression compared to untreated controls 48 hours after treatment (n = 1–4 biological replicates) (Figure 6).

[0269] Example 6 PMO treatment induces upregulation of OPA1 protein in ADOA fibroblasts The selected refined PMO was transfected in triplicate into ADOA patient fibroblasts carrying the OPA1 mutation (c.2708_2711delTTAG) using the NEON™ electroporation system (ThermoFisher) and incubated for 120 hours. Total protein was harvested from the transfected cells using RIPA buffer (ThermoFisher) according to the manufacturer's instructions and assessed by Western blot assay using rabbit anti-OPA1 monoclonal antibody (Cell Signaling, Cat. No. 67589) at a dilution of 1:250 in 5% BSA in TBST buffer, followed by goat anti-rabbit IgG H&L antibody (Abcam, Cat. No. ab216773, IRDye® 800CW). HPRT1 served as a loading control and was detected using HPRT1 polyclonal antibody (ProteinTech, Cat. No. 15059-1-AP). The expression levels of OPA1 protein were compared between non-ASO-transfected cells (UT) and OPA1 NMD ASO-transfected cells. Figure 7 shows that PMO upregulated OPA1 expression compared to untreated patient fibroblasts (n = 1 biological replicate).

[0270] Example 7 PMO-mediated OPA1 upregulation and maintaining OPA1 isoform balance Selected parental and refined PMOs were screened for their efficacy in upregulating total OPA1 and expression of both OPA1 isoforms with and without exon 7. PMOs were transfected into ADOA patient fibroblasts carrying the OPA1 mutation (c.2708_2711delTTAG) and incubated for 48 hours. Total RNA was extracted using a MagMAX™-96. Total RNA isolation kit and OPA1 transcript levels were assessed by digital droplet PCR (Qiagen, probe catalog number: dHsaCPE5043545). OPA1 transcript expression was normalized to HPRT1 (Qiagen, probe catalog number: dHsaCPE5192872). OPA1 isoform expression was analyzed by RT-PCR to determine the proportion of OPA1 isoforms with and without exon 7. Figure 8 shows that PMOs mediated total OPA1 upregulation while maintaining the proportion of OPA1 isoforms.

[0271] Example 8 Quantification of OPA1 mRNA isoforms correlates with total OPA1 levels. The PMO OPA1_Ex7xA(-134-105)2mmA>G (25 μM and 50 μM) was incubated in fibroblasts derived from a patient carrying the OPA1 mutation (c.2708_2711delTTAG), and samples were collected for quantification of total and OPA1 isoform upregulation using ddPCR assays. Primers targeting exons 22-23 and exons 6-8 were used to determine the levels of total OPA1 mRNA and the OPA1 isoform without exon 7, respectively, and normalized to HPRT1 levels. As shown in Figure 9, expression of the OPA1 isoform without exon 7 increased 1.5-1.6-fold, correlating with the level of total OPA1 mRNA, confirming the ability of the PMO to increase OPA1 upregulation while maintaining OPA1 isoform balance.

[0272] Example 9 Peptide-conjugated PMO (PPMO) treatment induces total OPA1 mRNA in RGC-enriched cultures from ADOA patients. The PMO hOPA1_Ex7xA(-100-76)1mmA-G was conjugated to a cell-permeable peptide and incubated in triplicate with iPSC-RGCs derived from ADOA patients carrying the OPA1 mutation (c.985-1G>A) for 120 hours. Total RNA was extracted using a MagMAX™-96. Total RNA isolation kit and OPA1 transcript levels were assessed by digital droplet PCR (Qiagen, probe catalog number: dHsaCPE5043545). OPA1 transcript expression was normalized to HPRT1 levels (Qiagen, probe catalog number: dHsaCPE5192872). Figure 10 shows that the PMO hOPA1_Ex7xA(-100-76)1mmA-G mediated upregulation of total OPA1 transcripts by up to 1.7-fold at 15 μM compared to untreated patient fibroblasts.

Claims

1. An isolated or purified antisense oligonucleotide that binds to intron 7 of the OPA1 gene pre-mRNA.

2. a) inducing increased production of a functional OPA1 protein or a portion thereof; b) increasing the translation of a functional OPA1 protein by excluding nonsense-mediated RNA decay-inducing exons; and / or c) The isolated or purified antisense oligonucleotide of claim 1, which increases the translation of functional OPA1 protein by excluding nonsense-mediated RNA decay-induced (NMD) exon 7x.

3. 3. The isolated or purified antisense oligonucleotide of claim 1 or 2, wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide.

4. The antisense oligonucleotide is a) Table 1, Table 3 and Table 4, b) SEQ ID NOs: 1 to 31; c) SEQ ID NOs: 33 to 54; d) SEQ ID NOs: 1, 2, 3, 4, 7 and 33 to 44; e) SEQ ID NOs: 1, 3, 4, 37, 40 and 44, and f) combinations or cocktails thereof; The isolated or purified antisense oligonucleotide of any one of claims 1 to 3, selected from the group consisting of:

5. 5. The isolated or purified antisense oligonucleotide of any one of claims 1 to 4, wherein the antisense oligonucleotide, when administered to a cell, does not alter the relative expression level or normal physiological ratio of an OPA1 gene transcript that includes exon 7 or lacks exon 7, compared to the level or ratio of the transcript in a cell to which the antisense oligonucleotide is not administered.

6. 1. A method for manipulating translation of an OPA1 gene transcript, the method comprising: i. A method comprising the steps of providing one or more of the antisense oligonucleotides of any one of claims 1 to 5 and allowing said oligonucleotides to bind to a target nucleic acid site.

7. 1. A method for treating, preventing, or alleviating the effects of a disease associated with OPA1 expression, the method comprising: i. A method comprising the step of administering to a patient an effective amount of one or more antisense oligonucleotides according to any one of claims 1 to 5.

8. 1. A pharmaceutical, prophylactic, or therapeutic composition for treating, preventing, or alleviating the effects of a disease associated with OPA1 expression in a patient, the composition comprising: a) one or more antisense oligonucleotides according to any one of claims 1 to 5; b) one or more pharma- ceutically acceptable carriers and / or diluents.

9. 13. Use of the purified and isolated antisense oligonucleotide of any one of claims 1 to 5 for the manufacture of a medicament for treating, preventing, or alleviating the effects of a disease associated with OPA1 expression.

10. 1. A kit for treating, preventing, or alleviating the effects of a disease associated with OPA1 expression in a patient, the kit comprising at least a) an antisense oligonucleotide according to any one of claims 1 to 5, packaged in a suitable container; and b) instructions for use.

11. The disease associated with OPA1 expression is a) is associated with a decrease in the level of functional OPA1 protein expression; b) in patients with autosomal dominant optic atrophy (ADOA), c) is associated with a reduced level of functional OPA1 protein expression in patients with ADOA; and / or d) A method according to claim 6 or 7, a composition according to claim 8, a use according to claim 9, or a kit according to claim 10, which is associated with a reduced level of functional OPA1 protein expression in patients with ADOA associated with OPA1 haploinsufficiency.

12. The antisense oligonucleotide of any one of claims 1 to 5, wherein the antisense oligonucleotide comprises a backbone modification.

13. The antisense oligonucleotide of claim 12, wherein the antisense oligonucleotide comprises a backbone modification comprising a phosphorothioate or phosphorodiamidate linkage.

14. The antisense oligonucleotide of claim 12 or 13, wherein the antisense oligonucleotide comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

15. The antisense oligonucleotide of any one of claims 12 to 14, wherein the antisense oligonucleotide comprises at least one modified sugar moiety.

16. The antisense oligonucleotide of claim 15, wherein each sugar moiety in the antisense oligonucleotide is a modified sugar moiety.

17. The antisense oligonucleotide of any one of claims 12 to 16, wherein the antisense oligonucleotide comprises a 2'-O-methoxyethyl moiety.

18. 18. The antisense oligonucleotide of claim 17, wherein each nucleotide of the antisense oligonucleotide comprises a 2'-O-methoxyethyl moiety.

19. A vector for expressing the antisense RNA (AR) according to any one of claims 1 to 5 in a mammalian cell.

20. 20. The vector of claim 19, wherein the expression vector comprises a cell type-selective or tissue-selective promoter to drive expression of the antisense RNA in the mammalian cell.

21. 20. The vector of claim 19, wherein the cell type-selective or tissue-selective promoter is selective for expression in a cell type or tissue selected from the list consisting of ocular tissue, retinal ganglion cells, neurons, glial cells, astrocytes, and photoreceptors.

22. The vector of any one of claims 19 to 21, wherein the vector comprises an inducible promoter.

23. The vector according to any one of claims 19 to 21, wherein the vector is a non-viral vector.

24. The vector according to any one of claims 19 to 21, wherein the vector is a viral vector.

25. 25. The vector of claim 24, wherein the viral vector is a recombinant virus selected from the group consisting of an adeno-associated virus (AAV), an adenovirus, a lentivirus, and anellovirus.

26. The antisense oligonucleotide or vector according to any one of claims 1 to 5, 12 to 18, wherein the nucleotide sequence of the antisense oligonucleotide or antisense RNA 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, 24 to 30 nucleotides, 25 to 30 nucleotides, or 26 to 30 nucleotides.

27. The antisense oligonucleotide or vector according to claims 1 to 5, wherein the nucleotide sequence of the antisense oligonucleotide or antisense RNA consists of 20 to 30 nucleotides.

28. 28. The antisense oligonucleotide of claim 27, wherein the antisense oligonucleotide comprises one or more phosphorodiamidate morpholino moieties.

29. The antisense oligonucleotide of any one of claims 1 to 5 or 12 to 16, wherein the antisense oligonucleotide further comprises a linked functional moiety.

30. 30. The antisense oligonucleotide of claim 29, wherein the functional moiety comprises a delivery moiety.

31. 30. The antisense oligonucleotide of claim 29, wherein the delivery moiety is selected from the group consisting of a lipid, a peptide, a carbohydrate, and an antibody.

32. 31. The antisense oligonucleotide of claim 29 or 30, wherein the delivery moiety comprises a cell penetrating peptide (CPP).

33. The antisense oligonucleotide of claim 29 or 30, wherein the delivery moiety comprises an N-acetylgalactosamine (GalNAc) moiety.

34. 30. The antisense oligonucleotide of claim 29, wherein the functional portion comprises a stabilizing portion.

35. The antisense oligonucleotide of any one of claims 29 to 34, wherein the functional moiety is covalently linked to the antisense oligonucleotide.

36. The antisense oligonucleotide of any one of claims 29 to 34, wherein the functional moiety is non-covalently attached to the antisense oligonucleotide.

37. The antisense oligonucleotide of any one of claims 29 to 34, wherein the functional moiety is linked to the 5' end of the antisense oligonucleotide.

38. The antisense oligonucleotide of any one of claims 29 to 34, wherein the functional moiety is linked to the 3' end of the antisense oligonucleotide.

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