OPA1 antisense oligomers for treating diseases and conditions

Abstract

Alternative splicing events in genes can induce unproductive mRNA transcripts, which can subsequently lead to abnormal protein expression. Therefore, therapeutic agents that can target alternative splicing events in genes can regulate functional protein expression levels and / or inhibit abnormal protein expression in patients. These therapeutic agents can be used to treat conditions or diseases caused by protein deficiencies and / or mitochondrial dysfunction.

Description

Technical Field

[0001] Cross - reference to Related Art This application claims the benefit of U.S. Provisional Patent Application No. 63 / 505,333, filed on May 31, 2023, which is incorporated herein by reference in its entirety.

Background Art

[0002] Alternative splicing events in genes can induce non - productive mRNA transcripts, which can then induce abnormal protein expression or reduced protein expression. Thus, therapeutic agents that can target alternative splicing events in genes can regulate functional protein expression levels and / or inhibit abnormal protein expression in patients. Such therapeutic agents can be used to treat conditions or diseases caused by protein deficiencies.

[0003] Autosomal dominant optic atrophy (ADOA) is one of the most commonly diagnosed optic neuropathies. This optic nerve disease is associated with structural and functional mitochondrial dysfunction leading to degeneration of retinal ganglion cells and progressive irreversible vision loss. Most ADOA patients have mutations in OPA1, and most mutations lead to haploinsufficiency (see Lenaers G. et al. Orphanet J Rare Dis 2012). OPA1 encodes a mitochondrial GTPase that plays important roles in mitochondrial fusion, ATP synthesis, and apoptosis. Currently, there is no approved disease - modifying treatment for ADOA patients, and there is a need for such treatment.

Summary of the Invention

[0004] In some aspects, described herein is a method of treating a subject having a disease or condition, or a method of reducing the likelihood that a subject will develop a disease or condition, the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer. The antisense oligomer contains a nucleotide sequence that has at least 80% sequence identity with any one of the sequences described in SEQ ID NOs: 6-275 or 280-299. The method involves administering a pharmaceutical composition to one eye of the subject in a dose of approximately 0.005 to 20 mg of antisense oligomer.

[0005] In some embodiments, this specification describes methods for treating a subject having a disease or condition, or for reducing the likelihood of the subject developing a disease or condition, the methods comprising administering a pharmaceutical composition comprising an antisense oligomer to the subject. The antisense oligomer contains a nucleotide sequence having at least 80% sequence identity with any one of the sequences described in SEQ ID NOs: 6-275 or 280-299. The disease or condition includes autosomal dominant optic atrophy (ADOA), and the subjects are: (i) Heterozygous OPA1 gene variant, (ii) A transparent ocular medium that enables proper visualization of the vitreous humor and fundus and achieves proper quality of all ophthalmic evaluations. (iii) Regarding posterior subcapsular (PSC) opacity, <1 AREDS clinical lens standard, (iv) Each eye administered with the pharmaceutical composition individually has a BCVA EDTRS letter score of ≥35 and ≤70, or (v) is characterized by having any combination of (i) to (viii).

[0006] In some embodiments, this specification describes methods for treating a subject having a disease or condition, or for reducing the likelihood of the subject developing a disease or condition, the methods comprising administering a pharmaceutical composition comprising an antisense oligomer to the subject. The antisense oligomer contains a nucleotide sequence having at least 80% sequence identity with any one of the sequences described in SEQ ID NOs: 6-275 or 280-299. The disease or condition includes autosomal dominant optic atrophy (ADOA), and the subjects are: (1) The OPA1 gene does not have a gain-of-function mutant, or a compound heterozygous or homozygous pathogenic or pathogenic mutant. (2) The OPA1 gene does not contain a benign variant or only potentially a benign variant. (3) Not exhibiting extraocular phenotypic expression of (symptomatic) ADOA (ADOA-plus), (4) Never been diagnosed with Behr syndrome, (5) Not having known pathogenic mutations in another gene involved in optic nerve atrophy or retinal disease, (6) Not having diabetic retinopathy that may lead to proliferative diabetic retinopathy, diabetic macular edema, or optic neuropathy. (7) Neither eye has any eye condition, nor any history of any eye condition. (8) No history of intraocular surgery, including refractive surgery, or corneal surgery in either eye within 12 weeks prior to administration. (9) No history of retinal photocoagulation, (10) No history of or presence of retinal vein occlusion, (11) Having had an active relapse of non-infectious uveitis in either eye, or an episode of infectious uveitis or another eye infection, within 12 months prior to administration, does not mean that the patient is at risk of uveitis or eye infection. (12) Neither eye has age-related macular degeneration in dry eyes. (13) No high myopia (>6 diopters) (14) No history of cancer (excluding a diagnosis of successful treatment for basal cell carcinoma, squamous cell carcinoma, or cervical intraepithelial neoplasia), (15) You have never taken or ingested any medication or treatment that may cause or could cause optic neuropathy. (16) No history of nutritional deficiencies (including B12 and / or folic acid deficiency), no known serum B12 or folic acid deficiency, no history of bariatric surgery, or (17) It has the characteristics of any combination of (1) to (16).

[0007] This specification describes, in some embodiments, methods for treating a subject having a disease or condition, or for reducing the likelihood of a subject developing a disease or condition, the method comprising administering a pharmaceutical composition comprising an antisense oligomer to the subject, the antisense oligomer having a chemical structure:

[0008] [ka] [ka] [ka] The method involves a compound consisting of one of the pharmaceutically acceptable salts thereof.

[0009] In some embodiments, the method includes administering the pharmaceutical composition to one eye of a subject in a dose of about 0.005 to about 20 mg of an antisense oligomer.

[0010] In some embodiments, the method involves antisense oligomers of approximately 0.005 mg to 15 mg, approximately 0.005 mg to 10 mg, approximately 0.005 mg to 5 mg, approximately 0.005 mg to 1 mg, approximately 0.01 mg to 15 mg, approximately 0.01 mg to 10 mg, approximately 0.01 mg to 5 mg, approximately 0.01 mg to 2.5 mg, approximately 0.01 mg to 1.0 mg, approximately 0.01 mg to 0.5 mg, approximately 0.01 mg to 0.25 mg, approximately 0.01 mg to 0.1 mg, approximately 0.01 mg to 0.05 mg, and approximately This includes administering the pharmaceutical composition to one eye of the subject in doses of 0.05 mg to approximately 10 mg, approximately 0.05 mg to approximately 5 mg, approximately 0.05 mg to approximately 2.5 mg, approximately 0.05 mg to approximately 1.0 mg, approximately 0.05 mg to approximately 0.5 mg, approximately 0.05 mg to approximately 0.25 mg, approximately 0.05 mg to approximately 0.1 mg, approximately 0.1 mg to approximately 5 mg, approximately 0.1 mg to approximately 2.5 mg, approximately 0.1 mg to approximately 1.5 mg, approximately 0.1 mg to approximately 1.0 mg, approximately 0.1 mg to approximately 0.5 mg, or approximately 0.1 mg to approximately 0.25 mg.

[0011] In some embodiments, the method involves approximately 0.005 mg, 0.01 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.75 mg of antisense oligomers. This includes administering the pharmaceutical composition to one eye of the target in doses of 2.0 mg, approximately 2.25 mg, approximately 2.5 mg, approximately 2.75 mg, approximately 3 mg, approximately 3.5 mg, approximately 4.0 mg, approximately 4.5 mg, approximately 5.0 mg, approximately 5.5 mg, approximately 6.0 mg, approximately 7.0 mg, approximately 8.0 mg, approximately 9.0 mg, approximately 10 mg, approximately 12.5 mg, approximately 15 mg, approximately 17.5 mg, or approximately 20 mg.

[0012] In some embodiments, the method involves antisense oligomers in amounts of approximately 0.1 mg to 1.5 mg, 0.1 mg to 1.4 mg, 0.1 mg to 1.2 mg, 0.1 mg to 1.0 mg, 0.1 mg to 0.8 mg, 0.1 mg to 0.7 mg, 0.1 mg to 0.5 mg, 0.1 mg to 0.3 mg, 0.2 mg to 1.5 mg, and 0.2 mg to 0. 1.4mg, about 0.2mg to about 1.2mg, about 0.2mg to about 1.0mg, about 0.2mg to about 0.8mg, about 0.2mg to about 0.7mg, about 0.2mg to about 0.5mg, about 0 .3mg to about 1.5mg, about 0.3mg to about 1.4mg, about 0.3mg to about 1.2mg, about 0.3mg to about 1.0mg, about 0.3mg to about 0.8mg, about 0.3mg to about 0.7 mg, about 0.3mg to about 0.5mg, about 0.5mg to about 1.5mg, about 0.5mg to about 1.4mg, about 0.5mg to about 1.2mg, about 0.5mg to about 1.0mg, about 0.5m g ~ about 0.8mg, about 0.5mg - about 0.7mg, about 0.7mg - about 1.5mg, about 0.7mg - about 1.4mg, about 0.7mg - about 1.2mg, about 0.7mg - about 1.0mg, This includes administering the pharmaceutical composition to one eye of the subject in doses of approximately 0.8 mg to 1.5 mg, approximately 0.8 mg to 1.4 mg, approximately 0.8 mg to 1.2 mg, approximately 0.8 mg to 1.0 mg, approximately 1.0 mg to 1.5 mg, approximately 1.0 mg to 1.4 mg, approximately 1.0 mg to 1.2 mg, approximately 1.2 mg to 1.5 mg, or approximately 1.2 mg to 1.4 mg.

[0013] In some embodiments, the method involves administering a pharmaceutical composition to one eye of a subject in doses of approximately 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, or 1.5 mg of an antisense oligomer.

[0014] In some embodiments, the method involves approximately 5 μL to 250 μL of antisense oligomer, approximately 10 μL to 250 μL, approximately 20 μL to 250 μL, approximately 30 μL to 250 μL, approximately 40 μL to 250 μL, approximately 50 μL to 250 μL, approximately 60 μL to 250 μL, approximately 70 μL to 250 μL, approximately 80 μL to 250 μL, approximately 100 μL to 250 μL, approximately 120 μL to 250 μL, approximately 150 μL to 250 μL, approximately 160 μL to 250 μL, approximately 180 μL to 500 μL, approximately 200 μL to 250 μL, approximately 220 μL to 250 μL, approximately 5 μL to 220 μL, and approximately 10μL to approximately 220μL, approximately 20μL to approximately 220μL, approximately 30μL to approximately 220μL, approximately 40μL to approximately 220μL, approximately 50μL to approximately 220μL, approximately 60μL to approximately 220μL, approximately 70μL to approximately 220μL, approximately 80μL to approximately 220μL, approximately 100μL to approximately 220μL, approximately 120μL to approximately 220μL, about 150μL to about 220μL, about 160μL to about 220μL, about 180μL to about 220μL, about 5μL to about 200μL, about 1 0μL to about 200μL, about 20μL to about 200μL, about 30μL to about 200μL, about 40μL to about 200μL, about 50μL to about 200μL, Approximately 60μL to approximately 200μL, approximately 70μL to approximately 200μL, approximately 80μL to approximately 200μL, approximately 100μL to approximately 200μL, approximately 120μL to approximately 2 00μL, about 150μL to about 200μL, about 160μL to about 200μL, about 180μL to about 200μL, about 5μL to about 180μL, about 10μL L ~ approx. 180 μL, approx. 20 μL ~ approx. 180 μL, approx. 30 μL ~ approx. 180 μL, approx. 40 μL ~ approx. 180 μL, approx. 60μL ~ approx. 180μL, approx. 70μL ~ approx. 180μL, approx. 80μL ~ approx. 180μL, approx. 100μL ~ approx. 180μL, approx. 120μL ~ approx. 180 μL, approximately 150 μL to approximately 180 μL, approximately 5 μL to approximately 150 μL, approximately 10 μL to approximately 150 μL, approximately 20 μL to approximately 150 μL, approximately 30 μL to approximately 150μL, about 40μL to about 150μL, about 50μL to about 150μL, about 60μL to about 150μL, about 70μL to about 150μL, about 80μL ~150μL, approx. 100μL~approx. 150μL, approx. 120μL~approx. 150μL, approx. 5μL~approx. 150μL, approx. 10μL~approx. 120μL, approx. 20μL~approx. 120μL, approx. 30μL~approx. 120μL, approx. 40μL~approx. 120μL, approx. 50μL~approx. 120μL, approx. 60μL~approx. 120μL,Administering the pharmaceutical composition to one eye of the subject in a volume of about 70 μL to about 120 μL, about 80 μL to about 120 μL, about 100 μL to about 120 μL, about 5 μL to about 100 μL, about 10 μL to about 100 μL, about 20 μL to about 100 μL, about 30 μL to about 100 μL, about 40 μL to about 100 μL, about 50 μL to about 100 μL, about 60 μL to about 100 μL, about 70 μL to about 100 μL, about 80 μL to about 100 μL, about 5 μL to about 80 μL, about 10 μL to about 80 μL, about 20 μL to about 80 μL, about 30 μL to about 80 μL, about 40 μL to about 80 μL, about 50 μL to about 80 μL, about 60 μL to about 80 μL, about 5 μL to about 60 μL, about 10 μL to about 60 μL, about 20 μL to about 60 μL, about 30 μL to about 60 μL, about 40 μL to about 60 μL, or about 50 μL to about 60 μL.

[0015] In some embodiments, the method comprises administering the pharmaceutical composition to one eye of the subject in a volume of about 5 μL, about 8 μL, about 10 μL, about 12 μL, about 15 μL, about 18 μL, about 20 μL, about 25 μL, about 28 μL, about 30 μL, about 35 μL, about 40 μL, about 45 μL, about 48 μL, about 50 μL, about 55 μL, about 60 μL, about 65 μL, about 70 μL, about 75 μL, about 80 μL, about 90 μL, about 100 μL, about 120 μL, about 150 μL, about 160 μL, about 180 μL, about 200 μL, about 220 μL, or about 250 μL.

[0016] In some embodiments, the method comprises administering the pharmaceutical composition to both the left and right eyes of the subject.

[0017] In some embodiments, the method comprises administering the pharmaceutical composition to both the left and right eyes of the subject at the same dosage.

[0018] In some embodiments, the method comprises administering the pharmaceutical composition to the left and right eyes of the subject at different dosages.

[0019] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 90%, or 100% sequence identity with a sequence set forth in any one of SEQ ID NOs: 6 to 275 or 280 to 299.

[0020] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity with any one of the sequences described in SEQ ID NOs. 36, 236, 242, 250, 280-283, 288, or 290-292.

[0021] In some embodiments, the antisense oligomer modulates the splicing of nonsense-mediated RNA decay-inducing exons (NMD exons) from premRNA in the target cell, the premRNA encoding the OPA1 protein and containing NMD exons, thereby modulating the processing level of the mRNA treated from the premRNA and regulating OPA1 protein expression in the cell.

[0022] In some forms, antisense oligomers are (a) Binds to the target region of premRNA, (b) modulate the binding of factors involved in splicing NMD exons, or (c) This is a combination of (a) and (b).

[0023] In some embodiments, the target region of the premRNA is located proximal to the NMD exon.

[0024] In some embodiments, the target region of the premRNA is approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides upstream of the 5' end of the NMD exon.

[0025] In some embodiments, the target region of the premRNA is at least approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, 50 nucleotides, 40 nucleotides, 30 nucleotides, 20 nucleotides, 10 nucleotides, 5 nucleotides, 4 nucleotides, 2 nucleotides, and 1 nucleotide upstream of the 5' end of the NMD exon.

[0026] In some embodiments, the target region of the premRNA is approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides downstream of the 3' end of the NMD exon.

[0027] In some embodiments, the target region of the premRNA is at least approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, 50 nucleotides, 40 nucleotides, 30 nucleotides, 20 nucleotides, 10 nucleotides, 5 nucleotides, 4 nucleotides, 2 nucleotides, and 1 nucleotide downstream of the 3' end of the NMD exon.

[0028] In some embodiments, the target region of premRNA is approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides upstream of the genomic site GRCh38 / hg38:chr3 193628509.

[0029] In some embodiments, the target region of the premRNA is approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides upstream of the genomic site GRCh38 / hg38:chr3 193628509.

[0030] In some embodiments, the target region of premRNA is approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides downstream of the genomic site GRCh38 / hg38:chr3 193628616.

[0031] In some embodiments, the target region of the premRNA is approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides downstream of the genomic site GRCh38 / hg38:chr3 193628616.

[0032] In some embodiments, the target region of the premRNA is located within an intronic region between two standard exonic regions of the premRNA, and the intronic region contains an NMD exon.

[0033] In some embodiments, the target region of the premRNA at least partially overlaps with the NMD exon.

[0034] In some embodiments, the target region of the premRNA at least partially overlaps with an intron upstream or downstream of the NMD exon.

[0035] In some embodiments, the target region of the premRNA includes a 5'NMD exon-intron junction or a 3'NMD exon-intron junction.

[0036] In some embodiments, the target region of the premRNA is located within the NMD exon.

[0037] In some embodiments, the target portion of the premRNA contains approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides of the NMD exon.

[0038] In some embodiments, the NMD exon comprises a sequence having at least 80%, at least 90%, or 100% sequence identity with sequence number 279.

[0039] In some embodiments, the NMD exon contains the sequence of sequence number 279.

[0040] In some embodiments, the target region of the premRNA is located within the nonsense-mediated RNA decay-inducing exon GRCh38 / hg38:chr3 193628509~193628616.

[0041] In some embodiments, the target region of the premRNA is either upstream or downstream of the nonsense-mediated RNA decay-inducing exon GRCh38 / hg38:chr3 193628509~193628616.

[0042] In some embodiments, the target region of the premRNA includes the exon-intron junction of exon GRCh38 / hg38:chr3 193628509~193628616.

[0043] In some embodiments, the OPA1 protein expressed from the treated mRNA is either the full-length OPA1 protein or the wild-type OPA1 protein.

[0044] In some embodiments, the OPA1 protein expressed from the treated mRNA is a functional OPA1 protein.

[0045] In some embodiments, the OPA1 protein expressed from treated mRNA is at least partially functional compared to the wild-type OPA1 protein.

[0046] In some embodiments, the OPA1 protein expressed from treated mRNA is at least partially functional compared to the full-length wild-type OPA1 protein.

[0047] In some embodiments, antisense oligomers facilitate the elimination of NMD exons from premRNA.

[0048] In some embodiments, excluding NMD exons from intracellular premRNA in contact with antisense oligomers is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, and approximately compared to the absence of antisense oligomers. It increased by 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0049] In some embodiments, the method results in an increase in the level of processed mRNA within the cell.

[0050] In some embodiments, the level of treated mRNA in cells in contact with antisense oligomers is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, and 2 to 6 times compared to the absence of antisense oligomers. It increases by approximately 2 to 7 times, approximately 2 to 8 times, approximately 2 to 9 times, approximately 3 to 6 times, approximately 3 to 7 times, approximately 3 to 8 times, approximately 3 to 9 times, approximately 4 to 7 times, approximately 4 to 8 times, approximately 4 to 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times.

[0051] In some embodiments, the method results in increased intracellular OPA1 protein expression.

[0052] In some embodiments, the level of OPA1 protein expressed from processed mRNA in cells in contact with the antisense oligomer is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, and 2 to 5 times higher compared to the absence of the antisense oligomer. It increased by a factor of two, approximately 2 to six times, approximately 2 to seven times, approximately 2 to eight times, approximately 2 to nine times, approximately 3 to six times, approximately 3 to seven times, approximately 3 to eight times, approximately 3 to nine times, approximately 4 to seven times, approximately 4 to eight times, approximately 4 to nine times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times.

[0053] In some embodiments, the antisense oligomer includes modifications to the skeleton, such as a phosphorothioate bond or a phosphorodiamidate bond.

[0054] In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a loc nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-ON-methylacetamide (2'-NMA), or a 2'-O-methoxyethyl moiety.

[0055] In some embodiments, the antisense oligomer comprises at least one modified sugar moiety.

[0056] In some embodiments, each sugar moiety is a modified sugar moiety.

[0057] In some embodiments, the antisense oligomer contains 5'-methylcytosine (5'-MeC). In some embodiments, each cytosine in the antisense oligomer is 5'-methylcytosine (5'-MeC).

[0058] In some embodiments, the antisense oligomer contains 5'-methyluracil (5'-MeU).

[0059] In some embodiments, the antisense oligomer contains a phosphorothioate linkage.

[0060] In some embodiments, the internucleoside bonds in ASO are phosphorothioate bonds.

[0061] In some embodiments, the antisense oligomer contains LOK nucleic acid (LNA).

[0062] In some embodiments, the antisense oligomers are of a length of 8-50 nucleic acid bases, 8-40 nucleic acid bases, 8-35 nucleic acid bases, 8-30 nucleic acid bases, 8-25 nucleic acid bases, 8-20 nucleic acid bases, 8-15 nucleic acid bases, 9-50 nucleic acid bases, 9-40 nucleic acid bases, 9-35 nucleic acid bases, 9-30 nucleic acid bases, 9-25 nucleic acid bases, 9-20 nucleic acid bases, 9-15 nucleic acid bases, 10-50 nucleic acid bases, 10-40 nucleic acid bases, 10-35 nucleic acid bases, and 10-3 It consists of 0 nucleic acid bases, 10-25 nucleic acid bases, 10-20 nucleic acid bases, 10-15 nucleic acid bases, 11-50 nucleic acid bases, 11-40 nucleic acid bases, 11-35 nucleic acid bases, 11-30 nucleic acid bases, 11-25 nucleic acid bases, 11-20 nucleic acid bases, 11-15 nucleic acid bases, 12-50 nucleic acid bases, 12-40 nucleic acid bases, 12-35 nucleic acid bases, 12-30 nucleic acid bases, 12-25 nucleic acid bases, 12-20 nucleic acid bases, or 12-15 nucleic acid bases.

[0063] In some embodiments, antisense oligomers have a chemical structure:

[0064] [ka] [ka] [ka] It is a compound comprising either one of the following: or a pharmaceutically acceptable salt thereof.

[0065] In some embodiments, antisense oligomers have the following structure:

[0066] [ka] [ka] [ka] It is a compound that conforms to one of the following conditions.

[0067] In some embodiments, the pharmaceutical composition is a liquid composition.

[0068] In some embodiments, the method includes administering the pharmaceutical composition as a bolus injection.

[0069] In some embodiments, the method includes administering the pharmaceutical composition as a bolus injection over a period of 1 to 60 minutes, 1 to 50 minutes, 1 to 40 minutes, 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes, 1 to 5 minutes, or 1 to 3 minutes.

[0070] In some embodiments, the antisense oligomer is solubilized or diluted in a solution containing one or more of sodium chloride, sodium diphosphate, potassium monophosphate, or water for injection.

[0071] In some embodiments, the antisense oligomer is solubilized or diluted in a solution containing one or more of sodium chloride, sodium diphosphate, potassium monophosphate, and water for injection.

[0072] In some embodiments, the antisense oligomer is solubilized or diluted in an isotonic solution.

[0073] In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate buffer at pH 5.8.

[0074] In some embodiments, the antisense oligomer is solubilized or diluted in phosphate buffer (pH 6.6-7.6).

[0075] In some embodiments, the pharmaceutical preparation does not contain preservatives.

[0076] In some embodiments, the antisense oligomer is present in the pharmaceutical composition at a concentration of approximately 2 mg / mL to approximately 200 mg / mL.

[0077] In some embodiments, the antisense oligomer is approximately 2 mg / mL to 200 mg / mL, approximately 5 mg / mL to 200 mg / mL, approximately 10 mg / mL to 200 mg / mL, approximately 15 mg / mL to 200 mg / mL, approximately 20 mg / mL to 200 mg / mL, approximately 25 mg / mL to 200 mg / mL, approximately 30 mg / mL to 200 mg / mL, approximately 35 mg / mL to 200 mg / mL, approximately 40 mg / mL to 200 mg / mL, approximately 50 mg / mL to 200 mg / mL, approximately 60 mg / mL to 200 mg / mL, and approximately 80 mg / mL to 200 mg / mL. , about 100 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, 2 mg / mL to about 150 mg / mL, about 5 mg / mL to about 150 mg / mL, about 10 mg / mL to about 150 mg / mL, about 15 mg / mL ~150mg / mL, 20mg / mL~150mg / mL, 25mg / mL~150mg / mL, 30mg / mL~150mg / mL, 35mg / mL~150mg / mL, 40mg / mL~150mg / mL, 50mg / mL~150mg / mL , about 60 mg / mL to about 150 mg / mL, about 80 mg / mL to about 150 mg / mL, about 100 mg / mL to about 150 mg / mL, 2 mg / mL to about 100 mg / mL, about 5 mg / mL to about 100 mg / mL, about 10 mg / mL to about 100 mg / mL, about 15 mg / mL to about 100mg / mL, approximately 20mg / mL to approximately 100mg / mL, approximately 25mg / mL to approximately 100mg / mL, approximately 30mg / mL to approximately 100mg / mL, approximately 35mg / mL to approximately 100mg / mL, approximately 40mg / mL to approximately 100mg / mL, approximately 50mg / mL to approximately 100mg / mL, approximately 60mg / mL to about 100mg / mL, about 80mg / mL to about 100mg / mL, 2mg / mL to about 80mg / mL, about 5mg / mL to about 80mg / mL, about 10mg / mL to about 80mg / mL, about 15mg / mL to about 80mg / mL, about 20mg / mL to about 80mg / mL , about 25 mg / mL to about 80 mg / mL, about 30 mg / mL to about 80 mg / mL, about 35 mg / mL to about 80 mg / mL, about 40 mg / mL to about 80 mg / mL, about 60 mg / mL to about 80 mg / mL, 2 mg / mL to about 60 mg / mL, about 5 mg / mL to about 60 mg / mL,It is present in the pharmaceutical composition at concentrations of approximately 10 mg / mL to 60 mg / mL, 15 mg / mL to 60 mg / mL, 20 mg / mL to 60 mg / mL, 25 mg / mL to 60 mg / mL, 30 mg / mL to 60 mg / mL, 35 mg / mL to 60 mg / mL, 40 mg / mL to 60 mg / mL, 2 mg / mL to 40 mg / mL, 5 mg / mL to 40 mg / mL, 10 mg / mL to 40 mg / mL, 15 mg / mL to 40 mg / mL, 20 mg / mL to 40 mg / mL, or 25 mg / mL to 40 mg / mL.

[0078] In some embodiments, the antisense oligomer is present in the pharmaceutical composition at concentrations of approximately 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 22 mg / mL, 24 mg / mL, 26 mg / mL, 28 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 140 mg / mL, 160 mg / mL, 180 mg / mL, or 200 mg / mL.

[0079] In some embodiments, the pharmaceutical composition is prepared by diluting a concentrate containing an antisense oligomer.

[0080] In some embodiments, the antisense oligomer is approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 200 mg / mL, approximately 100 mg / mL to approximately 200 mg / mL, approximately 150 mg / mL to approximately 200 mg / mL, approximately 180 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 150 mg / mL, approximately 35 mg / mL to approximately 150 mg / mL, approximately 40 mg / mL to approximately 150 mg / mL, approximately 50 mg / mL to approximately 150 mg / mL, approximately 60 mg / mL to approximately 150 mg / mL, and approximately 8 It is present in the anonymized material at concentrations of approximately 0 mg / mL to 150 mg / mL, approximately 100 mg / mL to 150 mg / mL, approximately 30 mg / mL to 100 mg / mL, approximately 35 mg / mL to 100 mg / mL, approximately 40 mg / mL to 100 mg / mL, approximately 50 mg / mL to 100 mg / mL, approximately 60 mg / mL to 100 mg / mL, approximately 80 mg / mL to 100 mg / mL, approximately 30 mg / mL to 80 mg / mL, approximately 35 mg / mL to 80 mg / mL, approximately 40 mg / mL to 80 mg / mL, approximately 60 mg / mL to 80 mg / mL, approximately 30 mg / mL to 60 mg / mL, approximately 35 mg / mL to 60 mg / mL, or approximately 40 mg / mL to 60 mg / mL.

[0081] In some embodiments, the antisense oligomer is present in the concentrate at concentrations of approximately 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 120 mg / mL, 140 mg / mL, 160 mg / mL, 180 mg / mL, or 200 mg / mL.

[0082] In some embodiments, the concentrate is a phosphate buffer.

[0083] In some cases, the subject is a human subject.

[0084] In some embodiments, the disease or condition is a disease or condition associated with a deficiency in the amount or activity of the OPA1 protein.

[0085] In some embodiments, the disease or condition includes a disease or condition of the eye.

[0086] In some embodiments, the disease or condition includes cardiovascular diseases or conditions.

[0087] In some embodiments, the disease or condition includes neurological diseases or conditions.

[0088] In some manifestations, the disease or condition is ADOA-plus syndrome, mitochondrial dysfunction, glaucoma, normal-tension glaucoma, Charcot-Marie-Tooth disease, mitochondrial dysfunction, diabetic retinopathy, age-related macular degeneration, retinal ganglion cell death, mitochondrial fission-mediated mitochondrial dysfunction, progressive extraocular muscle palsy, hearing loss, ataxia, motor neuropathy, sensory neuropathy, myopathy, Baer's syndrome, brain dysfunction, encephalopathy, peripheral neuropathy, fatal infantile mitochondrial encephalomyopathy, hypertrophic cardiomyopathy, spastic ataxia syndrome, sensorimotor peripheral neuropathy, hypotonia, gastrointestinal motility disorders and dysphagia, optic nerve atrophy, optic nerve atrophy-plus syndrome. The group includes mitochondrial DNA depletion syndrome14, late-onset cardiomyopathy, diabetic cardiomyopathy, Alzheimer's disease, focal segmental glomerulosclerosis, kidney disease, Huntington's disease, cognitive decline associated with healthy aging, prion diseases, late-onset dementia and parkinsonism, mitochondrial myopathy, Leigh syndrome, Friedreich's ataxia, Parkinson's disease, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), pyruvate dehydrogenase complex deficiency, chronic kidney disease, Leber's hereditary optic neuropathy, obesity, age-related systemic neurodegeneration, skeletal muscle atrophy, ischemic injury of the heart and brain, or massive hepatocyte apoptosis.

[0089] In some embodiments, the disease or condition includes type 1 optic atrophy.

[0090] In some embodiments, the disease or condition includes autosomal dominant optic atrophy (ADOA).

[0091] In some cases, the subject is (i) Heterozygous OPA1 gene variant, (ii) A transparent ocular medium that enables proper visualization of the vitreous humor and fundus and achieves proper quality of all ophthalmic evaluations. (iii) Regarding posterior subcapsular (PSC) opacity, <1 AREDS clinical lens standard, (iv) Each eye administered with the pharmaceutical composition individually has a BCVA EDTRS letter score of ≥35 and ≤70, or (v) is characterized by having any combination of (i) to (viii).

[0092] In some cases, the subject is (1) The OPA1 gene does not have a gain-of-function mutant, or a compound heterozygous or homozygous pathogenic or pathogenic mutant. (2) The OPA1 gene does not have a benign variant or only the possibility of being a benign variant, (3) Not exhibiting extraocular phenotypic expression of (symptomatic) ADOA (ADOA-plus), (4) Never been diagnosed with Behr syndrome, (5) Not having known pathogenic mutations in another gene involved in optic nerve atrophy or retinal disease, (6) Not having diabetic retinopathy that may lead to proliferative diabetic retinopathy, diabetic macular edema, or optic neuropathy. (7) Neither eye has any eye condition, nor any history of any eye condition. (8) No history of intraocular surgery, including refractive surgery, or corneal surgery in either eye within 12 weeks prior to administration. (9) No history of retinal photocoagulation, (10) No history of or presence of retinal vein occlusion, (11) Having had an active relapse of non-infectious uveitis in either eye, or an episode of infectious uveitis or another eye infection, within 12 months prior to administration, does not mean that the patient is at risk of uveitis or eye infection. (12) Neither eye has age-related macular degeneration in dry eyes. (13) No high myopia (>6 diopters) (14) No history of cancer (excluding a diagnosis of successful treatment for basal cell carcinoma, squamous cell carcinoma, or cervical intraepithelial neoplasia), (15) You have never taken or ingested any medication or treatment that may cause or could cause optic neuropathy. (16) No history of nutritional deficiencies (including B12 and / or folic acid deficiency), no known serum B12 or folic acid deficiency, and no history of bariatric surgery. (17) It has the characteristics of any combination of (1) to (16).

[0093] In some embodiments, administration includes administering a pharmaceutical composition in multiple doses to a human subject.

[0094] In some embodiments, administration includes administering an initial dose of the pharmaceutical composition to a human subject, followed by administering subsequent doses of the pharmaceutical composition to the human subject.

[0095] In some embodiments, the subsequent dose is less than the previous dose, in accordance with instructions that the previous dose was not tolerated.

[0096] In some embodiments, the subsequent dose is the same as the previous dose, in accordance with instructions that the administration of the previous dose is tolerable.

[0097] In some embodiments, the subsequent dose is greater than the previous dose, in accordance with instructions that the administration of the previous dose was tolerable.

[0098] In some embodiments, the subsequent dose is the same as the previous dose, in accordance with instructions that the previous dose was effective.

[0099] In some embodiments, the subsequent dose is less than the previous dose, in accordance with instructions that the previous dose was effective.

[0100] In some embodiments, the subsequent dose is greater than the previous dose, in accordance with instructions that the previous dose was ineffective.

[0101] In some embodiments, the pharmaceutical composition is administered by intraventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreous administration, subretinal injection, topical application, implantation, or intravenous injection.

[0102] In some embodiments, the pharmaceutical composition is administered via intravitreous injection.

[0103] In some embodiments, the method further includes administering an additional therapeutic agent.

[0104] In some embodiments, the additional therapeutic agent includes small molecules.

[0105] In some embodiments, the additional therapeutic agent includes an antisense oligomer.

[0106] In some embodiments, the additional therapeutic agent includes ophthalmic drugs.

[0107] In some embodiments, the structure described herein is:

[0108] [ka] [ka] [ka] It is an antisense oligomer, or a pharmaceutically acceptable salt thereof, that is a compound that follows any one of the following.

[0109] In some embodiments, the structure is as follows:

[0110] [ka] [ka] [ka] An antisense oligomer having any of the following characteristics is described.

[0111] In some embodiments, what is described herein is: (a) an antisense oligomer comprising an antisense oligomer having at least 80% sequence identity with one of sequence numbers 6-275 or 280-299, (b) A pharmaceutical preparation comprising a pharmaceutically acceptable diluent, Antisense oligomers are dissolved or suspended in solution at concentrations ranging from approximately 1 mg / mL to approximately 200 mg / mL.

[0112] In some embodiments, what is described herein is: (a) Antisense oligomers and (b) A pharmaceutical preparation comprising a pharmaceutically acceptable diluent, Antisense oligomers are dissolved or suspended in solution. Antisense oligomers have the following chemical structure:

[0113] [ka] [ka] [ka] It is a compound consisting of one of the pharmaceutically acceptable salts thereof.

[0114] In some embodiments, what is described herein is: (a) Antisense oligomers and (b) A pharmaceutical preparation comprising a pharmaceutically acceptable diluent, Antisense oligomers are dissolved or suspended in solution. Antisense oligomers have the following chemical structure:

[0115] [ka] [ka] [ka] It has one of the following.

[0116] In some embodiments, the antisense oligomer is present in the solution at a concentration of approximately 1 mg / mL to approximately 200 mg / mL.

[0117] In some embodiments, the antisense oligomer is approximately 5 mg / mL to approximately 200 mg / mL, approximately 10 mg / mL to approximately 200 mg / mL, approximately 15 mg / mL to approximately 200 mg / mL, approximately 20 mg / mL to approximately 200 mg / mL, approximately 25 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 200 mg / mL, approximately 100 mg / mL to approximately 200 mg / mL mL, approximately 150 mg / mL to approximately 200 mg / mL, approximately 180 mg / mL to approximately 200 mg / mL, 2 mg / mL to approximately 150 mg / mL, approximately 5 mg / mL to approximately 150 mg / mL, approximately 10 mg / mL to approximately 150 mg / mL, approximately 15 mg / mL to approximately 150 mg / mL, approximately 20 mg / m L ~ about 150mg / mL, about 25mg / mL - about 150mg / mL, about 30mg / mL - about 150mg / mL, about 35mg / mL - about 150mg / mL, about 40mg / mL - about 150mg / mL, about 50mg / mL - about 150mg / mL, about 60mg / mL - about 150mg / m L, about 80 mg / mL to about 150 mg / mL, about 100 mg / mL to about 150 mg / mL, 2 mg / mL to about 100 mg / mL, about 5 mg / mL to about 100 mg / mL, about 10 mg / mL to about 100 mg / mL, about 15 mg / mL to about 100 mg / mL, about 20 mg / mL to Approximately 100mg / mL, approximately 25mg / mL to approximately 100mg / mL, approximately 30mg / mL to approximately 100mg / mL, approximately 35mg / mL to approximately 100mg / mL, approximately 40mg / mL to approximately 100mg / mL, approximately 50mg / mL to approximately 100mg / mL, approximately 60mg / mL to approximately 100mg / mL, Approximately 80 mg / mL to approximately 100 mg / mL, 2 mg / mL to approximately 80 mg / mL, approximately 5 mg / mL to approximately 80 mg / mL, approximately 10 mg / mL to approximately 80 mg / mL, approximately 15 mg / mL to approximately 80 mg / mL, approximately 20 mg / mL to approximately 80 mg / mL, approximately 25 mg / mL to approximately 80 mg / mL , about 30 mg / mL to about 80 mg / mL, about 35 mg / mL to about 80 mg / mL, about 40 mg / mL to about 80 mg / mL, about 60 mg / mL to about 80 mg / mL, 2 mg / mL to about 60 mg / mL, about 5 mg / mL to about 60 mg / mL, about 10 mg / mL to about 60 mg / mL,It is present in the solution at concentrations of approximately 15 mg / mL to 60 mg / mL, 20 mg / mL to 60 mg / mL, 25 mg / mL to 60 mg / mL, 30 mg / mL to 60 mg / mL, 35 mg / mL to 60 mg / mL, 40 mg / mL to 60 mg / mL, 2 mg / mL to 40 mg / mL, 5 mg / mL to 40 mg / mL, 10 mg / mL to 40 mg / mL, 15 mg / mL to 40 mg / mL, 20 mg / mL to 40 mg / mL, or 25 mg / mL to 40 mg / mL.

[0118] In some embodiments, the antisense oligomer is present in the solution at concentrations of approximately 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 22 mg / mL, 24 mg / mL, 26 mg / mL, 28 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 140 mg / mL, 160 mg / mL, 180 mg / mL, or 200 mg / mL.

[0119] In some embodiments, the pharmaceutical composition is prepared by diluting a concentrate containing an antisense oligomer.

[0120] In some embodiments, the antisense oligomer is approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 200 mg / mL, approximately 100 mg / mL to approximately 200 mg / mL, approximately 150 mg / mL to approximately 200 mg / mL, approximately 180 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 150 mg / mL, approximately 35 mg / mL to approximately 150 mg / mL, approximately 40 mg / mL to approximately 150 mg / mL, approximately 50 mg / mL to approximately 150 mg / mL, approximately 60 mg / mL to approximately 150 mg / mL, and approximately 8 It is present in the anonymized material at concentrations of approximately 0 mg / mL to 150 mg / mL, approximately 100 mg / mL to 150 mg / mL, approximately 30 mg / mL to 100 mg / mL, approximately 35 mg / mL to 100 mg / mL, approximately 40 mg / mL to 100 mg / mL, approximately 50 mg / mL to 100 mg / mL, approximately 60 mg / mL to 100 mg / mL, approximately 80 mg / mL to 100 mg / mL, approximately 30 mg / mL to 80 mg / mL, approximately 35 mg / mL to 80 mg / mL, approximately 40 mg / mL to 80 mg / mL, approximately 60 mg / mL to 80 mg / mL, approximately 30 mg / mL to 60 mg / mL, approximately 35 mg / mL to 60 mg / mL, or approximately 40 mg / mL to 60 mg / mL.

[0121] In some embodiments, the antisense oligomer is present in the concentrate at concentrations of approximately 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 120 mg / mL, 140 mg / mL, 160 mg / mL, 180 mg / mL, or 200 mg / mL.

[0122] In some embodiments, the concentrate is phosphate-buffered.

[0123] In some embodiments, the antisense oligomer is solubilized or diluted in a solution containing one or more of sodium chloride, sodium diphosphate, potassium monophosphate, or water for injection.

[0124] In some embodiments, the antisense oligomer is solubilized or diluted in a solution containing sodium chloride, sodium diphosphate, potassium monophosphate, and water for injection.

[0125] In some embodiments, the antisense oligomer is solubilized or diluted in an isotonic solution.

[0126] In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate buffer at pH 5.8.

[0035] In some embodiments, the antisense oligomer is solubilized or diluted in phosphate buffer (pH 6.6-7.6).

[0127] In some embodiments, the pharmaceutical preparation does not contain preservatives.

[0128] In some embodiments, the pharmaceutical formulation is suitable for intravitreal injection.

[0129] In some embodiments, the pharmaceutical product is packaged in a single-use vial.

[0130] In some embodiments, what is described herein is: (i) A concentrate containing an antisense oligomer (ASO), wherein the ASO contains a sequence having at least 80% sequence identity with one of sequence numbers 6-275 or 280-299, (ii) A diluent, wherein the concentrate is miscible with the diluent, (iii) A kit including instructions for diluting the concentrate with a diluent.

[0131] In some embodiments, the antisense oligomer is approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 200 mg / mL, approximately 100 mg / mL to approximately 200 mg / mL, approximately 150 mg / mL to approximately 200 mg / mL, approximately 180 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 150 mg / mL, approximately 35 mg / mL to approximately 150 mg / mL, approximately 40 mg / mL to approximately 150 mg / mL, approximately 50 mg / mL to approximately 150 mg / mL, approximately 60 mg / mL to approximately 150 mg / mL, and approximately 8 It is present in the anonymized material at concentrations of approximately 0 mg / mL to 150 mg / mL, approximately 100 mg / mL to 150 mg / mL, approximately 30 mg / mL to 100 mg / mL, approximately 35 mg / mL to 100 mg / mL, approximately 40 mg / mL to 100 mg / mL, approximately 50 mg / mL to 100 mg / mL, approximately 60 mg / mL to 100 mg / mL, approximately 80 mg / mL to 100 mg / mL, approximately 30 mg / mL to 80 mg / mL, approximately 35 mg / mL to 80 mg / mL, approximately 40 mg / mL to 80 mg / mL, approximately 60 mg / mL to 80 mg / mL, approximately 30 mg / mL to 60 mg / mL, approximately 35 mg / mL to 60 mg / mL, or approximately 40 mg / mL to 60 mg / mL.

[0132] In some embodiments, the antisense oligomer is present in the concentrate at concentrations of approximately 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 120 mg / mL, 140 mg / mL, 160 mg / mL, 180 mg / mL, or 200 mg / mL.

[0133] In some embodiments, the concentrate is phosphate-buffered.

[0134] In some embodiments, the diluent is a solution containing one or more of sodium chloride, sodium diphosphate, potassium monophosphate, or water for injection.

[0135] In some embodiments, the diluent is a solution comprising sodium chloride, sodium diphosphate, potassium monophosphate, and water for injection.

[0136] In some embodiments, the diluent includes an isotonic solution.

[0137] In some embodiments, the diluent includes a phosphate buffer with a pH of at least 5.8.

[0138] In some embodiments, the diluent includes phosphate buffer (pH 6.6-7.6).

[0139] In some embodiments, the concentrate or diluent does not contain preservatives.

[0140] In some embodiments, instructions for diluting the concentrate with a diluent include instructions for diluting or solubilizing the ASO in the diluent to a concentration of approximately 2 mg / mL to 200 mg / mL.

[0141] In some embodiments, instructions for diluting the concentrate with a diluent describe diluting or solubilizing the ASO in the diluent to approximately 5 mg / mL to 200 mg / mL, 10 mg / mL to 200 mg / mL, 15 mg / mL to 200 mg / mL, 20 mg / mL to 200 mg / mL, 25 mg / mL to 200 mg / mL, 30 mg / mL to 200 mg / mL, 35 mg / mL to 200 mg / mL, 40 mg / mL to 200 mg / mL, 50 mg / mL to 200 mg / mL, 60 mg / mL to 200 mg / mL, and 80 mg / mL to 20 0mg / mL, about 100mg / mL to about 200mg / mL, about 150mg / mL to about 200mg / mL, about 180mg / mL to about 200mg / mL, 2mg / mL to about 150mg / mL, about 5mg / mL to about 150mg / mL, about 10mg / mL to about 150mg / mL, about 15 mg / mL~about 150mg / mL, about 20mg / mL~about 150mg / mL, about 25mg / mL~about 150mg / mL, about 30mg / mL~about 150mg / mL, about 35mg / mL~about 150mg / mL, about 40mg / mL~about 150mg / mL, about 50mg / mL~about 150m g / mL, about 60 mg / mL to about 150 mg / mL, about 80 mg / mL to about 150 mg / mL, about 100 mg / mL to about 150 mg / mL, 2 mg / mL to about 100 mg / mL, about 5 mg / mL to about 100 mg / mL, about 10 mg / mL to about 100 mg / mL, about 15 mg / m L ~ about 100mg / mL, about 20mg / mL - about 100mg / mL, about 25mg / mL - about 100mg / mL, about 30mg / mL - about 100mg / mL, about 35mg / mL - about 100mg / mL, about 40mg / mL - about 100mg / mL, about 50mg / mL - about 100mg / mL , about 60 mg / mL to about 100 mg / mL, about 80 mg / mL to about 100 mg / mL, 2 mg / mL to about 80 mg / mL, about 5 mg / mL to about 80 mg / mL, about 10 mg / mL to about 80 mg / mL, about 15 mg / mL to about 80 mg / mL, about 20 mg / mL to about 80 mg / m L, about 25 mg / mL to about 80 mg / mL, about 30 mg / mL to about 80 mg / mL, about 35 mg / mL to about 80 mg / mL, about 40 mg / mL to about 80 mg / mL, about 60 mg / mL to about 80 mg / mL, 2 mg / mL to about 60 mg / mL, about 5 mg / mL to about 60 mg / mL,Includes instructions for adjusting concentrations to approximately 10 mg / mL to 60 mg / mL, 15 mg / mL to 60 mg / mL, 20 mg / mL to 60 mg / mL, 25 mg / mL to 60 mg / mL, 30 mg / mL to 60 mg / mL, 35 mg / mL to 60 mg / mL, 40 mg / mL to 60 mg / mL, 2 mg / mL to 40 mg / mL, 5 mg / mL to 40 mg / mL, 10 mg / mL to 40 mg / mL, 15 mg / mL to 40 mg / mL, 20 mg / mL to 40 mg / mL, or 25 mg / mL to 40 mg / mL.

[0142] In some embodiments, instructions for diluting the concentrate with a diluent describe diluting or solubilizing the antisense oligomer in the diluent at concentrations of approximately 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 18 mg / mL, and 2 mg / mL. Includes instructions for adjusting concentrations to 0 mg / mL, approximately 22 mg / mL, approximately 24 mg / mL, approximately 26 mg / mL, approximately 28 mg / mL, approximately 30 mg / mL, approximately 35 mg / mL, approximately 40 mg / mL, approximately 50 mg / mL, approximately 60 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 140 mg / mL, approximately 160 mg / mL, approximately 180 mg / mL, or approximately 200 mg / mL.

[0143] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 90% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 6-275 or 280-299.

[0144] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 36, 236, 242, 250, 280-283, 288, and 290-292.

[0145] In some embodiments, the antisense oligomer includes modifications to the skeleton, such as a phosphorothioate bond or a phosphorodiamidate bond.

[0146] In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a loc nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-ON-methylacetamide (2'-NMA), or a 2'-O-methoxyethyl moiety.

[0147] In some embodiments, the antisense oligomer comprises at least one modified sugar moiety.

[0148] In some embodiments, each sugar moiety is a modified sugar moiety.

[0149] In some embodiments, the antisense oligomer contains 5'-methylcytosine (5'-MeC).

[0150] In some embodiments, each cytosine in the antisense oligomer is 5'-methylcytosine (5'-MeC).

[0151] In some embodiments, the antisense oligomer contains 5'-methyluracil (5'-MeU).

[0152] In some embodiments, the antisense oligomer contains a phosphorothioate linkage.

[0153] In some embodiments, the internucleoside bonds in ASO are phosphorothioate bonds.

[0154] In some embodiments, the antisense oligomer contains LOK nucleic acid (LNA).

[0155] In some embodiments, the antisense oligomers are of a length of 8-50 nucleic acid bases, 8-40 nucleic acid bases, 8-35 nucleic acid bases, 8-30 nucleic acid bases, 8-25 nucleic acid bases, 8-20 nucleic acid bases, 8-15 nucleic acid bases, 9-50 nucleic acid bases, 9-40 nucleic acid bases, 9-35 nucleic acid bases, 9-30 nucleic acid bases, 9-25 nucleic acid bases, 9-20 nucleic acid bases, 9-15 nucleic acid bases, 10-50 nucleic acid bases, 10-40 nucleic acid bases, 10-35 nucleic acid bases, and 10-3 It consists of 0 nucleic acid bases, 10-25 nucleic acid bases, 10-20 nucleic acid bases, 10-15 nucleic acid bases, 11-50 nucleic acid bases, 11-40 nucleic acid bases, 11-35 nucleic acid bases, 11-30 nucleic acid bases, 11-25 nucleic acid bases, 11-20 nucleic acid bases, 11-15 nucleic acid bases, 12-50 nucleic acid bases, 12-40 nucleic acid bases, 12-35 nucleic acid bases, 12-30 nucleic acid bases, 12-25 nucleic acid bases, 12-20 nucleic acid bases, or 12-15 nucleic acid bases.

[0156] In some embodiments, antisense oligomers have a chemical structure:

[0157] [ka] [ka] [ka] It is a compound comprising either one of the following: or a pharmaceutically acceptable salt thereof.

[0158] In some embodiments, antisense oligomers have a chemical structure:

[0159] [ka] [ka] [ka] It has one of the following.

[0160] This specification describes the use of antisense oligomers for manufacturing a pharmaceutical to treat a disease or condition characterized by reduced OPA1 protein expression or function in a human subject requiring such treatment, or to reduce the likelihood of the subject developing a disease or condition, wherein the pharmaceutical is administered to one eye of the subject in a dose of about 0.005 mg to about 20 mg, and the antisense oligomer comprises a sequence having at least 80% sequence identity with one of SEQ ID NOs: 6-275 or 280-299.

[0161] In some embodiments, the pharmaceutical product is an antisense oligomer in the following concentrations: approximately 0.005 mg to 15 mg, approximately 0.005 mg to 10 mg, approximately 0.005 mg to 5 mg, approximately 0.005 mg to 1 mg, approximately 0.01 mg to 15 mg, approximately 0.01 mg to 10 mg, approximately 0.01 mg to 5 mg, approximately 0.01 mg to 2.5 mg, approximately 0.01 mg to 1.0 mg, approximately 0.01 mg to 0.5 mg, approximately 0.01 mg to 0.25 mg, approximately 0.01 mg to 0.1 mg, and approximately 0.01 mg to 0.05 mg. This includes administering the drug to one eye of the subject in doses of approximately 0.05 mg to 10 mg, approximately 0.05 mg to 5 mg, approximately 0.05 mg to 2.5 mg, approximately 0.05 mg to 1.0 mg, approximately 0.05 mg to 0.5 mg, approximately 0.05 mg to 0.25 mg, approximately 0.05 mg to 0.1 mg, approximately 0.1 mg to 5 mg, approximately 0.1 mg to 2.5 mg, approximately 0.1 mg to 1.5 mg, approximately 0.1 mg to 1.0 mg, approximately 0.1 mg to 0.5 mg, or approximately 0.1 mg to 0.25 mg.

[0162] In some embodiments, the pharmaceutical product is an antisense oligomer in the following concentrations: approximately 0.005 mg, 0.01 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, and 1.7 mg. It is administered to one eye of the patient in doses of 5 mg, approximately 2.0 mg, approximately 2.25 mg, approximately 2.5 mg, approximately 2.75 mg, approximately 3 mg, approximately 3.5 mg, approximately 4.0 mg, approximately 4.5 mg, approximately 5.0 mg, approximately 5.5 mg, approximately 6.0 mg, approximately 7.0 mg, approximately 8.0 mg, approximately 9.0 mg, approximately 10 mg, approximately 12.5 mg, approximately 15 mg, approximately 17.5 mg, or approximately 20 mg.

[0163] In some embodiments, the pharmaceutical product contains antisense oligomers in amounts of approximately 0.1 mg to 1.5 mg, 0.1 mg to 1.4 mg, 0.1 mg to 1.2 mg, 0.1 mg to 1.0 mg, 0.1 mg to 0.8 mg, 0.1 mg to 0.7 mg, 0.1 mg to 0.5 mg, 0.1 mg to 0.3 mg, 0.2 mg to 1.5 mg, and 0.2 mg. g ~ about 1.4mg, about 0.2mg - about 1.2mg, about 0.2mg - about 1.0mg, about 0.2mg - about 0.8mg, about 0.2mg - about 0.7mg, about 0.2mg - about 0.5m g, about 0.3mg to about 1.5mg, about 0.3mg to about 1.4mg, about 0.3mg to about 1.2mg, about 0.3mg to about 1.0mg, about 0.3mg to about 0.8mg, about 0.3mg ~0.7mg, approx. 0.3mg~0.5mg, approx. 0.5mg~1.5mg, approx. 0.5mg~1.4mg, approx. 0.5mg~1.2mg, approx. 0.5mg~1.0mg , about 0.5mg to about 0.8mg, about 0.5mg to about 0.7mg, about 0.7mg to about 1.5mg, about 0.7mg to about 1.4mg, about 0.7mg to about 1.2mg, about 0.7mg The drug is administered to one eye of the patient in the following doses: approximately 1.0 mg, 0.8 mg to 1.5 mg, 0.8 mg to 1.4 mg, 0.8 mg to 1.2 mg, 0.8 mg to 1.0 mg, 1.0 mg to 1.5 mg, 1.0 mg to 1.4 mg, 1.0 mg to 1.2 mg, 1.2 mg to 1.5 mg, or 1.2 mg to 1.4 mg.

[0164] In some embodiments, the drug is administered to one eye of the subject in doses of approximately 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, or 1.5 mg of antisense oligomer.

[0165] Built-in by reference All published documents, patents, and patent applications referenced herein are incorporated herein by reference to the same extent that each individual published document, patent, or patent application is specifically and individually indicated as if it were incorporated by reference. [Brief explanation of the drawing]

[0166] The patent or application file must include at least one drawing in color. A copy of the published patent or patent application containing the color drawing will be provided by the Patent and Trademark Office upon request and payment of the required fees.

[0167] Novel features of this disclosure are described in detail in the attached claims. A deeper understanding of the characteristics and advantages of this disclosure will be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of this disclosure are utilized, and to the attached drawings below.

[0168] [Figure 1A]Figure 1A shows a schematic diagram of a target mRNA containing a nonsense-mediated mRNA decay-inducing exon (NMD exon mRNA), and a schematic diagram of the therapeutic-mediated elimination of the nonsense-mediated mRNA decay-inducing exon to increase full-length target protein expression or functional RNA expression. In particular, Figure 1A shows a schematic example of the inclusion of an alternative exon (designated as exon X) identified in the OPA1 gene, which results in the introduction of an immature stop codon (PTC) and leads to a nonproductive mRNA transcript that is degraded by nonsense mutation-dependent degradation (NMD). The schematic diagram also shows the positions of two primers used to detect both productive and nonproductive mRNA transcripts by RT-PCR. [Figure 1B] Figure 1B shows the expression of OPA1 transcripts containing NMD exons in HEK293 cells treated with increased doses of cycloheximide. Three hours of cycloheximide treatment of HEK293 cells increases the abundance of unproductive mRNA transcripts. The abundance of unproductive transcripts was calculated as the intensity of (unproductive transcripts / [unproductive + productive transcripts]) and expressed as a percentage. Representative gel images are shown, and the abundance of unproductive transcripts shown below is the average of three biological replications. [Figure 1C] Figure 1C shows RT-PCR data from non-productive transcripts in RNA isolated from retinal tissue punches from the macular and peripheral regions of both eyes of a human donor. The relative abundance of non-productive transcripts in each sample is included at the bottom of each lane. OD: Oculus dexter (right eye), OS: Oculus sinister (left eye). [Figure 2A] Figure 2A shows RT-PCR data of non-productive OPA1 mRNA in HEK293 cells after in vitro treatment with ASO-1 and cycloheximide. [Figure 2B] Figure 2B shows the quantification of productive OPA1 mRNA in HEK293 cells after treatment with ASO-1 in the absence of cycloheximide. [Figure 2C]Figure 2C shows OPA1 protein expression in HEK293 cells after treatment with ASO-1 in the absence of cycloheximide. [Figure 2D] Figure 2D shows the EC50 evaluation of ASO-1 in HEK293 cells, illustrating a representative dose-response curve fit based on the levels of unproductive transcripts measured when various doses of ASO-1 were applied to HEK293 cells. [Figure 2E] Figure 2E is a Western blot gel image showing OPA1 protein expression after transfection of HEK293 cells with the vehicle (first 3 lanes), non-targeted ASO (NT ASO) at various concentrations (1 nM, 5 nM, and 20 nM), and ASO-1 at various concentrations (1 nM, 5 nM, and 20 nM). Each condition included 6 biological replicas. Cells were lysed in RIPA buffer after transfection, and Western blots were probed with antibodies targeting OPA1 and β-tubulin. Multiple bands at the top of each gel correspond to different isoforms of OPA1. [Figure 2F] Figure 2F is a line graph showing the relative cell viability of HEK293 cells over 72 hours after treatment with various doses (1 μM, 3 μM, 10 μM, and 20 μM) of either ASO-1 or non-targeted ASO (NT ASO). [Figure 2G] Figure 2G is a line graph showing the relative cell viability 72 hours after treatment of HEK293 cells with various concentrations of cycloheximide (8 ng / ml, 40 ng / ml, 200 ng / ml, 1000 ng / ml, and 5000 ng / ml). [Figure 3-1]Figures 3A–3E represent data from Western blot analysis showing a decrease in OPA1 protein levels in OPA1+ / + HEK293 cells compared to isogenic OPA1+ / + HEK293 cells. Western blot gel images are shown in Figure 3A. Histograms of the quantitative Western blots are shown in Figures 3B and 3E, the latter showing the relevant data points. Figure 3C is a histogram showing the relative OPA1 protein expression when isogenic OPA1+ / + HEK293 and OPA1+ / - HEK293 cells were treated with vehicle (without ASO) and ASO-1, respectively, corresponding to the quantification of the Western blot data in Figure 3D. Figure 3D shows exemplary Western blot gel images of OPA1+ / + HEK293 and OPA1+ / - HEK293 cells treated with vehicle or ASO-1 and probed with antibodies targeting OPA1 or β-actin. [Figure 3-2] Figures 3A–3E show data from Western blot analysis demonstrating reduced OPA1 protein levels in OPA1+ / -HEK293 cells compared to isogenic OPA1+ / +HEK293 cells. [Figure 3-3] Figure 3F is a gel image of RT-PCR data from OPA1+ / + HEK293 cells and OPA1+ / - HEK293 cells treated with either DMSO or cycloheximide (CHX), showing the levels of unproductive (upper band) and productive (lower band) OPA1 transcripts. Figure 3G is a histogram of quantitative data from the Western blot of Figure 3F, showing the percentage of unproductive transcripts under test conditions including OPA1+ / + HEK293 cells and OPA1+ / - HEK293 cells treated with either DMSO or CHX. Figure 3H is a histogram of qPCR data showing the relative levels of unproductive transcripts under test conditions including OPA1+ / + HEK293 cells and OPA1+ / - HEK293 cells treated with either DMSO or CHX. [Figure 4]Figures 4A–4E show that ASO-1 increases OPA1 mRNA and OPA1 protein expression in OPA1+ / - HEK293 cells. Figures 4A–4D are histograms of data from OPA1+ / + or OPA1+ / - HEK293 cells treated with a vehicle or 10 μM ASO-1 by gymnotic delivery for 72 hours, and the effect on OPA1 mRNA expression (Figures 4A–4B) and OPA1 protein (Figures 4C–4D) 72 hours after treatment is evaluated. Figure 4A is a histogram showing data for OPA1 mRNA expression, and Figure 4C is a histogram showing data for OPA1 protein levels when OPA1+ / + HEK293 cells were treated with a vehicle or 10 μM ASO-1 by gymnotic delivery. Figure 4B is a histogram showing OPA1 mRNA expression data, and Figure 4D is a histogram showing OPA1 protein level data when OPA1+ / + HEK293 cells were treated with a vehicle or by gymnotic delivery with 10 μM ASO-1. Figure 4E is a representative Western blot image for the data in Figures 4C-4D. [Figure 5-1] Figures 5A–5D show that ASO-1 reduces unproductive exon inclusion and increases OPA1 expression in fibroblasts from ADOA patients. The upper panel of Figure 5A shows source and identification information for the wild-type OPA1+ / + fibroblasts tested (WT(ATCC), GM02036, and GM03234), and the lower panel of Figure 5A contains OPA1 variant information for three ADOA patients designated as F34, F35, and F36. Figures 5B–5C show histograms (Figure 5B) and OPA1 mRNA levels (Figure 5C) representing the WT(ATCC) data and all three patient fibroblasts treated with ASO-1 and collected for analysis of unproductive splicing. Figure 5D shows a histogram of OPA1 protein levels, determined by Western blotting after transfection and normalized to β-actin. [Figure 5-2]Figures 5E–5F are histograms representing data from three different types of wild-type cells (WT(ATCC), GM02036, and GM03234) from the upper panel of Figure 5A, and three patient fibroblasts with different OPA1 pathogenic mutants from the lower panel of Figure 5A. These were collected for analysis of OPA1 mRNA levels (Figure 5E) and relative OPA1 protein levels (Figure 5F) before ASO-1 treatment. [Figure 5-3] Figure 5G is an exemplary Western blot showing OPA1 protein levels in three different wild-type fibroblasts with different OPA1 pathogenicity variants and dermal fibroblasts from three ADOA patients before treatment with ASO-1, where cells were probed with antibodies targeting either OPA1 or β-actin. The multiple bands at the top of the gel image in Figure 5G correspond to different isoforms of OPA1, and the bands at the bottom correspond to β-actin. Figure 5H shows histograms of relative unproductive OPA1 transcript levels when dermal fibroblasts from three wild-type cells (WT(ATCC), GM02036, and GM03234), as well as dermal fibroblasts from three ADOA patients with different OPA1 pathogenicity variants (F34, F35, and F36), were treated with either DMSO or CHX. [Figure 5-4] Figure 5I is an exemplary Western blot gel image showing OPA1 protein levels when fibroblasts from wild-type and ADOA patients were treated with either the vehicle (without ASO) or ASO-1, and the cells were probed with antibodies targeting either OPA1 or β-actin. Each gel image in Figure 5I represents a separate experiment, and each lane on each gel represents an individual biological replica. [Figure 6-1] Figures 6A–6D are exemplary histograms of oxygen consumption rates in fibroblasts derived from ADOA patients, illustrating the presence of reduced mitochondrial function. Basal respiration (Figure 6A), ATP-bound respiration (Figure 6B), maximal respiration (Figure 6C), and respiratory reserve (Figure 6D) are shown. [Figure 6-2]Figures 6E–6H are exemplary histograms from additional experiments evaluating oxygen consumption rates in fibroblasts derived from ADOA patients, demonstrating reduced mitochondrial function compared to three wild-type cells (WT(ATCC), GM02036, and GM03234). They show basal respiration (Figure 6E), ATP-bound respiration (Figure 6F), maximal respiration (Figure 6G), and respiratory reserve capacity (Figure 6H). [Figure 7-1] Figures 7A–7D are histograms showing that ASO-1 increases mitochondrial function in fibroblasts from all three ADOA patients. Oxygen consumption rates were measured from fibroblasts of patients treated with 20, 40, or 60 nM of ASO-1 or vehicle. Basal respiration (Figure 7A), ATP-bound respiration (Figure 7B), maximal respiration (Figure 7C), and respiratory reserve capacity (Figure 7D) are shown. [Figure 7-2] Figures 7E–7H are histograms showing the effect of untargeted ASO controls on mitochondrial function in F35 fibroblasts from ADOA patients. Oxygen consumption rates were measured from fibroblasts of patients treated with 20, 40, or 60 nM ASO-1 or vehicle (without ASO). Basal respiration (Figure 7E), ATP-bound respiration (Figure 7F), maximal respiration (Figure 7G), and respiratory reserve capacity (Figure 7H) are shown. [Figure 8-1] Figures 8A–8D show that the surrogate antisense oligonucleotide (ASO) ST-1102 increases OPA1 protein in wild-type (WT) rabbit retinal tissue after a single intravitreal (IVT) injection. Figure 8A shows the experimental design of the in vivo rabbit experiment in Example 9. [Figure 8-2] Figure 8B is a histogram representing RT-PCR data measuring nonproductive splicing using rabbit retinal RNA. Figure 8C shows a histogram of quantitative Western blot data for OPA1 protein normalized to actin. Figure 8D shows a histogram of ST-1102 detected in retinal tissue at day 29 by HELISA at all three dose levels. [Figure 9] Figure 9 shows RT-PCR gel images from the retina of rabbits injected with ST-1102. [Figure 10] Figure 10 shows a Western blot image from the retina of a rabbit injected with ST-1102. [Figure 11-1] Figures 11A–11C show histograms demonstrating that intravitreous administration of ASO-1 induces a dose-related decrease in nonsense mutation-dependent degradation exon inclusion and an increase in OPA1 protein in the retina of cynomolgus monkeys with sustained tissue exposure. Figure 11A is a histogram of qPCR data measuring the presence of unproductive exon-containing RNA transcripts in retinal tissue relative to total OPA1 transcript. Figure 11B is a histogram representing OPA1 protein quantified in retinal lysates by ELISA and normalized to total protein in the sample. Figure 11C is a histogram representing ASO-1 levels in retinal tissue quantified by HELISA. [Figure 11-2] Figure 11D is a gel image showing RT-PCR data from the right eye (oculus dexter, OD) and left eye (oculus sinister, OS) of three cynomolgus macaques. The upper band represents non-productive transcripts, and the lower band represents productive transcripts. The numbers at the bottom of the gel indicate the amount of AXB relative to the total (AX-B+AB). [Figure 12-1] Figures 12A–12D are images representing ASO-1 and OPA1 proteins in retinal ganglion cells (RGCs) after intravitreous (IVT) administration to cynomolgus monkeys. Figure 12A shows miRNAscope® staining with a specific probe for ASO-1 performed on MDF-fixed paraffin-embedded cynomolgus monkey eyes to demonstrate the cellular location of ASO-1 (red) in RGCs. The increase in ASO-1 signal correlates with increasing dose. Nuclei were counterstained with hematoxylin (blue). Figure 12B shows immunofluorescence staining with OPA1 antibody, showing a clear dose-related increase in protein (red) in intrafoveal / proximal RGCs. Nuclei were stained with DAPI (blue) at 40X magnification. [Figure 12-2]Figure 12C shows immunohistochemical (IHC) stained images of RGCs after intravitreous administration of PBS to cynomolgus monkeys. Figure 12D shows in-situ hybridization (ISH) of RGCs after intravitreous administration of ASO-1 to cynomolgus monkeys. [Figure 12-3] Figure 12E is a schematic diagram showing the experimental procedure involving in vitro administration of either PBS or ASO-1 to cynomolgus monkeys and evaluation after 4 weeks. The right eye of the cynomolgus monkeys was fixed for in-situ hybridization (ISH) and immunohistochemistry (IHC). [Figure 13] Figures 13A–13D show a dose-related increase in ASO-1 in retinal ganglion cells (RGCs) after intravitreous (IVT) administration to cynomolgus monkeys. Figures 13A–13B are histograms representing the quantified copies of mRNA per cell and the percentage of positive RGCs in or near the foveal RGC. Figures 13B–13C are histograms representing the quantified copies of mRNA per cell and the percentage of positive RGCs in the peripheral retina. [Figure 14] Figures 14A-14B show the quantification of OPA1 protein immunofluorescence in cynomolgus monkey retinal ganglion cells (RGCs) after intravitreous ASO-1 administration. Fluorescence (FL) intensity was quantified in the RGC layer (Figure 14A) and peripheral retina (Figure 14B) within / near the fovea. [Figure 15-1] Figure 15A is a schematic diagram showing the experimental procedure involving in vitro administration of 50 μl aliquots of either PBS or ASO-1 to cynomolgus monkeys, followed by 8 weeks of tissue collection and retinal detachment. Figure 15B shows the treatment conditions for each group in the cynomolgus monkey study. Figure 15C is a schematic diagram of retinal detachment showing a quadrant map for downstream evaluation of OPA1 protein expression (Q1), degree of ASO-1 exposure (Q2 and Q4), and OPA1 mRNA expression (Q3). [Figure 15-2]Figure 15D is a histogram of retinal ASO-1 exposure in the five dose groups of Figure 15B, evaluated by the measured concentration of ASO-1 (units / g) derived from hybridization ELISA (hELISA). Figure 15E is a histogram of retinal NMD exon inclusion in the five dose groups of Figure 15B, evaluated by OPA1 NMD exon inclusion compared to a control from qPCR data. [Figure 15-3] Figure 15F is a histogram of retinal OPA1 protein levels in the five dose groups shown in Figure 15B, evaluated by OPA1 measured in units / mg of total protein, compared to a control derived from ELISA. [Modes for carrying out the invention]

[0169] Alternative splicing events in the OPA1 gene can produce unproductive mRNA transcripts, which can subsequently lead to abnormal protein expression. Therapeutic agents that can target alternative splicing events in the OPA1 gene can regulate functional protein expression levels and / or inhibit abnormal protein expression in DS patients. Such therapeutic agents can be used to treat conditions caused by OPA1 protein deficiency.

[0170] One alternative splicing event that can induce unproductive mRNA transcripts is the inclusion of excess exons in the mRNA transcript, which can trigger nonsense-mediated mRNA degradation. This disclosure provides compositions and methods for modulating alternative splicing of OPA1 to increase the production of mature mRNA encoding the protein, and consequently, the production of functional OPA1 protein that is translated. These compositions and methods include antisense oligomers (ASOs) that can induce exon skipping, e.g., pseudoexon skipping, and promote structural splicing of OPA1 premRNA. In various embodiments, functional OPA1 protein can be increased using the methods of this disclosure to treat conditions caused by OPA1 protein deficiency.

[0171] mRNA splicing Intervening sequences or introns in RNA sequences are removed by a large, highly dynamic RNA-protein complex called a spliceosome, which regulates complex interactions between the primary transcript, nuclear small RNA (snRNA), and numerous proteins. The spliceosome assembles each intron in an orderly, ad-hoc manner, starting with the recognition of the 5' splice site (5'ss) by U1 snRNA, or the recognition of the 3' splice site (3'ss) by the U2 pathway, which involves binding the U2 cofactor (U2AF) to the 3'ss region to facilitate U2 binding to the branching point sequence (BPS). U2AF is a stable heterodimer consisting of a 65kD subunit (U2AF65) encoding U2AF-2, which binds to polypyrimidine tract (PPT), and a 35kD subunit (U2AF35) encoding U2AF-1, which stabilizes U2AF65 binding by interacting with the highly conserved AG dinucleotide via 3'ss. In addition to the BPS / PPT unit and 3'ss / 5'ss, accurate splicing requires auxiliary sequences or structures known as intron or exon splicing enhancers or splicing silencers that activate or suppress splice site recognition. These elements enable the recognition of true splice sites from among the numerous and excessive hidden or false splice sites in higher-order eukaryotic genomes, which have the same sequence as standard sites but are orders of magnitude more numerous. While these elements often have regulatory functions, the exact mechanisms of their activation or suppression are not well understood.

[0172] The decision of whether or not to splice is often designed as a probabilistic rather than a deterministic process, and therefore even the most obvious splicing signals can lead to false splicing. However, under normal conditions, premRNA splicing proceeds remarkably faithfully. This is partly due to the activity of adjacent cis-acting auxiliary exon-splicing and intron-splicing regulators (ESRs or ISRs). These functional elements are usually classified as either exon-splicing enhancers or intron-splicing enhancers (ESEs or ISEs) or exon-splicing silencers or intron-splicing silencers (ESSs or ISSs), based on their ability to stimulate or inhibit splicing, respectively. While there is evidence that some auxiliary cis-acting elements may act by influencing the dynamics of spliceosome assembly, such as the arrangement of the complex between U1 snRNP and 5'ss, many elements appear to function very likely in cooperation with trans-acting RNA-binding proteins (RBPs). For example, the serine and arginine-rich family of RBPs (SR proteins) is a conserved family of proteins that plays a crucial role in exon determination. SR proteins facilitate exon recognition by recruiting prespliceosome components to adjacent splice sites or by weakening the effect of ESS in the vicinity. The suppressive effect of ESS can be mediated by members of the heterologous intranuclear ribonucleoprotein (hnRNP) family, which can alter the recruitment of core splicing factors to adjacent splice sites. In addition to their roles in splicing regulation, silencer elements have been suggested to play a role in the suppression of pseudoexons, which are collections of decoy intron splice sites that have typical exon spacing but lack a functional open reading frame.ESEs and ESSs, in conjunction with similar trans-acting RBPs, are key components in a series of splicing controls that determine how, where, and when mRNA is assembled from its precursor.

[0173] Alternative splicing is a controlled process during gene expression that can result in multiple isoforms of mature mRNA transcripts being processed from a single primary mRNA transcript transcribed from a single gene, and multiple proteins resulting from translation from at least some of these mature mRNA isoforms. In this process, specific exons of a gene may or may not be included in the final processed mRNA produced from that gene. As a result, proteins translated from alternatively spliced ​​mRNA will have differences in their amino acid sequences and, in some cases, differences in their biological functions.

[0174] As described herein, “alternatively spliced ​​exons” may refer to exons of genes that may be naturally included in or excluded from a mature mRNA transcript, thus resulting in different protein products translated from different mature mRNA transcripts. The inclusion or skipping of alternatively spliced ​​exons can occur spontaneously in cells, either randomly or in a controlled manner, subject to, for example, external physiological or pathological stimuli or regulation by intracellular signaling. In some cases, the production of alternatively spliced ​​mRNA, e.g., the splicing of alternatively spliced ​​exons, is regulated by a system of trans-acting proteins that bind to cis-acting sites on the primary transcript itself. In some cases, alternatively spliced ​​exons are coding exons, e.g., exons that, if included in a mature mRNA transcript, are translated into an amino acid sequence as part of a protein product translated from the mature mRNA transcript. In some cases, including alternatively spliced ​​exons in a mature mRNA transcript maintains the standard open reading frame compared to a mature mRNA transcript without alternatively spliced ​​exons; for example, the number of nucleotides in the alternatively spliced ​​exon is divided into 3.

[0175] The sequences characterizing the exon-intron boundaries are degenerate signals of varying intensities that can occur frequently within human genes. In multi-exon genes, numerous and varied combinations of splice site pairs are joined to generate a diverse array of transcripts from a single gene. This is commonly referred to as alternative premRNA splicing. While the majority of mRNA isoforms produced by alternative splicing can be exported from the nucleus and translated into functional polypeptides, the translation efficiency of various mRNA isoforms from a single gene can vary significantly. mRNA isoforms with a premature termination codon (PTC) at least 50 bp upstream of the exon junction complex are likely to be targeted for degradation by the nonsense-mediated mRNA degradation (NMD) pathway. Mutations in conventional (BPS / PPT / 3'ss / 5'ss) and auxiliary splicing motifs can lead to abnormal splicing, such as exon skipping, inclusion of hidden exons (or pseudoexons), or activation of splice sites, which can have a significant impact on human morbidity and mortality. Both abnormal and alternative splicing patterns can be influenced by native DNA variants in exons and introns.

[0176] Considering that exon-intron boundaries can occur at any of the three codon positions, it is clear that only a subset of alternative splicing events can maintain a standard open reading frame. For example, only exons divisible by 3 can be skipped without altering the reading frame or can be included in the mRNA. Splicing events that do not have a matching phase induce a frameshift. Unless reversed by a downstream event, a frameshift deterministically leads to one or more PTCs, which can then lead to the degradation of an NMD. The NMD is a translation-coupled mechanism that excludes mRNA containing PTCs. The NMD can function as a surveillance pathway and is present in all eukaryotes. By excluding mRNA transcripts containing immature stop codons, the NMD can reduce errors in gene expression. When these abnormal mRNAs are translated, the resulting protein may, in some cases, acquire harmful functions or exhibit dominant-negative activity. NMD targets not only transcripts containing PTCs, but also diverse mRNA isoforms expressed from numerous endogenous genes. This suggests that NMD is a master regulator that induces both fine and coarse adjustments at the RNA level in the steady state of cells.

[0177] NMD-inducing exons ("NIEs" or "NMD exons") are exons or pseudoexons located within introns that, when present in mature RNA transcripts, can activate the NMD pathway. In constitutive splicing events, introns containing NMD exons are typically removed during splicing; however, during alternative or abnormal splicing events, the intron or a portion thereof (e.g., an NMD exon) may be retained. Mature mRNA transcripts containing such NMD exons may become unproductive due to frameshifts that induce the NMD pathway. The presence of NMD exons in mature RNA transcripts may result in downregulation of gene expression. mRNA transcripts containing NMD exons may be referred to in this disclosure as "NIE-containing mRNA" or "NMD exon mRNA."

[0178] Hidden (or false splice) sites have the same splicing recognition sequence as legitimate splice sites but are not used in the splicing reaction. Such false splice sites are orders of magnitude more numerous than true splice sites in the human genome and are usually suppressed by molecular mechanisms that are still largely unknown. Hidden 5' splice sites have a consensus sequence NNN / GUNNNN or NNN / GCNNNN, where N is any nucleotide and / is the exon-intron boundary. Hidden 3' splice sites have a consensus sequence NAG / N. Their activity is positively influenced by the surrounding nucleotides, which are made to be more similar to the optimal consensus sequences of standard splice sites, namely MAG / GURAGU and YAG / G, respectively, where M is C or A, R is G or A, and Y is C or U.

[0179] The splicing sites and their regulatory sequences can be readily identified by those skilled in the art using publicly available and suitable algorithms, for example, listed in Kralovicova, J. and Vorechovsky, I. (2007) Global control of aberrant splice site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition. Nucleic Acids Res., 35, 6399-6413 (http: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC2095810 / pdf / gkm680.pdf).

[0180] Hidden splice sites or splicing regulatory sequences may compete with splice sites on NMD exons for RNA-binding proteins, such as U2AF. In some embodiments, a drug may bind to a hidden splice site or splicing regulatory sequence, interfering with the binding of RNA-binding proteins and thereby favoring the binding of RNA-binding proteins to splice sites on NMD exons.

[0181] In some embodiments, the hidden splice site may not include the 5' or 3' splice site of the NMD exon. In some embodiments, the hidden splice site may be at least 10 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, or at least 200 nucleotides upstream of the NMD exon 5' splice site. In some embodiments, the hidden splice site may be at least 10 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, or at least 200 nucleotides downstream of the NMD exon 3' splice site.

[0182] Target transcript In some embodiments, the methods and compositions of this disclosure utilize the presence of NMD exons in premRNA transcribed from the OPA1 gene. Splicing the identified OPA1 NMD exon premRNA species to produce functionally mature OPA1 mRNA may be induced using agents that stimulate exon skipping of NMD exons, such as ASO. Induction of exon skipping can inhibit the NMD pathway. The resulting mature OPA1 mRNA can be translated normally without activating the NMD pathway, thereby increasing the amount of OPA1 protein in the patient's cells and symptoms of conditions or diseases associated with OPA1 deficiency, such as eye diseases or conditions, type 1 optic atrophy, autosomal dominant optic atrophy (ADOA), ADOA-plus syndrome, mitochondrial dysfunction, glaucoma, normal-tension glaucoma, Charcot-Marie-Tooth disease, mitochondrial dysfunction, diabetic retinopathy, age-related macular degeneration, retinal ganglion cell death, mitochondrial fission-mediated mitochondrial dysfunction, progressive extraocular muscle palsy, hearing loss, ataxia, motor neuropathy, sensory neuropathy, myopathy, Beer syndrome, brain dysfunction, encephalopathy, peripheral neuropathy, fatal infantile mitochondrial encephalomyopathy, hypertrophic cardiomyopathy, and spastic movement. It alleviates ataxia syndrome, sensorimotor peripheral neuropathy, hypotonia, gastrointestinal motility and dysphagia, optic nerve atrophy, optic nerve atrophy plus syndrome, mitochondrial DNA depletion syndrome14, late-onset cardiomyopathy, diabetic cardiomyopathy, Alzheimer's disease, focal segmental glomerulosclerosis, renal disease, Huntington's disease, cognitive decline in healthy aging, prion disease, late-onset dementia and parkinsonism, mitochondrial myopathy, Leigh syndrome, Friedreich's ataxia, Parkinson's disease, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), pyruvate dehydrogenase complex deficiency, chronic kidney disease, Leber's hereditary optic neuropathy, obesity, age-related systemic neurodegeneration, skeletal muscle atrophy, cardiac and cerebral ischemic injury, or massive hepatocyte apoptosis.

[0183] In some embodiments, the methods and compositions of this disclosure utilize alternative splicing of premRNA transcribed from the OPA1 gene. In some cases, splicing of a coding exon, e.g., an alternatively spliced ​​exon, e.g., OPA1 exon 7 (or the exon encoded by the genomic region extending GRCh38 / hg38:chr3 193626092~193626202), can regulate the level of OPA1 protein expressed from the OPA1 gene. As described herein, the term “OPA1 exon 7” or its grammatical equivalent is used interchangeably with the term “exon (GRCh38 / hg38:chr3 193626092~193626202)” or the exon encoded by the genomic region extending “GRCh38 / hg38:chr3 193626092~193626202”. While we do not wish to be bound by any particular theory, the presence or absence of the amino acid sequence encoded by exon 7 or exon (GRCh38 / hg38:chr3 193626092~193626202) can modulate the stability of the OPA1 protein. For example, in some cases, the OPA1 protein encoded by a mature mRNA transcript lacking exon 7 may have fewer proteolytic cleavage sites compared to the OPA1 protein encoded by the corresponding mature mRNA transcript containing exon 7. In some cases, the OPA1 protein encoded by a mature mRNA transcript lacking exon 7 is a functional protein compared to the OPA1 protein encoded by the corresponding mature mRNA transcript containing exon 7. The OPA1 protein encoded by a mature mRNA transcript lacking exon 7 may be at least partially functional compared to the OPA1 protein encoded by the corresponding mature mRNA transcript containing exon 7. In some cases, the OPA1 protein encoded by a mature mRNA transcript lacking exon 7 is at least partially functional compared to the full-length wild-type OPA1 protein.In some cases, an increase in OPA1 protein encoded by mature mRNA transcripts lacking exon 7 in cells may result in a more functional OPA1 protein in cells, due to the higher stability and at least partially functional equivalence of the exon 7-lacking OPA1 protein.

[0184] In other embodiments, coding exons of OPA1 premRNA other than exon 7 are targeted by agents disclosed herein that promote the elimination of coding exons other than exon 7. In these other embodiments, agents that promote the elimination of coding exons other than exon 7 increase OPA1 protein expression encoded by mature mRNA transcripts lacking the eliminated exon.

[0185] Alternative splicing of the OPA1 premRNA species, such as skipping of coding exons, or alternatively spliced ​​exons, such as exon 7, can be induced using drugs that stimulate exon skipping, such as ASO, to produce a functionally mature OPA1 protein. Induction of exon skipping can result in regulation of the levels of differently alternatively spliced ​​mRNA transcripts. The resulting mature OPA1 mRNA can be successfully translated into different OPA1 proteins, thereby increasing the amount of OPA1 protein in the patient's cells and leading to symptoms of conditions or diseases associated with OPA1 deficiency, such as eye diseases or conditions, type 1 optic atrophy, autosomal dominant optic atrophy (ADOA), ADOA-plus syndrome, mitochondrial dysfunction, glaucoma, normal-tension glaucoma, Charcot-Marie-Tooth disease, mitochondrial dysfunction, diabetic retinopathy, age-related macular degeneration, retinal ganglion cell death, mitochondrial fission-mediated mitochondrial dysfunction, progressive extraocular muscle palsy, hearing loss, ataxia, motor neuropathy, sensory neuropathy, myopathy, Beale syndrome, brain dysfunction, encephalopathy, peripheral neuropathy, fatal infantile mitochondrial encephalomyopathy, hypertrophic cardiomyopathy, and spastic ataxia. It alleviates syndromes, sensorimotor peripheral neuropathy, hypotonia, gastrointestinal motility and dysphagia, optic nerve atrophy, optic nerve atrophy plus syndrome, mitochondrial DNA depletion syndrome14, late-onset cardiomyopathy, diabetic cardiomyopathy, Alzheimer's disease, focal segmental glomerulosclerosis, renal disease, Huntington's disease, cognitive decline in healthy aging, prion disease, late-onset dementia and parkinsonism, mitochondrial myopathy, Leigh syndrome, Friedreich's ataxia, Parkinson's disease, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), pyruvate dehydrogenase complex deficiency, chronic kidney disease, Leber's hereditary optic neuropathy, obesity, age-related systemic neurodegeneration, skeletal muscle atrophy, cardiac and cerebral ischemic injury, or massive hepatocyte apoptosis.

[0186] In some embodiments, the diseases or conditions that can be treated or improved using the methods or compositions disclosed herein are not directly related to the target proteins (genes) targeted by the therapeutic agents. In some embodiments, the therapeutic agents provided herein may target proteins (genes) that are not directly related to the disease or condition, but the regulation of the expression of the target proteins (genes) can treat or improve the disease or condition.

[0187] In some embodiments, the antisense oligomer modulates the splicing of nonsense-mediated RNA attenuation-inducing exons (NMD exons) from premRNA in the target cell, the premRNA encoding the OPA1 protein and containing NMD exons, thereby modulating the processing level of the mRNA treated from the premRNA and regulating OPA1 protein expression in the cell.

[0188] In some embodiments, the antisense oligomer either (a) binds to a target region of premRNA, (b) modulates the binding of factors involved in NMD exon splicing, or (c) a combination of (a) and (b).

[0189] In some embodiments, the target region of the premRNA is located proximal to the NMD exon.

[0190] In various embodiments, this disclosure provides therapeutic agents that can target OPA1 mRNA transcripts to modulate splicing or protein expression levels. The therapeutic agents may be small molecules, polynucleotides, or polypeptides. In some embodiments, the therapeutic agent is an ASO. Various regions or sequences on OPA1 premRNA may be targeted by therapeutic agents such as ASOs. In some embodiments, the ASO targets an OPA1 premRNA transcript containing an NMD exon. In some embodiments, the ASO targets a sequence within the NMD exon of the OPA1 premRNA transcript. In some embodiments, the ASO targets a sequence upstream (or 5' side) of the 5' end of the NMD exon (3'ss) of the OPA1 premRNA transcript. In some embodiments, the ASO targets a sequence downstream (or 3' side) of the 3' end of the NMD exon (5'ss) of the OPA1 premRNA transcript. In some embodiments, the ASO targets a sequence within the intron adjacent to the 5' end of the NMD exon of the OPA1 premRNA transcript. In some embodiments, the ASO targets a sequence within an intron adjacent to the 3' end of an NMD exon in the OPA1 premRNA transcript. In some embodiments, the ASO targets a sequence containing the NMD exon-intron boundary in the OPA1 premRNA transcript. The NMD exon-intron boundary can refer to the junction between an intron sequence and an NMD exon region. The intron sequence can be adjacent to the 5' end or the 3' end of an NMD exon. In some embodiments, the ASO targets a sequence within an exon in the OPA1 premRNA transcript. In some embodiments, the ASO targets a sequence within an intron in the OPA1 premRNA transcript. In some embodiments, the ASO targets a sequence containing both a portion of an intron and a portion of an exon in the OPA1 premRNA transcript.

[0191] In some embodiments, the ASO targets a sequence approximately 4 to 300 nucleotides upstream (or 5' side) from the 5' end of the NMD exon. In some embodiments, the ASO targets a sequence approximately 1 to 20 nucleotides, 20 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, or 250 to 300 nucleotides upstream (or 5' side) from the 5' end of the NMD exon region. In some embodiments, the ASO may target a sequence more than 300 nucleotides upstream from the 5' end of the NMD exon. In some embodiments, the ASO targets a sequence approximately 4 to 300 nucleotides downstream (or 3' side) from the 3' end of the NMD exon. In some embodiments, the ASO targets sequences approximately 1 to 20 nucleotides, 20 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, or 250 to 300 nucleotides downstream from the 3' end of the NMD exon. In some embodiments, the ASO targets sequences more than 300 nucleotides downstream from the 3' end of the NMD exon.

[0192] In some embodiments, the OPA1 NMD exon-containing premRNA transcript is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1. In some embodiments, the OPA1 NMD exon premRNA transcript contains a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 2-5.

[0193] In some embodiments, the OPA1 NMD exon-containing premRNA transcript (or NMD exon mRNA) contains a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs. In some embodiments, the OPA1 NMD exon-containing premRNA transcript (or NMD exon mRNA) is encoded by a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs. In some embodiments, the target portion of the NMD exon mRNA contains a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region containing at least eight consecutive nucleic acids of any one of SEQ ID NOs.

[0194] In some embodiments, the ASO targets exon 6x of OPA1 NMD exon-containing premRNA containing NIE exon 6, exon 7x of OPA1 NMD exon-containing premRNA containing NIE exon 7, or exon 28x of OPA1 NMD exon-containing premRNA containing NIE exon 28. In some embodiments, the ASO targets an exon of OPA1 premRNA (GRCh38 / hg38:chr3 193628509 193628616) or an exon of OPA1 (GRCh38 / hg38:chr3 193603500 193603557). In some embodiments, the ASO targets an NMD exon of OPA1 premRNA other than an NMD exon (GRCh38 / hg38:chr3 193628509 193628616).

[0195] In some embodiments, the ASO targets sequences approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides upstream (or 5') from the 5' end of exon 6x, exon 7x, or exon 28x of OPA1. In some embodiments, the ASO targets the upstream (or 5') sequences of approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides from GRCh38 / hg38:chr3 193603500 of OPA1.

[0196] In some embodiments, the ASO targets a sequence up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, or approximately 50 nucleotides upstream (or 5') of the 5' end of exon 6x, exon 7x, or exon 28x of OPA1. In some embodiments, the ASO targets the upstream (or 5') sequence of up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides from GRCh38 / hg38:chr3 193603500 of OPA1.

[0197] In some embodiments, the ASO targets sequences approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides downstream (or 3') of the 3' end of exon 6x, exon 7x, or exon 28x of OPA1. In some embodiments, the ASO targets the downstream (or 3') sequences of approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides from GRCh38 / hg38:chr3 193603557 of OPA1.

[0198] In some embodiments, the ASO targets a sequence up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, or approximately 50 nucleotides downstream (or 3') of the 3' end of exon 6x, exon 7x, or exon 28x of OPA1. In some embodiments, the ASO targets the downstream (or 3') sequence of up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides from GRCh38 / hg38:chr3 193603557 of OPA1.

[0199] In some embodiments, the ASO has a sequence complementary to the target region of the NMD exon mRNA by one of sequence numbers 2-5 or 279.

[0200] In some embodiments, the ASO targets a sequence upstream of the 5' end of an NMD exon. For example, an ASO targeting a sequence upstream of the 5' end of an NMD exon (exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1) includes a sequence that is at least approximately 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least eight consecutive nucleic acids of SEQ ID NO: 2 or 3. For example, an ASO targeting a sequence upstream of the 5' end of an NMD exon (e.g., an exon of OPA1 (GRCh38 / hg38:chr3 193628509~193628616) or an exon of OPA1 (GRCh38 / hg38:chr3 193603500 193603557)) may contain a sequence that has at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with sequence number 4 or 5.

[0201] In some embodiments, the ASO targets sequences containing exon-intron boundaries (or junctions). For example, an ASO targeting sequences containing exon-intron boundaries may include sequences that are at least approximately 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least eight consecutive nucleic acids of any one of sequence numbers 2-5. In some embodiments, the ASO targets sequences downstream from the 3' end of an NMD exon. For example, an ASO targeting sequences downstream from the 3' end of an NMD exon (e.g., exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1) may include sequences with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to sequence number 2 or 3, or sequences having at least eight consecutive nucleic acids of sequence number 2 or 3. For example, an ASO targeting the OPA1 of an NMD exon (e.g., exon (GRCh38 / hg38:chr3 193628509~193628616) or the sequence downstream from the 3' end of an OPA1 exon (GRCh38 / hg38:chr3 193603500~193603557) may include a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 4 or 5, or a sequence having at least eight consecutive nucleic acids of SEQ ID NO: 4 or 5. In some embodiments, the ASO targets a sequence within an NMD exon.

[0202] In some embodiments, the ASO targets exon 6x of OPA1 NMD exon-containing premRNA containing NIE exon 6, exon 7x of OPA1 NMD exon-containing premRNA containing NIE exon 7, or exon 28x of OPA1 NMD exon-containing premRNA containing NIE exon 28. In some embodiments, the ASO targets the sequence downstream (or 3') from the 5' end of exon 6x, exon 7x, or exon 28x of OPA1 premRNA. In some embodiments, the ASO targets the sequence upstream (or 5') from the 3' end of exon 6x, exon 7x, or exon 28x of OPA1 premRNA.

[0203] In some embodiments, the target region of the OPA1 NMD exon-containing premRNA is located within introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, hybridization of the ASO NMD exon premRNA to the target region results in exon skipping of at least one NMD exon within introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, which then increases the production of the OPA1 protein. In some embodiments, the target region of the OPA1 NMD exon-containing premRNA is located within intron 6 of OPA1, or intron 28 of OPA1. In some embodiments, the target region of the OPA1 NMD exon-containing premRNA is either an OPA1 intron (GRCh38 / hg38:chr3 193626203~193631611) or an OPA1 intron (GRCh38 / hg38:chr3 193593374~193614710).

[0204] In some embodiments, the methods and compositions of this disclosure are used to increase OPA1 expression by inducing exon skipping of pseudoexons in OPA1 NMD exon-containing premRNA. In some embodiments, the pseudoexon is a sequence located within any of introns 1-50. In some embodiments, the pseudoexon is a sequence located within any of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the pseudoexon may be an OPA1 intron or a part thereof. In some embodiments, the pseudoexon is located within intron 6 of OPA1 or intron 28 of OPA1. In some embodiments, the pseudoexon is located within intron (GRCh38 / hg38:chr3 193626203~193631611) of OPA1 or intron (GRCh38 / hg38:chr3 193593374~193614710) of OPA1.

[0205] In some embodiments, the ASO targets the OPA1 premRNA transcript to induce exon skipping of coding exons, such as alternatively spliced ​​exons. In some embodiments, the ASO targets sequences within coding exons of the OPA1 premRNA transcript, such as alternatively spliced ​​exons. In some embodiments, the ASO targets sequences upstream (or 5') of the 5' end of the coding exon (3'ss) of the OPA1 premRNA transcript. In some embodiments, the ASO targets sequences downstream (or 3') of the 3' end of the coding exon (5'ss) of the OPA1 premRNA transcript. In some embodiments, the ASO targets sequences within the intron adjacent to the 5' end of the coding exon of the OPA1 premRNA transcript. In some embodiments, the ASO targets sequences within the intron adjacent to the 3' end of the coding exon of the OPA1 premRNA transcript. In some embodiments, the ASO targets sequences containing exon-intron boundaries in the OPA1 premRNA transcript. An exon-intron boundary can refer to the junction of an intron sequence and an exon sequence. The intron sequence may be adjacent to the 5' end or the 3' end of a coding exon. In some embodiments, the ASO targets sequences within exons of the OPA1 premRNA transcript. In some embodiments, the ASO targets sequences within introns of the OPA1 premRNA transcript. In some embodiments, the ASO targets sequences containing both portions of introns and exons of the OPA1 premRNA transcript.

[0206] In some embodiments, the ASO targets a sequence approximately 4 to 300 nucleotides upstream (or 5' side) from the 5' end of a code exon (e.g., an alternatively spliced ​​exon). In some embodiments, the ASO targets a sequence approximately 1 to 20 nucleotides, 20 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, or 250 to 300 nucleotides upstream (or 5') from the 5' end of the code exon region. In some embodiments, the ASO may target a sequence more than 300 nucleotides upstream from the 5' end of the code exon. In some embodiments, the ASO targets a sequence approximately 4 to 300 nucleotides downstream (or 3' side) from the 3' end of the code exon. In some embodiments, the ASO targets sequences approximately 1 to 20 nucleotides, 20 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, or 250 to 300 nucleotides downstream from the 3' end of the code exon. In some embodiments, the ASO targets sequences more than 300 nucleotides downstream from the 3' end of the code exon.

[0207] In some embodiments, the OPA1 premRNA transcript is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1. In some embodiments, the OPA1 premRNA transcript contains a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 2-5.

[0208] In some embodiments, the OPA1 premRNA transcript (or NMD exon mRNA) contains a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs. In some embodiments, the OPA1 premRNA transcript (or NMD exon mRNA) is encoded by a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs. In some embodiments, the target portion of the OPA1 premRNA contains a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region containing at least eight consecutive nucleic acids of any one of SEQ ID NOs.

[0209] In some embodiments, the ASO targets exon 7 of the OPA1 premRNA, that is, the ASO targets the exon (GRCh38 / hg38:chr3 193626092~193626202) of the OPA1 premRNA.

[0210] In some embodiments, the ASO targets coding exons of OPA1 premRNA other than exon 7, i.e., the ASO targets exons of OPA1 premRNA other than the exon defined by (GRCh38 / hg38:chr3 193626092~193626202).

[0211] In some embodiments, the ASO targets sequences approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, approximately 50 nucleotides upstream (or 5') from the 5' end of exon 7 of OPA1. In some embodiments, the ASO targets sequences approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, approximately 50 nucleotides upstream (or 5') from GRCh38 / hg38:chr3 193626092 of OPA1.

[0212] In some embodiments, the ASO targets sequences up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, approximately 50 nucleotides upstream (or 5') from the 5' end of exon 7 of OPA1. In some embodiments, the ASO targets sequences up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, approximately 50 nucleotides upstream (or 5') from GRCh38 / hg38:193626092 of OPA1.

[0213] In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream (or 3') from the 3' end of exon 7 of OPA1. In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream (or 3') from GRCh38 / hg38:chr3 193626202 of OPA1.

[0214] In some embodiments, the ASO targets a sequence up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream (or 3') from the 3' end of exon 7 of OPA1. In some embodiments, the ASO targets a sequence up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream (or 3') from GRCh38 / hg38:chr3 193626202 of OPA1.

[0215] In some embodiments, the ASO has a sequence complementary to the target portion of the NMD exon mRNA according to any one of SEQ ID NOs: 2-5 or 277.

[0216] In some embodiments, the ASO targets a coding exon, for example, a sequence upstream of the 5' end of an alternatively spliced ​​exon. For example, an ASO targeting a sequence upstream of the 5' end of a coding exon (e.g., exon 7 of OPA1) includes a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least eight consecutive nucleic acids of SEQ ID NO: 2 or 3. For example, an ASO targeting a sequence upstream of the 5' end of a coding exon of OPA1 (e.g., exon (GRCh38 / hg38:193626092~193626202)) may include a sequence that has at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with SEQ ID NO: 4 or 5.

[0217] In some embodiments, the ASO targets sequences containing exon-intron boundaries (or junctions). For example, an ASO targeting sequences containing exon-intron boundaries may include sequences that are at least approximately 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least eight consecutive nucleic acids of any one of sequence numbers 2-5. In some embodiments, the ASO targets sequences downstream of the 3' end of a coding exon, such as an alternatively spliced ​​exon. For example, an ASO targeting sequences downstream of the 3' end of a coding exon (e.g., exon 7 of OPA1) may include sequences with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to sequence number 2 or 3, or sequences having at least eight consecutive nucleic acids of sequence number 2 or 3. For example, an ASO targeting a sequence downstream from the 3' end of a coding exon (e.g., exon 7 of OPA1) may include a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 4 or 5, or a sequence having at least eight consecutive nucleic acids of SEQ ID NO: 4 or 5. In some embodiments, the ASO targets a sequence within the coding exon, for example, an alternatively spliced ​​exon.

[0218] Protein expression In some embodiments, the methods described herein are used to increase the production of functional OPA1 protein or RNA. As used herein, the term “functional” refers to the amount of activity or function of OPA1 protein or RNA required to eliminate any one or more symptoms of a treated condition or disease, e.g., type 1 optic atrophy. In some embodiments, the methods are used to increase the production of partially functional OPA1 protein or RNA. As used herein, the term “partially functional” refers to any amount of activity or function of OPA1 protein or RNA that is less than the amount of activity or function required to eliminate or prevent any one or more symptoms of a disease or condition. In some embodiments, partially functional protein or RNA has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower activity compared to fully functional protein or RNA.

[0219] In some embodiments, the method is a method for increasing OPA1 protein expression by target cells having OPA1 premRNA, the target having a disease or condition (e.g., type 1 optic atrophy) resulting from insufficient activity of OPA1 protein, the insufficient amount of OPA1 protein being due to haploinsufficiency of OPA1 protein. In such embodiments, the target has a first allele encoding a functional OPA1 protein and a second allele that does not produce OPA1 protein. In another such embodiment, the target has a first allele encoding a functional OPA1 protein and a second allele encoding a non-functional OPA1 protein. In yet another such embodiment, the target has a first allele encoding a functional OPA1 protein and a second allele encoding a partially functional OPA1 protein. In any of these embodiments, an antisense oligomer binds to a target region of OPA1 premRNA transcribed from the second allele, thereby inducing exon skipping of pseudoexons from the premRNA, increasing the level of mature mRNA encoding the functional OPA1 protein, and increasing OPA1 protein expression in the target cells.

[0220] In some embodiments, the method is a method for increasing OPA1 protein expression by target cells having OPA1 premRNA, the target having a disease or condition resulting from insufficient activity of the OPA1 protein, and the insufficient amount of OPA1 protein is due to autosomal recessive inheritance of the OPA1 protein.

[0221] In some embodiments, the method is a method for increasing OPA1 protein expression by target cells having OPA1 premRNA, the target having a disease or condition (e.g., type 1 optic atrophy) resulting from insufficient activity of the OPA1 protein, and the insufficient amount of OPA1 protein is due to autosomal dominant inheritance of the OPA1 protein.

[0222] In relevant embodiments, the method involves using ASO to increase the expression of a protein or functional RNA. In some embodiments, ASO may be used to increase OPA1 protein expression in cells of a subject having OPA1 premRNA, the subject having a deficiency in the amount or function of OPA1 protein, for example, type 1 optic atrophy.

[0223] In some embodiments, a premRNA transcript encoding a protein that causes a disease or condition is targeted by a drug, e.g., an oligonucleotide, as described herein. In some cases, this is an NMD exon-containing premRNA transcript targeted by the drug, e.g., an oligonucleotide, as described herein. In some cases, the drug, e.g., an oligonucleotide, as described herein, is designed to target the coding exon of the premRNA. In some cases, the drug, e.g., an oligonucleotide, as described herein, may induce skipping of the NMD exon, the coding exon, or both. In some embodiments, an NMD exon-containing premRNA transcript encoding a protein that does not cause a disease is not targeted by the ASO. For example, a disease resulting from a mutation or deficiency of a first protein in a particular pathway may be mitigated by targeting a premRNA encoding a second protein, thereby increasing the production of the second protein. In some embodiments, the function of the second protein may compensate for the mutation or deficiency of the first protein (which causes the disease or condition).

[0224] In some embodiments, the subject is: (a) The first mutant allele, (i) OPA1 protein is produced at a lower level compared to production from the wild-type allele. (ii) A less functional OPA1 protein is produced compared to an equivalent wild-type protein, or (iii) The first mutant allele, which does not produce the OPA1 protein or functional RNA, (b) A second mutant allele, (i) OPA1 protein is produced at a lower level compared to production from the wild-type allele. (ii) A less functional OPA1 protein is produced compared to an equivalent wild-type protein, or (iii) Having a second mutant allele in which the OPA1 protein is not produced, and NMD exon-containing premRNA is transcribed from a first allele and / or a second allele. In these embodiments, ASO binds to a target region of the NMD exon-containing premRNA transcribed from the first or second allele, thereby inducing exon skipping of pseudoexons from the NMD exon-containing premRNA, increasing the level of mRNA encoding the OPA1 protein, and increasing the expression of the target protein or functional RNA in the target cell. In these embodiments, the target protein or functional RNA whose expression level is increased as a result of exon skipping of pseudoexons from the NMD exon-containing premRNA is either deficient in function compared to an equivalent wild-type protein (partially functional) or fully functional compared to an equivalent wild-type protein (fully functional).

[0225] In some embodiments, the subject is: (a) The first mutant allele, (i) OPA1 protein is produced at a lower level compared to production from the wild-type allele. (ii) A less functional OPA1 protein is produced compared to an equivalent wild-type protein, or (iii) The first mutant allele, which does not produce the OPA1 protein or functional RNA, (b) A second mutant allele, (i) OPA1 protein is produced at a lower level compared to production from the wild-type allele. (ii) A less functional OPA1 protein is produced compared to an equivalent wild-type protein, or (iii) Having a second mutant allele in which the OPA1 protein is not produced, OPA1 premRNA is transcribed from a first allele and / or a second allele. In these embodiments, ASO binds to a target region of the OPA1 premRNA transcribed from the first or second allele, thereby inducing exon skipping of the coding exon from the OPA1 premRNA and increasing the expression of the target OPA1 protein in the target cell. In these embodiments, the increased expression level of the target OPA1 protein resulting from exon skipping of the coding exon from the OPA1 premRNA is either a form with lower function compared to the equivalent full-length wild-type protein (partially functional) or a form with full function compared to the equivalent full-length wild-type protein (fully functional).

[0226] In some embodiments, the level of mRNA encoding the OPA1 protein is increased 1.1 to 10 times compared to the amount of mRNA encoding the OPA1 protein produced in control cells, such as cells not treated with the antisense oligomer or cells treated with an antisense oligomer that does not bind to the target region of the OPA1 premRNA.

[0227] In some embodiments, subjects treated using the method disclosed express a partially functional OPA1 protein from a single allele, in which case the partially functional OPA1 protein may result from a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion. In some embodiments, subjects treated using the method disclosed express a nonfunctional OPA1 protein from a single allele, in which case the nonfunctional OPA1 protein may result from a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion in a single allele. In some embodiments, subjects treated using the method disclosed have a complete gene deletion of OPA1 in a single allele.

[0228] Exon inclusion As used herein, "NMD exon-containing pre-mRNA" is a pre-mRNA transcript containing at least one pseudo-exon. Alternative splicing or aberrant splicing may result in the inclusion of at least one pseudo-exon in the mature mRNA transcript. The terms "mature mRNA" and "fully spliced mRNA" are used interchangeably herein to describe a fully processed mRNA. The inclusion of at least one pseudo-exon may result in a non-productive mRNA and may lead to NMD of the mature mRNA. NMD exon-containing mature mRNA may induce aberrant protein expression.

[0229] In some embodiments, the included pseudo-exon is the most abundant pseudo-exon in a population of NMD exon-containing pre-mRNAs transcribed from a gene encoding a target protein in a cell. In some embodiments, the included pseudo-exon is the most abundant pseudo-exon in a population of NMD exon-containing pre-mRNAs transcribed from a gene encoding a target protein in a cell, where the population of NMD exon-containing pre-mRNAs includes two or more included pseudo-exons. In some embodiments, an antisense oligomer targeting the most abundant pseudo-exon in a population of NMD exon-containing pre-mRNAs encoding a target protein induces exon skipping of one or more pseudo-exons in the population, including the pseudo-exon targeted or bound by the antisense oligomer. In some embodiments, the target region is within a pseudo-exon, and the pseudo-exon is the most abundant pseudo-exon in an NMD exon-containing pre-mRNA encoding the OPA1 protein.

[0230] The degree of exon inclusion can be expressed as a percentage of exon inclusion, for example, as the percentage of transcripts containing a given pseudoexon. Briefly, the exon inclusion rate may be calculated as the percentage of exon-inclusion RNA transcripts relative to the sum of the average amount of exon-inclusion RNA transcripts and the average amount of exon-exclusion RNA transcripts.

[0231] In some embodiments, the included pseudoexons are those identified as included pseudoexons based on a determination that they constitute at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% of the exon.In multiple implementations, the included pseudoexons are approximately 5% to 100%, 5% to 95%, 5% to 90%, 5% to 85%, 5% to 80%, 5% to 75%, 5% to 70%, 5% to 65%, 5% to 60%, 5% to 55%, 5% to 50%, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, 5% to 15%, 10% to 100%, 10% to 95%, and 10%. ~90%, 10%~85%, 10%~80%, 10%~75%, 10%~70%, 10%~65%, 10%~60%, 10%~55%, 10%~50%, 10%~45%, 10%~40%, 10%~35%, 10%~30%, 10%~25%, 10%~20%, 15%~100%, 15%~95%, 15%~90%, 15%~85%, 15%~80%, 15%~75%, 15%~ 70%, approximately 15% to 65%, approximately 15% to 60%, approximately 15% to 55%, approximately 15% to 50%, approximately 15% to 45%, approximately 15% to 40%, approximately 15% to 35%, approximately 15% to 30%, approximately 15% to 25%, approximately 20% to 100%, approximately 20% to 95%, approximately 20% to 90%, approximately 20% to 85%, approximately 20% to 80%, approximately 20% to 75%, approximately 20% to 70%, approximately 20% to 65%, approximately 20% to 60%, approximately 20% to 55%, approximately 20% to 50%, approximately 20% to 45% These are exons identified as pseudoexons that are present based on a determination of content of approximately 20% to 40%, 20% to 35%, 20% to 30%, 25% to 100%, 25% to 95%, 25% to 90%, 25% to 85%, 25% to 80%, 25% to 75%, 25% to 70%, 25% to 65%, 25% to 60%, 25% to 55%, 25% to 50%, 25% to 45%, 25% to 40%, or 25% to 35%.ENCODE data (described in Tilgner, et al., 2012, “Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs,” Genome Research 22(9):1616-25) can be used to assist in identifying exon inclusion.

[0232] In some embodiments, contacting cells with an ASO complementary to the target region of the OPA1 premRNA transcript results in an increase of at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% in the amount of OPA1 protein produced compared to the amount of protein produced by cells in the absence of the ASO / lack of treatment. In some embodiments, the total amount of OPA1 protein produced by cells in contact with the antisense oligomer is approximately 20% to 300%, 50% to 300%, 100% to 300%, 150% to 300%, 20% to 50%, 20% to 100%, 20% to 150%, 20% to 200%, 20% to 250%, 50% to 100%, and 50%. Increases by approximately 150%, 200%, 250%, 150%, 250%, 200%, 200%, 100%, 250%, 200%, 200%, 200%, 250%, 200%, 200%, 250%, 100%, 200%, 200%, 200%, 200%, 200%, 200%, 200%, 200%, 200%, 200%, 200%, 200%, 200%, 200%, 300%, 100%, 150%, 200%, 200%, 250%, or 300%. In some embodiments, the total amount of OPA1 protein produced by cells in contact with the antisense oligomer is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, and 2 to 5 times, compared to the amount of target protein produced by the control compound. The increase is approximately 2 to 6 times, 2 to 7 times, 2 to 8 times, 2 to 9 times, 3 to 6 times, 3 to 7 times, 3 to 8 times, 3 to 9 times, 4 to 7 times, 4 to 8 times, 4 to 9 times, at least 1.1 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 5 times, or at least 10 times. The control compound may be, for example, an oligonucleotide that is not complementary to the target region of premRNA.

[0233] In some embodiments, contact between the cell and an ASO complementary to the target region of the OPA1 premRNA transcript results in an increase in the amount of OPA1-coding mRNA, including mature mRNA encoding the target protein. In some embodiments, the amount of OPA1 protein-coding mRNA, or mature mRNA encoding the OPA1 protein, results in an increase of at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the amount of protein produced by the cell in the absence of the ASO / treatment. In some embodiments, the total amount of mRNA encoding the OPA1 protein, or the total amount of mature mRNA encoding the OPA1 protein, produced in cells that come into contact with the antisense oligomer is, for example, about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, and about 20% to about 150% compared to the amount of mature RNA produced in untreated cells, which are untreated cells or cells treated with a control compound. Increases by approximately 200%, 20% to 250%, 50% to 100%, 50% to 150%, 50% to 200%, 50% to 250%, 100% to 150%, 100% to 200%, 100% to 250%, 150% to 200%, 150% to 250%, 200% to 250%, at least approximately 10%, at least approximately 20%, at least approximately 50%, at least approximately 100%, at least approximately 150%, at least approximately 200%, at least approximately 250%, or at least approximately 300%.In some embodiments, the total amount of mRNA encoding the OPA1 protein produced in cells that come into contact with the antisense oligomer, or the total amount of mature mRNA encoding the OPA1 protein, is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, and 1.1 to 6 times, compared to the amount of mature RNA produced in untreated cells, such as untreated cells or cells treated with a control compound. The increase is approximately 1.1 to 7 times, approximately 1.1 to 8 times, approximately 1.1 to 9 times, approximately 2 to 5 times, approximately 2 to 6 times, approximately 2 to 7 times, approximately 2 to 8 times, approximately 2 to 9 times, approximately 3 to 6 times, approximately 3 to 7 times, approximately 3 to 8 times, approximately 3 to 9 times, approximately 4 to 7 times, approximately 4 to 8 times, approximately 4 to 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times. The control compound may be, for example, an oligonucleotide that is not complementary to the target portion of the OPA1 NMD exon-containing premRNA.

[0234] NMD exons may be of any length. In some embodiments, an NMD exon may contain the entire sequence of an intron, in which case it may be referred to as an intron retention. In some embodiments, an NMD exon may be part of an intron. In some embodiments, an NMD exon may be the 5' end portion of an intron containing a 5'ss sequence. In some embodiments, an NMD exon may be the 3' end portion of an intron containing a 3'ss sequence. In some embodiments, an NMD exon may not contain a 5'ss sequence and may be part of an intron. In some embodiments, an NMD exon may not contain a 3'ss sequence and may be part of an intron. In some embodiments, an NMD exon may not contain a 5'ss sequence or a 3'ss sequence and may be part of an intron. In some embodiments, the NMD exons may be 5 to 10 nucleotides long, 10 to 15 nucleotides long, 15 to 20 nucleotides long, 20 to 25 nucleotides long, 25 to 30 nucleotides long, 30 to 35 nucleotides long, 35 to 40 nucleotides long, 40 to 45 nucleotides long, 45 to 50 nucleotides long, 50 to 55 nucleotides long, 55 to 60 nucleotides long, 60 to 65 nucleotides long, 65 to 70 nucleotides long, 70 to 75 nucleotides long, 75 to 80 nucleotides long, 80 to 85 nucleotides long, 85 to 90 nucleotides long, 90 to 95 nucleotides long, or 95 to 100 nucleotides long. In some embodiments, the NMD exon may be at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, or at least 100 nucleotides in length.In some embodiments, the NMD exon may be 100-200 nucleotides long, 200-300 nucleotides long, 300-400 nucleotides long, 400-500 nucleotides long, 500-600 nucleotides long, 600-700 nucleotides long, 700-800 nucleotides long, 800-900 nucleotides long, or 900-1,000 nucleotides long. In some embodiments, the NMD exon may be longer than 1,000 nucleotides long.

[0235] The inclusion of pseudoexons causes a frameshift, introducing immature stop codons (PTCs) into mature mRNA transcripts, making the transcripts targets for NMDs. Mature mRNA transcripts containing NMD exons may be non-productive mRNA transcripts that do not result in protein expression. PTCs can be located at any position downstream of an NMD exon. In some embodiments, PTCs can be located in any exon downstream of an NMD exon. In some embodiments, PTCs can be located within an NMD exon. For example, including OPA1 exon 6x, OPA1 exon 7x, and OPA1 exon 28x in an mRNA transcript encoded by the OPA1 gene can induce PTCs in the mRNA transcript. For example, this involves including an OPA1 exon (GRCh38 / hg38:chr3 193628509 193628616) or an OPA1 exon (GRCh38 / hg38:chr3 193603500 193603557) in the mRNA transcript encoded by OPA1.

[0236] In some embodiments, methods for modulating OPA1 protein expression by promoting the inclusion of a coding exon are provided herein. The method may include contacting cells having OPA1 premRNA with a drug, the drug comprising (a) a target portion of premRNA in the intron region immediately upstream of the 5' end of the coding exon of premRNA, or (b) a target portion of premRNA in the intron region immediately downstream of the 3' end of the coding exon of premRNA, thereby increasing the level of treated mRNA containing the coding exon in the cell after being treated from premRNA. In some cases, the included coding exon is an alternatively spliced ​​exon. In some cases, the method promotes the inclusion of the coding exon in the treated mRNA during splicing of premRNA in the cell.

[0237] In some of these embodiments for code exon inclusion, the target portion of the premRNA is located within a region extending 100–50, 100–60, 100–70, 100–80, or 100–90 nucleotides upstream of the 5' end of the code exon. In some cases, the target portion of the premRNA is located within a region extending 40–100, 50–100, 60–100, 70–100, 80–100, or 90–100 nucleotides downstream of the 3' end of the code exon. In some cases, the code exon is exon 7 of OPA1. In some cases, the code exon contains a sequence having at least 80%, at least 90%, or 100% sequence identity with SEQ ID NO: 277. In some cases, the code exon contains SEQ ID NO: 277. The target region of premRNA may be located within a region spanning 100–50, 100–60, 100–70, 100–80, or 100–90 nucleotides upstream of genomic site GRCh38 / hg38:chr3 193626092. In some cases, the target region of premRNA is located within a region spanning 40–100, 50–100, 60–100, 70–100, 80–100, or 90–100 nucleotides downstream of genomic site GRCh38 / hg38:chr3 193626202.

[0238] In some cases, including coding exons within processed mRNA in cells that have come into contact with the drug is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, and 2 to Increase by 7 times, approximately 2 to 8 times, approximately 2 to 9 times, approximately 3 to 6 times, approximately 3 to 7 times, approximately 3 to 8 times, approximately 3 to 9 times, approximately 4 to 7 times, approximately 4 to 8 times, approximately 4 to 9 times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times.

[0239] Elimination of both NMD exons and code exons In some embodiments, this specification provides methods for modulating target protein expression by targeting premRNA and modulating the exclusion of both coding exons and nonsense-mediated RNA decay-inducing exons (NMD exons) from the premRNA. In some cases, the method involves contacting cells with a drug, the drug promoting the exclusion of both coding exons and NMD exons from the premRNA, thereby increasing the level of treated mRNA that has been treated from the premRNA and lacks both coding exons and NMD exons. In some cases, the drug binds to a target region of the premRNA or modulates the binding of factors involved in the splicing of coding exons, NMD exons, or both. In some cases, the drug prevents the binding of factors involved in the splicing of coding exons, NMD exons, or both to a region of the target region. In some cases, the NMD exons are located in an intron region adjacent to the coding exon. In some cases, the NMD exons are located in an intron region immediately upstream of the coding exon. In some cases, the NMD exons are located in an intron region immediately downstream of the coding exon. In some cases, code exons are alternatively spliced ​​exons.

[0240] In some cases, the target region of premRNA is located proximal to the coding exon. The target region of premRNA may be located within an intron region immediately upstream of the coding exon. The target region of premRNA may be located within an intron region immediately downstream of the coding exon. In some cases, the target region of premRNA may be located within the coding exon. In some cases, the target region of premRNA is located within a region extending 49–1, 39–1, 29–1, or 19–1 nucleotides upstream of the 5' end of the coding exon. In some cases, the target region of premRNA is located within a region extending 100 nucleotides upstream to 100 nucleotides downstream of the coding exon. In some cases, the target portion contains approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides of the code exon.

[0241] In some cases, the target region of premRNA is located proximal to the NMD exon. In some cases, the target region of premRNA is located in an intron region immediately upstream of the NMD exon. In some cases, the target region of premRNA is located in an intron region immediately downstream of the NMD exon. In some cases, the target region of premRNA is located within the NMD exon. In some cases, the target region of premRNA is located within a region extending from 100 nucleotides upstream to 100 nucleotides downstream of the NMD exon.

[0242] In some embodiments, the methods described herein are applicable to the regulation of OPA1 protein expression by modulating the exclusion of both exon 7 and NMD exon (e.g., exon 7x) of OPA1 premRNA containing both exon 7 and exon 7x. In some cases, the coding exon contains a sequence having at least 80%, at least 90%, or 100% sequence identity with SEQ ID NO: 277. In some cases, the coding exon contains SEQ ID NO: 277. In some cases, the target region of the premRNA is located immediately upstream of the coding exon GRCh38 / hg38:chr3 193626092~193626202. In some cases, the target region of the premRNA is located immediately downstream of the coding exon GRCh38 / hg38:chr3 193626092~193626202. In some cases, the premRNA target region is located within a region extending from 49 to 1, 39 to 1, 29 to 1, or 19 to 1 nucleotides upstream of GRCh38 / hg38:chr3 193626092. In some cases, the premRNA target region is located within a region extending from 100 nucleotides upstream of genomic site GRCh38 / hg38:chr3 193626092 to 100 nucleotides downstream of genomic site GRCh38 / hg38:chr3 193626202. In some cases, the premRNA target region is located within the coding exon GRCh38 / hg38:chr3 193626092 to 193626202. In some cases, the target region of the premRNA includes the exon-intron junction of the coding exon GRCh38 / hg38:chr3 193626092~193626202. In some cases, the NMD exon contains a sequence with at least 80%, at least 90%, or 100% sequence identity with SEQ ID NO: 279. In some cases, the NMD exon contains SEQ ID NO: 279. In some cases, the target region of the premRNA is located immediately upstream of the NMD exon GRCh38 / hg38:chr3 193628509~193628616. In some cases, the target region of the premRNA is located immediately downstream of the NMD exon GRCh38 / hg38:chr3 193628509~193628616.In some cases, the target region of premRNA is located within a region extending from 100 nucleotides upstream of genomic site GRCh38 / hg38:chr3 193628509 to 100 nucleotides downstream of genomic site GRCh38 / hg38:chr3 193628616.

[0243] In some cases, the target region of the premRNA is within the range of NMD exon GRCh38 / hg38:chr3 193628509~193628616. In some cases, the target region of the premRNA includes the exon-intron junction of NMD exon GRCh38 / hg38:chr3 193628509~193628616. In some cases, the target region includes approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides of the NMD exon.

[0244] In some cases, the elimination of coding exons into premRNA within cells that have come into contact with the drug is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, and 2 to 8 times, compared to cases without drug contact. Increase by approximately 7 times, 2 to 8 times, 2 to 9 times, 3 to 6 times, 3 to 7 times, 3 to 8 times, 3 to 9 times, 4 to 7 times, 4 to 8 times, 4 to 9 times, at least 1.1 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 5 times, or at least 10 times. In some cases, the elimination of NMD exons from premRNA within cells that have come into contact with the drug is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, and 2 to 8 times, compared to cases without drug contact. It increases by approximately 7 times, 2 to 8 times, 2 to 9 times, 3 to 6 times, 3 to 7 times, 3 to 8 times, 3 to 9 times, 4 to 7 times, 4 to 8 times, 4 to 9 times, at least 1.1 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 5 times, or at least 10 times. In some cases, the method results in an increase in the level of processed mRNA in the cell.The level of processed mRNA in cells in contact with the drug is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, 2 to 7 times, and 2 to 5 times, compared to cells without drug contact. It increases by approximately 8 times, 2 to 9 times, 3 to 6 times, 3 to 7 times, 3 to 8 times, 3 to 9 times, 4 to 7 times, 4 to 8 times, 4 to 9 times, at least 1.1 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 5 times, or at least 10 times.

[0245] In some cases, the method results in increased intracellular OPA1 protein expression. The level of OPA1 protein expressed from treated mRNA in cells in contact with the drug is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, and approximately It increased by 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0246] In some cases, the level of OPA1 protein expressed from treated mRNA in cells that came into contact with the drug increased by at least approximately 1.5 times compared to cells that did not come into contact with the active ingredient.

[0247] In some cases, OPA1 protein expressed from treated mRNA lacking exon 7 and exon 7x is a functional OPA1 protein. OPA1 protein expressed from treated mRNA lacking exon 7 and exon 7x may be at least partially functional compared to wild-type OPA1 protein. OPA1 protein expressed from treated mRNA lacking exon 7 and exon 7x may be at least partially functional compared to full-length wild-type OPA1 protein.

[0248] Therapeutic drugs Various embodiments of this disclosure provide compositions and methods comprising therapeutic agents for modulating the protein expression level of OPA1. In some embodiments, compositions and methods for modulating alternative splicing of OPA1 premRNA are provided herein. In some embodiments, compositions and methods for inducing exon skipping in the splicing of OPA1 premRNA, for example, compositions and methods for inducing pseudoexon skipping during the splicing of OPA1 premRNA are provided herein. In other embodiments, the therapeutic agent may be used to induce exon contentation and thereby reduce the protein expression level.

[0249] The therapeutic agents disclosed herein may be NIE inhibitors. The therapeutic agents may include polynucleic acid polymers.

[0250] According to one aspect of the present disclosure, a method for treating or preventing a condition or disease associated with functional OPA1 protein deficiency is provided herein, the method comprising administering an NIE inhibitor to a target to increase the level of functional OPA1 protein, wherein the agent binds to a region of a premRNA transcript to reduce the content of NMD exons in the mature transcript. For example, the present disclosure provides a method for treating or preventing a condition associated with functional OPA1 protein deficiency, the method comprising administering an NIE inhibitor to a target to increase the level of functional OPA1 protein, wherein the agent binds to a region of an intron containing an NMD exon of a premRNA transcript (e.g., exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1) or to an NMD exon activation regulatory sequence in the same intron. For example, this specification provides a method for treating or preventing a condition associated with functional OPA1 protein deficiency, the method comprising administering an NIE inhibitor to a target to increase the level of functional OPA1 protein, the agent binding to a region of an intron containing an NMD exon of a premRNA transcript (e.g., an exon of OPA1 (GRCh38 / hg38:chr3 193628509 193628616) or an exon of OPA1 (GRCh38 / hg38:chr3 193603500 193603557)) or to an NMD exon activation regulatory sequence of the same intron. In some embodiments, the method involves administering an NIE inhibitor to a target to increase the level of functional OPA1 protein, wherein the agent binds to a region of an intron containing an NMD exon of the premRNA transcript (e.g., an exon of OPA1 other than exon 7x defined by (GRCh38 / hg38:chr3 193628509 193628616) or an exon defined by (GRCh38 / hg38:chr3 193603500 193603557), or to an NMD exon activation regulatory sequence within the same intron.In some embodiments, the therapeutic agent promotes the elimination of NMD exons of OPA1 premRNA other than exon 7x defined by (GRCh38 / hg38:chr3 193628509 193628616) or exon defined by (GRCh38 / hg38:chr3 193603500 193603557). In some embodiments, the compositions disclosed herein include agents that promote the elimination of NMD exons of OPA1 premRNA other than exon 7x defined by (GRCh38 / hg38:chr3 193628509 193628616) or exon defined by (GRCh38 / hg38:chr3 193603500 193603557).

[0251] When considering reducing NMD exon content in mature mRNA, the reduction may be complete, such as 100%, or partial. The reduction may be clinically significant. The reduction / correction may be relative to the level of NMD exon content in untreated subjects, or relative to the amount of NMD exon content in similar target populations. The reduction / correction may be at least 10% less NMD exon content compared to the average subject or pre-treatment subjects. The reduction may be at least 20% less NMD exon content compared to the average subject or pre-treatment subjects. The reduction may be at least 40% less NMD exon content compared to the average subject or pre-treatment subjects. The reduction may be at least 50% less NMD exon content compared to the average subject or pre-treatment subjects. The reduction may be at least 60% less NMD exon content compared to the average subject or pre-treatment subjects. The reduction may be at least 80% less NMD exon content compared to the average subject or pre-treatment subjects. The reduction may be at least 90% less NMD exon content compared to the average subject or the subject before treatment.

[0252] According to one aspect of the present disclosure, a method for treating or preventing a condition or disease associated with functional OPA1 protein deficiency is provided herein, the method comprising administering a drug to a target to increase the level of functional OPA1 protein, the drug binding to a region of a premRNA transcript to reduce the content of a coding exon (e.g., exon 7) in the mature transcript. For example, the present disclosure provides a method for treating or preventing a condition associated with functional OPA1 protein deficiency, the method comprising administering a drug to a target to increase the level of functional OPA1 protein, the drug binding to a region of a coding exon (e.g., exon 7 of OPA1) in a premRNA transcript. For example, this specification provides a method for treating or preventing a condition associated with functional OPA1 protein deficiency, the method comprising administering a drug to a subject to increase the level of functional OPA1 protein, the drug binding to a region of the coding exon of a premRNA transcript (e.g., an exon of OPA1 (GRCh38 / hg38:chr3 193626092~193626202)). In some embodiments, the method comprises administering a drug to a subject to increase the level of functional OPA1 protein, the drug binding to a region containing the coding exon of a premRNA transcript (e.g., an exon of OPA1 other than exon 7 as defined by (GRCh38 / hg38:chr3 193626092~193626202)). In some embodiments, the therapeutic agent promotes the elimination of the coding exon of OPA1 premRNA other than exon 7, as defined by (GRCh38 / hg38:chr3 193626092~193626202). In some embodiments, the compositions disclosed herein include agents that promote the elimination of the coding exon of OPA1 premRNA other than exon 7, as defined by (GRCh38 / hg38:chr3 193626092~193626202).

[0253] When considering increasing the level of active OPA1 protein, the increase may be clinically significant. The increase may be relative to the level of active OPA1 protein in untreated subjects, or relative to the amount of active OPA1 protein in a similar population of subjects. The increase may be at least 10% higher than the average subject or the subject before treatment. The increase may be at least 20% higher than the average subject or the subject before treatment. The increase may be at least 40% higher than the average subject or the subject before treatment. The increase may be at least 50% higher than the average subject or the subject before treatment. The increase may be at least 80% higher than the average subject or the subject before treatment. The increase may be at least 100% higher than the average subject or the subject before treatment. The increase may be at least 200% more active OPA1 protein compared to the average subject or the subject before treatment. The increase may be at least 500% more active OPA1 protein compared to the average subject or the subject before treatment.

[0254] In embodiments where the NIE inhibitor includes a polynucleotide polymer, the polynucleotide polymer may be about 50 nucleotides long. The polynucleotide polymer may be about 45 nucleotides long. The polynucleotide polymer may be about 40 nucleotides long. The polynucleotide polymer may be about 35 nucleotides long. The polynucleotide polymer may be about 30 nucleotides long. The polynucleotide polymer may be about 24 nucleotides long. The polynucleotide polymer may be about 25 nucleotides long. The polynucleotide polymer may be about 20 nucleotides long. The polynucleotide polymer may be about 19 nucleotides long. The polynucleotide polymer may be about 18 nucleotides long. The polynucleotide polymer may be about 17 nucleotides long. The polynucleotide polymer may be about 16 nucleotides long. The polynucleotide polymer may be about 15 nucleotides long. The polynucleotide polymer may be about 14 nucleotides long. The polynucleotide polymer may be about 13 nucleotides long. The polynucleotide polymer may be about 12 nucleotides long. The polynucleotide polymer may be about 11 nucleotides long. The polynucleotide polymer may be about 10 nucleotides long. Polynucleotide polymers can be approximately 10 to 50 nucleotides in length. Polynucleotide polymers can be approximately 10 to 45 nucleotides in length. Polynucleotide polymers can be approximately 10 to 40 nucleotides in length. Polynucleotide polymers can be approximately 10 to 35 nucleotides in length. Polynucleotide polymers can be approximately 10 to 30 nucleotides in length. Polynucleotide polymers can be approximately 10 to 25 nucleotides in length. Polynucleotide polymers can be approximately 10 to 20 nucleotides in length. Polynucleotide polymers can be approximately 15 to 25 nucleotides in length. Polynucleotide polymers can be approximately 15 to 30 nucleotides in length. Polynucleotide polymers can be approximately 12 to 30 nucleotides in length.

[0255] The sequence of the polynucleic acid polymer may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% complementary to the target sequence of the mRNA transcript, for example, a partially treated mRNA transcript. The sequence of the polynucleic acid polymer may be 100% complementary to the target sequence of the premRNA transcript.

[0256] The sequence of a polynucleic acid polymer may have four or fewer inappropriate base pairs relative to the target sequence of the premRNA transcript. The sequence of a polynucleic acid polymer may have three or fewer inappropriate base pairs relative to the target sequence of the premRNA transcript. The sequence of a polynucleic acid polymer may have two or fewer inappropriate base pairs relative to the target sequence of the premRNA transcript. The sequence of a polynucleic acid polymer may have one or fewer inappropriate base pairs relative to the target sequence of the premRNA transcript. The sequence of a polynucleic acid polymer may have no inappropriate base pairs relative to the target sequence of the premRNA transcript.

[0257] Polynucleotide polymers can specifically hybridize to target sequences of premRNA transcripts. For example, polynucleotide polymers may have sequence complementarity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% to target sequences of premRNA transcripts. Hybridization can be carried out under highly stringent hybridization conditions.

[0258] The polynucleic acid polymer contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2 to 5. The polynucleic acid polymer may contain a sequence having 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2 to 5.

[0259] When considering polynucleotide polymer sequences, those skilled in the art will understand that one or more substitutions in the sequence may be tolerated, and optionally two substitutions may be tolerated, in order to maintain the ability to hybridize to a target sequence, or, if the substitutions are within the target sequence, the ability to be recognized as a target sequence. References to sequence identity may be determined by BLAST sequence alignment using standard / default parameters. For example, a sequence may have 99% identity and further possess the functionality compliant with this disclosure. In another embodiment, a sequence may have 98% identity and further possess the functionality compliant with this disclosure. In yet another embodiment, a sequence may have 95% identity and further possess the functionality compliant with this disclosure. In yet another embodiment, a sequence may have 90% identity and further possess the functionality compliant with this disclosure.

[0260] Antisense oligomers This specification provides compositions comprising antisense oligomers that induce exon skipping by binding to a target region of OPA1 premRNA, e.g., OPA1 NMD exon-containing premRNA. As used herein, the terms “ASO” and “antisense oligomer” are interchangeable and refer to oligomers such as polynucleotides containing nucleic acid bases that hybridize to a target nucleic acid (e.g., OPA1 premRNA, e.g., OPA1 NMD exon-containing premRNA) sequence by, for example, Watson-Crick base pairing or fluctuation base pairing (GU). The ASO may be strictly complementary or nearly complementary to the target sequence (e.g., sufficiently complementary to bind to the target sequence and improve splicing at the splice site). The ASO is designed to bind (hybridize) to a target nucleic acid (e.g., a target region of a premRNA transcript) and maintain the hybridized state under physiological conditions. Typically, when an ASO hybridizes to a site other than the target nucleic acid sequence, the number of non-target nucleic acid sequences to which it hybridizes is limited (it hybridizes to a small number of non-target nucleic acid sites). The design of the ASO can take into account the appearance of the target nucleic acid sequence in the premRNA transcript, or the appearance of sufficiently similar nucleic acid sequences at other locations in the genome or cell premRNA or transcriptome, so as to limit the possibility of the ASO binding to other sites and causing “off-target” effects. The methods described herein can be carried out using any antisense oligomer known in the art, for example, PCT application PCT / US2014 / 054151, published as WO 2015 / 035091, titled “Reducing Nonsense-Mediated mRNA Decay,” which is incorporated herein by reference.

[0261] In some embodiments, the ASO "specifically hybridizes" to or is "specific" to the target portion of the target nucleic acid or OPA1 premRNA, e.g., NMD exon-containing premRNA. Typically, such hybridization occurs at a Tm substantially higher than 37°C, preferably at least 50°C, typically 60°C to approximately 90°C. Such hybridization preferably corresponds to stringent hybridization conditions. Given ionic strength and pH, Tm is the temperature at which 50% of the target sequence hybridizes to complementary oligonucleotides.

[0262] For example, oligonucleotides are "complementary" to each other if hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration. Double-stranded polynucleotides can be "complementary" to another polynucleotide if hybridization can occur between one strand of the first polynucleotide and one strand of the second polynucleotide. Complementarity (the degree to which one polynucleotide is complementary to another) can be quantified in terms of the proportion (e.g., percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other according to generally accepted base pairing rules. The sequence of an antisense oligomer (ASO) does not need to be 100% complementary to the sequence of its target nucleic acid in order to hybridize. In certain embodiments, the ASO may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity with respect to the target region within the targeted nucleic acid sequence. For example, an ASO in which 18 of the 20 nucleic acid bases of an oligomer compound are complementary to the target region and thereby specifically hybridize will exhibit 90 percent complementarity. In this embodiment, the remaining non-complementary nucleic acid bases may cluster together with complementary nucleic acid bases, or be interspersed with complementary nucleic acid bases, and do not need to be continuous with each other or with complementary nucleic acid bases. The percentage of complementarity between the target nucleic acid region and the ASO can be routinely determined using the BLAST program (a basic local alignment search tool) and the PowerBLAST program (Altschul, et al., J. Mol. Biol., 1990, 215, 403-410, Zhang and Madden, Genome Res., 1997, 7, 649-656), which are known in the art.

[0263] ASO does not need to hybridize to all nucleic acid bases in the target sequence; the nucleic acid bases that hybridize may be continuous or discontinuous. ASO can hybridize across one or more segments of the premRNA transcript so that intermediate or adjacent segments do not participate in the hybridization event (e.g., a loop or hairpin structure may be formed). In certain embodiments, ASO hybridizes to discontinuous nucleic acid bases in the target premRNA transcript. For example, ASO can hybridize to nucleic acid bases in the premRNA transcript that are separated by one or more nucleic acid bases that ASO does not hybridize.

[0264] The ASOs described herein include nucleic acid bases that are complementary to the nucleic acid bases present in the target region of OPA1 premRNA (e.g., NMD exon-containing premRNA). The term ASO is a term that integrates oligonucleotides and any other oligomer molecules that include nucleic acid bases that can hybridize to complementary nucleic acid bases on the target mRNA, but do not contain a sugar moiety, such as peptide nucleic acid (PNA). An ASO may include native nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the foregoing. The term “native nucleotide” includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotide” includes nucleotides with modified or substituted sugar groups and / or having a modified backbone. In some embodiments, all nucleotides in the ASO are modified nucleotides. ASOs, or chemical modifications of ASO components, that are compatible with the methods and compositions described herein will become apparent to those skilled in the art, for example, in U.S. Patent No. 8,258,109B2, U.S. Patent No. 5,656,612, U.S. Patent Application Publication No. 2012 / 0190728, and Dias and Stein, Mol. Cancer Ther. 2002, 347-355, which are incorporated herein by reference as a whole.

[0265] One or more nucleic acid bases of the ASO may be any naturally occurring unmodified nucleic acid base, such as adenine, guanine, cytosine, thymine, and uracil, or any synthetic or modified nucleic acid base that is sufficiently similar to an unmodified nucleic acid base to be able to form hydrogen bonds with a nucleic acid base present on the target premRNA. Examples of modified nucleic acid bases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethoylcytosine.

[0266] ASOs described herein also include a skeletal structure that connects the components of the oligomer. The terms “skeletal structure” and “oligomer linkage” may be used interchangeably and may refer to the linkage between monomers of the ASO. In natural oligonucleotides, the skeletal structure includes a 3'-5' phosphodiester linkage that connects the sugar moieties of the oligomer. Examples of skeletal structures or oligomer links of ASOs described herein (but not limited to) include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniradiates, and phosphoramidates. For example, LaPlanche, et al., Nucleic Acids Res. 14:9081 (1986), Stec, et al., J. Am. Chem. Soc. 106:6077 (1984), Stein, et al., Nucleic Acids Res. 16:3209 (1988), Zon, et al., Anti-Cancer Drug Design 6:539 (1991), Zon, et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)), Stec, et al., US Pat. No. 5,151,510, Uhlmann and Peyman, Chemical Reviews 90:543 (1990). In some embodiments, the ASO skeleton does not contain phosphorus, but contains peptide bonds in, for example, peptide nucleic acid (PNA), or peptide bonds in linking groups including carbamate bases, amide groups, and linear and cyclic hydrocarbon groups. In some embodiments, the skeleton modification is a phosphorothioate bond. In some embodiments, the skeleton modification is a phosphoramidate bond.

[0267] In some embodiments, the stereochemistry of each phosphate nucleotide bond in the ASO skeleton is random. In some embodiments, the stereochemistry of each phosphate nucleotide bond in the ASO skeleton is controlled and not random. For example, U.S. Patent Application Publication 2014 / 0194610, “Methods for the Synthesis of Functionalized Nucleic Acids,” describes a method for independently selecting the chirality of each phosphate atom in a nucleic acid oligomer. In some embodiments, but not limited to, the ASOs used in the methods of this disclosure include any of the ASOs described herein in Tables 5 and 6, and include ASOs having non-random phosphate nucleotide bonds. In some embodiments, the compositions used in the methods of this disclosure include pure diastereomer ASOs. In some embodiments, the compositions used in the methods of the present disclosure include ASOs having a diastereomer 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%.

[0268] In some embodiments, the ASO has a non-random mixture of Rp and Sp configurations in its nucleotide-nucleotide linkages of phosphorus. For example, it has been suggested that a mixture of Rp and Sp is necessary in antisense oligonucleotides to achieve a good balance between activity and nuclease stability (Wan, et al., 2014, “Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages,” Nucleic Acids Research 42(22):13456-13468, incorporated herein by reference). In some embodiments, but not limited to, an ASO used in the method of the present disclosure, including any of the ASOs described herein in Sequence IDs 2-5, 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 as Sp, or contains about 100% Rp.In some embodiments, but not limited to, the ASO used in the methods of this disclosure includes any of the ASOs described herein, which include a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region containing at least eight consecutive nucleic acids of any one of SEQ ID NOs: about 10% to about 100% Rp, about 15% to about 100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp, and about 40% to about 100% Rp. , including approximately 45% to 100% Rp, approximately 50% to 100% Rp, approximately 55% to 100% Rp, approximately 60% to 100% Rp, approximately 65% ​​to 100% Rp, approximately 70% to 100% Rp, approximately 75% to 100% Rp, approximately 80% to 100% Rp, approximately 85% to 100% Rp, approximately 90% to 100% Rp, or approximately 95% to 100% Rp, approximately 20% to 80% Rp, approximately 25% to 75% Rp, approximately 30% to 70% Rp, approximately 40% to 60% Rp, or approximately 45% to 55% Rp, with the remainder as Sp.

[0269] In some embodiments, but not limited to, the ASO used in the method of the present disclosure includes any of the ASOs described herein, which include a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region containing at least eight consecutive nucleic acids of any one of SEQ ID NOs: about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, and at least about 25% S p, including at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, with the remainder as Rp, or including about 100% Sp. In embodiments, but not limited to, an ASO used in the method of the present disclosure includes any of the ASOs described herein, which include a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region containing at least eight consecutive nucleic acids of any one of SEQ ID NOs: about 10% to about 100% Sp, about 15% to about 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, about 40% to about 100% Sp, Approximately 45% to 100% Sp, approximately 50% to 100% Sp, approximately 55% to 100% Sp, approximately 60% to 100% Sp, approximately 65% ​​to 100% Sp, approximately 70% to 100% Sp, approximately 75% to 100% Sp, approximately 80% to 100% Sp, approximately 85% to 100% Sp, approximately 90% to 100% Sp, or approximately 95% to 100% Sp, approximately 20% to 80% Sp, approximately 25% to 75% Sp, approximately 30% to 70% Sp, approximately 40% to 60% Sp, or approximately 45% to 55% Sp, with the remainder as Rp.

[0270] Any of the ASOs described herein may contain a sugar moiety containing ribose or deoxyribose present in natural nucleotides, or a modified sugar moiety or sugar analog containing a morpholine ring. Non-limiting examples of modified sugar moieties include, for example, 2' substitutions such as 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F, N3'->P5' phosphoramidates, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamic acid-modified sugars, and bicyclic-modified sugars. In some embodiments, the modification of the sugar moiety is selected from 2'-O-Me, 2'F, and 2'MOE. In some embodiments, the modification of the sugar moiety is an excess crosslinking, such as loc nucleic acid (LNA). In some embodiments, the sugar analog includes a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety includes ribofuransyl or 2'deoxyribofuransyl modification. In some embodiments, the sugar moiety includes 2'4'-restricted 2'O-methyloxyethyl (cMOE) modification. In some embodiments, the sugar moiety includes BNA modification of 2',4'-restricted 2'-O-ethyl cEt. In some embodiments, the sugar moiety includes tricycloDNA (tcDNA) modification. In some embodiments, the sugar moiety includes ethylene nucleic acid (ENA) modification. In some embodiments, the sugar moiety includes MCE modification. The modifications are known in the art and are described in the literature, for example, Jarver, et al., 2014, “A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications,” Nucleic Acid Therapeutics 24(1):37-47, which is incorporated by reference to the present purpose of this specification.

[0271] In some embodiments, each monomer of ASO is modified in the same manner, for example, each bond in the ASO skeleton contains a phosphorothioate bond, or each ribose sugar moiety contains a 2'O-methyl modification. Such modifications present in each monomer component of ASO are referred to as “uniform modifications.” In some embodiments, combinations of different modifications may be desirable, for example, ASO may contain a combination of a phosphorodiamidate bond and a sugar moiety containing a morpholine ring (morpholino). Combinations of different modifications to ASO are referred to as “mixed modifications” or “mixed chemicals.”

[0272] In some embodiments, the ASO includes modifications to one or more skeletal structures. In some embodiments, the ASO includes modifications to one or more sugar moieties. In some embodiments, the ASO includes modifications to one or more skeletal structures and one or more sugar moieties. In some embodiments, the ASO includes a 2'MOE modification and a phosphorothioate skeleton. In some embodiments, the ASO includes phosphorodiamidate morpholino (PMO). In some embodiments, the ASO includes peptide nucleic acid (PNA). Any of the ASOs described herein, or any component of any ASO (e.g., nucleic acid bases, sugar moieties, skeletons), may be modified to achieve desired characteristics or activities of the ASO, or to reduce undesirable characteristics or activities of the ASO. For example, an ASO, or one or more components of any ASO, may be modified to enhance binding affinity to a target sequence on a premRNA transcript, reduce binding to any non-target sequence, reduce degradation by cellular nucleases (i.e., ribonuclease H), improve uptake of the ASO into the cell and / or nucleus, alter the pharmacokinetics or pharmacology of the ASO, and / or modulate the half-life of the ASO.

[0273] In some embodiments, the ASO is composed of 2'-O-(2-methoxyethyl)(MOE) phosphorothioate-modified nucleotides. ASOs composed of such nucleotides are particularly suitable for the methods disclosed herein, and oligomers having such modifications have been shown to significantly improve resistance to nuclease degradation and increase bioavailability, thereby making the ASO suitable for oral delivery in some embodiments herein. See, for example, Geary, et al., J Pharmacol Exp Ther. 2001; 296(3):890-7 and Geary, et al., J Pharmacol Exp Ther. 2001; 296(3):898-904.

[0274] In some embodiments, the ASO contains a 5'-methylcytosine (5'-MeC) nucleotide. In some embodiments, the ASO contains at least one 5'-methylcytosine (5'-MeC) nucleotide. In some embodiments, each cytosine in the ASO is 5'-methylcytosine (5'-MeC). In some embodiments, the ASO contains 5'-methyluracil (5'-MeU). In some embodiments, the ASO contains at least one 5'-methyluracil (5'-MeU). In some embodiments, at least one cytosine or thymidine in the antisense oligomer is 5'-methyluracil (5'-MeU). In some embodiments, each cytosine or thymidine in the antisense oligomer is 5'-methyluracil (5'-MeU).

[0275] In any of the structural formulas (illustrations of compounds) presented herein, two curves and a straight line between them are used to connect a phosphorus atom ("P") and an oxygen atom ("O"). The two curves and the straight line between them should be viewed as a single, integrated segment, representing a covalent bond between the phosphorus and oxygen atoms and part of a skeletal bond between two adjacent nucleotides (e.g., a phosphodiester bond or a phosphorothioate bond). In any of the structural formulas, the vertices (angles) where the curves and the straight line connect do not represent carbon atoms, nor do they represent the presence of -CH2- at the relevant positions in the compound shown in the structural formula.

[0276] In some embodiments, ASO has the following chemical structure:

[0277] [ka] [ka] [ka] One of the following, or a pharmaceutically acceptable salt thereof.

[0278] In some embodiments, the ASO has the following structure:

[0279] [ka] or a pharmaceutically acceptable salt thereof.

[0280] In some embodiments, the ASO has the following structure:

[0281] [ka] or a pharmaceutically acceptable salt thereof.

[0282] In some embodiments, the ASO has the following structure:

[0283] [ka] or a pharmaceutically acceptable salt thereof.

[0284] In some embodiments, the ASO has the following structure:

[0285] [ka] or a pharmaceutically acceptable salt thereof.

[0286] In some embodiments, the ASO has the following structure:

[0287] [ka] or a pharmaceutically acceptable salt thereof.

[0288] In some embodiments, the ASO has one of the following structures:

[0289] [ka] [ka] [ka] It holds.

[0290] In some embodiments, ASO is in salt form. In some embodiments, the salt form is a sodium salt. In some embodiments, the sodium salt form of ASO has the following structure:

[0291] [ka] It holds.

[0292] In some embodiments, ASO is in salt form. In some embodiments, the salt form is a sodium salt. In some embodiments, the sodium salt form of ASO has the following structure:

[0293] [ka] It holds.

[0294] In some embodiments, ASO is in salt form. In some embodiments, the salt form is a sodium salt. In some embodiments, the sodium salt form of ASO has the following structure:

[0295] [ka] It holds.

[0296] In some embodiments, ASO is in salt form. In some embodiments, the salt form is a sodium salt. In some embodiments, the sodium salt form of ASO has the following structure:

[0297] [ka] It holds.

[0298] In some embodiments, ASO is in salt form. In some embodiments, the salt form is a sodium salt. In some embodiments, the sodium salt form of ASO has the following structure:

[0299] [ka] It holds.

[0300] Methods for synthesizing ASO are known to those skilled in the art. Alternatively, ASO can be obtained from commercial suppliers.

[0301] Unless otherwise specified, the left end of a single-stranded nucleic acid sequence (e.g., pre-mRNA transcript, oligonucleotide, ASO, etc.) is the 5' end, and the left direction of a single-stranded or double-stranded nucleic acid sequence is referred to as the 5' direction. Similarly, the right end or right direction of a nucleic acid sequence (single-stranded or double-stranded) is the 3' end or 3' direction. Generally, a region or sequence that is 5' relative to a reference point in a nucleic acid is referred to as "upstream," and a region or sequence that is 3' relative to a reference point in a nucleic acid is referred to as "downstream." Generally, the 5' direction or 5' end of mRNA is where the start codon is located, while the 3' end or 3' direction is where the stop codon is located. In some embodiments, nucleotides upstream of a reference point in a nucleic acid may be referred to by negative numbers, while nucleotides downstream of a reference point may be referred to by positive numbers. For example, a reference point (e.g., an exon-exon junction in mRNA) may be called a "zero" site, and a nucleotide directly adjacent to and upstream of the reference point is called "minus 1," or "-1," while a nucleotide directly adjacent to and downstream of the reference point is called "plus 1," or "+1."

[0302] In some embodiments, the ASO is complementary to (and binds to) the target region of the OPA1 premRNA, e.g., OPA1 NMD exon-containing premRNA, i.e., located downstream (3' direction) of the 5' splice site (or 3' end of the NMD exon) of the exon contained within the OPA1 premRNA (e.g., in a direction specified by a positive number relative to the 5' splice site). In some embodiments, the ASO is complementary to the target region of the OPA1 premRNA, e.g., OPA1 NMD exon-containing premRNA, located in a region of approximately -1 to approximately -500 nucleotides relative to the 5' splice site (or 3' end) of the contained exon. In some embodiments, the ASO may be complementary to the target region of the OPA1 premRNA, e.g., OPA1 NMD exon-containing premRNA, located in a region of nucleotide +6 to +40,000 nucleotides relative to the 5' splice site (or 3' end) of the contained exon.In some embodiments, the ASO is approximately +1 to +40,000, +1 to +30,000, +1 to +20,000, +1 to +15,000, +1 to +10,000, +1 to +5,000, +1 to +4,000, +1 to +3,000, +1 to +2,000, and +1 to +1,000 relative to the 5' splice site (or 3' end) of the exon it contains. 00, approximately +1 to approximately +500, approximately +1 to approximately +490, approximately +1 to approximately +480, approximately +1 to approximately +470, approximately +1 to approximately +460, approximately +1 to approximately +450, approximately +1 to approximately +440, approximately +1 to approximately +430, approximately +1 to approximately +420, approximately +1 to approximately +410, approximately +1 to approximately +400, approximately +1 to approximately +390, approximately +1 to approximately +380, approximately +1 to approximately +370, approximately +1 to approximately +360, approximately +1 to approximately +350, approximately +1 to approximately +3 40, approximately +1 to approximately +330, approximately +1 to approximately +320, approximately +1 to approximately +310, approximately +1 to approximately +300, approximately +1 to approximately +290, approximately +1 to approximately +280, approximately +1 to approximately +270, approximately +1 to approximately +260, approximately +1 to approximately +250, approximately +1 to approximately +240, approximately +1 to approximately +230, approximately +1 to approximately +220, approximately +1 to approximately +210, approximately +1 to approximately +200, approximately +1 to approximately +190, approximately +1 to approximately +180, approximately +1 to approximately + It is complementary to the target portion located in the range of 170, approximately +1 to +160, approximately +1 to +150, approximately +1 to +140, approximately +1 to +130, approximately +1 to +120, approximately +1 to +110, approximately +1 to +100, approximately +1 to +90, approximately +1 to +80, approximately +1 to +70, approximately +1 to +60, approximately +1 to +50, approximately +1 to +40, approximately +1 to +30, or approximately +1 to +20. In some embodiments, the ASO is complementary to the target portion located approximately +1 to +100, approximately +100 to +200, approximately +200 to +300, approximately +300 to +400, or approximately +400 to +500 relative to the 5' splice site (or 3' end) of the exon containing it.

[0303] In some embodiments, the ASO is complementary to (and binds to) the target region of the OPA1 premRNA, e.g., OPA1 NMD exon-containing premRNA, i.e., located upstream (5' direction) of the 5' splice site (or 3' end) of the exon contained within the OPA1 premRNA, e.g., OPA1 NMD exon-containing premRNA (e.g., in a direction specified by a negative number relative to the 5' splice site). In some embodiments, the ASO is complementary to the target region of the OPA1 premRNA, e.g., OPA1 NMD exon-containing premRNA, located in a region of approximately -4 to approximately -270 nucleotides relative to the 5' splice site (or 3' end) of the contained exon. In some embodiments, the ASO may be complementary to the target region of the OPA1 premRNA, e.g., OPA1 NMD exon-containing premRNA, located in a region of nucleotide -1 to -40,000 relative to the 5' splice site (or 3' end) of the contained exon.In some embodiments, the ASO is distributed to the 5' splice site (or 3' end) of the exon containing it, with approximately -1 to -40,000, approximately -1 to -30,000, approximately -1 to -20,000, approximately -1 to -15,000, approximately -1 to -10,000, approximately -1 to -5,000, approximately -1 to -4,000, approximately -1 to -3,000, approximately -1 to -2,000, and approximately -1 to -1,0 00, approx. -1 to approx. -500, approx. -1 to approx. -490, approx. -1 to approx. -480, approx. -1 to approx. -470, approx. -1 to approx. -460, approx. -1 to approx. -450, approx. -1 to approx. -440, approx. -1 to approx. -430, approx. -1 to approx. -420, approx. -1 to approx. -410, approx. -1 to approx. -400, approx. -1 to approx. -390, approx. -1 to approx. -380, approx. -1 to approx. -370, approx. -1 to approx. -360, approx. -1 to approx. -350, approx. -1 to approx. -3 40, approx. -1 to approx. -330, approx. -1 to approx. -320, approx. -1 to approx. -310, approx. -1 to approx. -300, approx. -1 to approx. -290, approx. -1 to approx. -280, approx. -1 to approx. -270, approx. -1 to approx. -260, approx. -1 to approx. -250, approx. -1 to approx. -240, approx. -1 to approx. -230, approx. -1 to approx. -220, approx. -1 to approx. -210, approx. -1 to approx. -200, approx. -1 to approx. -190, approx. -1 to approx. -180, approx. -1 to approx. - It is complementary to target portions located in the regions of 170, approximately -1 to -160, approximately -1 to -150, approximately -1 to -140, approximately -1 to -130, approximately -1 to -120, approximately -1 to -110, approximately -1 to -100, approximately -1 to -90, approximately -1 to -80, approximately -1 to -70, approximately -1 to -60, approximately -1 to -50, approximately -1 to -40, approximately -1 to -30, or approximately -1 to -20.

[0304] In some embodiments, the ASO is complementary to the target region of the OPA1 premRNA, e.g., in the direction upstream (5' direction) of the 3' splice site (or 5' end) of the exon contained in the OPA1 NMD exon-containing premRNA (e.g., in a direction specified by a negative number). In some embodiments, the ASO is complementary to the target region of the OPA1 premRNA, e.g., in the region approximately -1 to approximately -500 relative to the 3' splice site (or 5' end) of the contained exon. In some embodiments, the ASO is complementary to the target region of the OPA1 premRNA, e.g., in the region approximately -1 to -40,000 relative to the 3' splice site of the contained exon.In some embodiments, the ASO is distributed to the 3' splice site of the included exon in approximately -1 to -40,000, approximately -1 to -30,000, approximately -1 to -20,000, approximately -1 to -15,000, approximately -1 to -10,000, approximately -1 to -5,000, approximately -1 to -4,000, approximately -1 to -3,000, approximately -1 to -2,000, approximately -1 to -1,000, and approximately -1 to -500, approx. -1 to approx. -490, approx. -1 to approx. -480, approx. -1 to approx. -470, approx. -1 to approx. -460, approx. -1 to approx. -450, approx. -1 to approx. -440, approx. -1 to approx. -430, approx. -1 to approx. -420, approx. -1 to approx. -410, approx. -1 to approx. -400, approx. -1 to approx. -390, approx. -1 to approx. -380, approx. -1 to approx. -370, approx. -1 to approx. -360, approx. -1 to approx. -350, approx. -1 to approx. -340, approx. - 1 to approximately -330, approximately -1 to approximately -320, approximately -1 to approximately -310, approximately -1 to approximately -300, approximately -1 to approximately -290, approximately -1 to approximately -280, approximately -1 to approximately -270, approximately -1 to approximately -260, approximately -1 to approximately -250, approximately -1 to approximately -240, approximately -1 to approximately -230, approximately -1 to approximately -220, approximately -1 to approximately -210, approximately -1 to approximately -200, approximately -1 to approximately -190, approximately -1 to approximately -180, approximately -1 to approximately -170 In some embodiments, the ASO is complementary to the target portion located in the region of approximately -1 to approximately -160, approximately -1 to approximately -150, approximately -1 to approximately -140, approximately -1 to approximately -130, approximately -1 to approximately -120, approximately -1 to approximately -110, approximately -1 to approximately -100, approximately -1 to approximately -100, approximately -1 to approximately -90, approximately -1 to approximately -80, approximately -1 to approximately -70, approximately -1 to approximately -60, approximately -1 to approximately -50, approximately -1 to approximately -40, approximately -1 to approximately -30, or approximately -1 to approximately -20 with respect to the 3' splice site of the included exon.

[0305] In some embodiments, the ASO is complementary to a target region downstream (3' direction) of the 3' splice site (5' end) of the OPA1 premRNA, e.g., OPA1 NMD exon-containing premRNA (e.g., direction indicated by a positive number), of the exon contained within the OPA1 premRNA, e.g., OPA1 NMD exon-containing premRNA. In some embodiments, the ASO is complementary to a target region of the OPA1 premRNA located approximately +1 to approximately +40,000 relative to the 3' splice site of the contained exon. In some embodiments, ASOs are approximately +1 to +40,000, +1 to +30,000, +1 to +20,000, +1 to +15,000, +1 to +10,000, +1 to +5,000, +1 to +4,000, +1 to +3,000, +1 to +2,000, +1 to +1,000, and +1 to +5 00, approximately +1 to approximately +490, approximately +1 to approximately +480, approximately +1 to approximately +470, approximately +1 to approximately +460, approximately +1 to approximately +450, approximately +1 to approximately +440, approximately +1 to approximately +430, approximately +1 to approximately +420, approximately +1 to approximately +410, approximately +1 to approximately +400, approximately +1 to approximately +390, approximately +1 to approximately +380, approximately +1 to approximately +370, approximately +1 to approximately +360, approximately +1 to approximately +350, approximately +1 to approximately +340, approximately +1 to approximately +3 30, approximately +1 to approximately +320, approximately +1 to approximately +310, approximately +1 to approximately +300, approximately +1 to approximately +290, approximately +1 to approximately +280, approximately +1 to approximately +270, approximately +1 to approximately +260, approximately +1 to approximately +250, approximately +1 to approximately +240, approximately +1 to approximately +230, approximately +1 to approximately +220, approximately +1 to approximately +210, approximately +1 to approximately +200, approximately +1 to approximately +190, approximately +1 to approximately +180, approximately +1 to approximately +170, approximately +1 to approximately +1 It is complementary to the target portion located in the range of 60, approximately +1 to +150, approximately +1 to +140, approximately +1 to +130, approximately +1 to +120, approximately +1 to +110, approximately +1 to +100, approximately +1 to +90, approximately +1 to +80, approximately +1 to +70, approximately +1 to +60, approximately +1 to +50, approximately +1 to +40, approximately +1 to +30, or approximately +1 to +20, or approximately +1 to +10.

[0306] In some embodiments, the target region of OPA1 premRNA, for example, OPA1 NMD exon-containing premRNA, is located in the region from +100 to -100 relative to the 5' splice site (3' end) of the contained exon. In some embodiments, the target region of OPA1 NMD exon-containing premRNA is located within the NMD exon. In some embodiments, the target region of OPA1 NMD exon-containing premRNA includes a pseudoexon and an intron boundary region.

[0307] The ASO may be of any length suitable for specific binding and improved effective splicing. In some embodiments, the ASO consists of 8 to 50 nucleic acid bases. For example, the ASO may be of a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleic acid bases. In some embodiments, the ASO consists of more than 50 nucleic acid bases. In some embodiments, the ASO consists of 8-50 nucleic acid bases, 8-40 nucleic acid bases, 8-35 nucleic acid bases, 8-30 nucleic acid bases, 8-25 nucleic acid bases, 8-20 nucleic acid bases, 8-15 nucleic acid bases, 9-50 nucleic acid bases, 9-40 nucleic acid bases, 9-35 nucleic acid bases, 9-30 nucleic acid bases, 9-25 nucleic acid bases, 9-20 nucleic acid bases, 9-15 nucleic acid bases, 10-50 nucleic acid bases, and 10-40 Nucleic acid bases, 10-35 nucleic acid bases, 10-30 nucleic acid bases, 10-25 nucleic acid bases, 10-20 nucleic acid bases, 10-15 nucleic acid bases, 11-50 nucleic acid bases, 11-40 nucleic acid bases, 11-35 nucleic acid bases, 11-30 nucleic acid bases, 11-25 nucleic acid bases, 11-20 nucleic acid bases, 11-15 nucleic acid bases, 12-50 nucleic acid bases, 12-40 nucleic acid bases, 12-35 nucleic acid bases, 12-3 0 nucleic acid bases, 12-25 nucleic acid bases, 12-20 nucleic acid bases, 12-15 nucleic acid bases, 13-50 nucleic acid bases, 13-40 nucleic acid bases, 13-35 nucleic acid bases, 13-30 nucleic acid bases, 13-25 nucleic acid bases, 13-20 nucleic acid bases, 14-50 nucleic acid bases, 14-40 nucleic acid bases, 14-35 nucleic acid bases, 14-30 nucleic acid bases, 14-25 nucleic acid bases, 14-20 nucleic acid bases, The length is 15-50 nucleic acid bases, 15-40 nucleic acid bases, 15-35 nucleic acid bases, 15-30 nucleic acid bases, 15-25 nucleic acid bases, 15-20 nucleic acid bases, 20-50 nucleic acid bases, 20-40 nucleic acid bases, 20-35 nucleic acid bases, 20-30 nucleic acid bases, 20-25 nucleic acid bases, 25-50 nucleic acid bases, 25-40 nucleic acid bases, 25-35 nucleic acid bases, or 25-30 nucleic acid bases.In some embodiments, the ASO is 18 nucleotides long. In some embodiments, the ASO is 15 nucleotides long. In some embodiments, the ASO is 25 nucleotides long.

[0308] In some embodiments, two or more ASOs are used that have different chemical structures but are complementary to the same target region of premRNA, e.g., NMD exon-containing premRNA. In some embodiments, two or more ASOs are used that are complementary to different target regions of premRNA, e.g., NMD exon-containing premRNA.

[0309] In some embodiments, the antisense oligonucleotides of this disclosure are chemically conjugated to one or more moieties or conjugates, for example, a targeting moiety that enhances the activity or cellular uptake of the oligonucleotide, or to other conjugates. Such moieties include, but are not limited to, lipid moieties, e.g., cholesterol moieties, cholesteryl moieties, aliphatic chains, e.g., dodecanediol or undecyl residues, polyamine or polyethylene glycol chains, or adamantane acetate. Oligonucleotides containing lipophilic moieties and methods for their preparation are described in the published literature. In embodiments, the antisense oligonucleotides are conjugated to moieties including, but are not limited to, debasalized nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, e.g., N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), lipids, or polyhydrocarbon compounds. The conjugates can be conjugated to one or more nucleotides, including the antisense oligonucleotide, at any of several positions on a sugar, base, or phosphate group, for example, using a linker, as understood in the art and described in the literature. The linker may include a divalent or trivalent branched linker. In embodiments, the conjugate is conjugated to the 3' end of an antisense oligonucleotide. For a method of preparing oligonucleotide conjugates, see, for example, U.S. Patent No. 8,450,467, “Carbohydrate conjugates as delivery agents for oligonucleotides,” which is incorporated herein by reference.

[0310] In some embodiments, the nucleic acid targeted by ASO is OPA1 premRNA (e.g., NMD exon-containing premRNA) expressed in cells, such as eukaryotic cells. In some embodiments, the term “cell” may refer to a population of cells. In some embodiments, the cells are present in the subject. In some embodiments, the cells are isolated from the subject. In some embodiments, the cells are ex vivo. In some embodiments, the cells are cells or cell lines associated with a condition or disease. In some embodiments, the cells are in vitro (e.g., in a cell culture medium).

[0311] Pharmaceutical composition Pharmaceutical compositions or formulations containing agents such as antisense oligonucleotides of the compositions described herein, for use in any of the methods described herein, are well known in the pharmaceutical industry and can be prepared according to the prior art described in the published literature. In embodiments, a pharmaceutical composition or formulation for treating a subject contains an effective amount of any antisense oligomer described herein, or a pharmaceutically acceptable salt, solvate, hydrate, or ester thereof. A pharmaceutical formulation containing an antisense oligomer may further contain a pharmaceutically acceptable excipient, diluent, or carrier.

[0312] Pharmaceutically acceptable salts are suitable for use in contact with human and lower animal tissues without adverse toxicity, irritation, or allelic reactions, and represent a reasonable benefit-risk ratio. (e.g., SM Berge, et al., J. Pharmaceutical Sciences, 66:1-19) See (1977), which is incorporated herein by reference for the purposes of this invention. Salts can be prepared in situ during the final isolation and purification of the compound, or separately by reacting its free base form with a suitable organic acid. Examples of pharmaceutically acceptable non-toxic acid addition salts include, for example, salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or salts of amino groups formed with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or salts of amino groups formed by the use of other demonstrated methods such as ion exchange. Other pharmaceutically acceptable salts include adipines, alginates, ascorbic acid, aspartates, benzenesulfons, benzoates, bisulfates, borates, butyrates, camphorates, camphosulfons, citrates, and cyclopentanes. Examples include propionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, 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, and valerate. Representative alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium.Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfons, and aryl sulfons.

[0313] In some embodiments, the composition is formulated into one of many possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. In some embodiments, the composition is formulated as a suspension in an aqueous, non-aqueous, or mixed medium. The aqueous suspension may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may contain stabilizers. In embodiments, the pharmaceutical formulations or pharmaceutical compositions of the present disclosure may include, but are not limited to, liquids, emulsions, microemulsions, foams, or liposome-containing formulations (e.g., cationic or non-cationic liposomes).

[0314] The pharmaceutical compositions or formulations described herein may optionally contain one or more penetration enhancers, carriers, excipients, or other active or inactive components that are well known to those skilled in the art or described in published literature. In embodiments, liposomes also include sterically stabilized liposomes, such as liposomes containing one or more specialized lipids. These specialized lipids result in liposomes with an extended lifespan in blood circulation. In embodiments, the sterically stabilized liposomes contain one or more glycolipids or are derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. In some embodiments, surfactants are included in the pharmaceutical formulation or pharmaceutical composition. The use of surfactants in pharmaceuticals, formulations, and emulsions is well known in the art. In embodiments, the disclosure uses penetration enhancers to cause efficient delivery of antisense oligonucleotides, for example, to aid diffusion across cell membranes, and / or to enhance the permeability of lipophilic drugs. In some embodiments, the penetration enhancer is a surfactant, a fatty acid, a bile salt, a chelating agent, or a non-chelating non-surfactant.

[0315] In some embodiments, the pharmaceutical formulation comprises multiple antisense oligonucleotides. In embodiments, the antisense oligonucleotides are administered in combination with another drug or therapeutic agent.

[0316] In some embodiments, the pharmaceutical composition is prepared by diluting a concentrate containing an antisense oligomer.

[0317] In some embodiments, the antisense oligomer is approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 200 mg / mL, approximately 100 mg / mL to approximately 200 mg / mL, approximately 150 mg / mL to approximately 200 mg / mL, approximately 180 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 150 mg / mL, approximately 35 mg / mL to approximately 150 mg / mL, approximately 40 mg / mL to approximately 150 mg / mL, approximately 50 mg / mL to approximately 150 mg / mL, approximately 60 mg / mL to approximately 150 mg / mL, approximately It is present in the anonymized material at concentrations of approximately 80 mg / mL to 150 mg / mL, approximately 100 mg / mL to 150 mg / mL, approximately 30 mg / mL to 100 mg / mL, approximately 35 mg / mL to 100 mg / mL, approximately 40 mg / mL to 100 mg / mL, approximately 50 mg / mL to 100 mg / mL, approximately 60 mg / mL to 100 mg / mL, approximately 80 mg / mL to 100 mg / mL, approximately 30 mg / mL to 80 mg / mL, approximately 35 mg / mL to 80 mg / mL, approximately 40 mg / mL to 80 mg / mL, approximately 60 mg / mL to 80 mg / mL, approximately 30 mg / mL to 60 mg / mL, approximately 35 mg / mL to 60 mg / mL, or approximately 40 mg / mL to 60 mg / mL.

[0318] In some embodiments, the antisense oligomer is present in the concentrate at concentrations of approximately 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 120 mg / mL, 140 mg / mL, 160 mg / mL, 180 mg / mL, or 200 mg / mL.

[0319] In some embodiments, the concentrate is a phosphate buffer.

[0320] In some embodiments, the pharmaceutical formulation comprises (a) an antisense oligomer comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 6-275 or 280-299, and (b) a pharmaceutically acceptable diluent, wherein the antisense oligomer is dissolved or suspended in a solution at a concentration of about 1 mg / mL to about 200 mg / mL.

[0321] In some embodiments, the pharmaceutical formulation comprises (a) an antisense oligomer, wherein the antisense oligomer contains a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 6-275 or 280-299, and (b) a pharmaceutically acceptable diluent, wherein the antisense oligomer is dissolved or suspended in a solution, and the antisense oligomer has a chemical structure:

[0322] [ka] [ka] [ka] It comprises a pharmaceutically acceptable diluent which is a compound of any one of the following.

[0323] In some embodiments, the pharmaceutical formulation comprises (a) an antisense oligomer, wherein the antisense oligomer contains a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 6-275 or 280-299, and (b) a pharmaceutically acceptable diluent, wherein the antisense oligomer is dissolved or suspended in a solution, and the antisense oligomer has a chemical structure:

[0324] [ka] [ka] [ka] It comprises a pharmaceutically acceptable diluent, which is a compound comprising any one of the above, or a pharmaceutically acceptable salt thereof.

[0325] In some embodiments, the antisense oligomer is present in the solution at a concentration of approximately 1 mg / mL to approximately 200 mg / mL.

[0326] In some embodiments, the antisense oligomer is approximately 5 mg / mL to approximately 200 mg / mL, approximately 10 mg / mL to approximately 200 mg / mL, approximately 15 mg / mL to approximately 200 mg / mL, approximately 20 mg / mL to approximately 200 mg / mL, approximately 25 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 200 mg / mL, and approximately 100 mg / mL to approximately 200 mg / mL, about 150mg / mL to about 200mg / mL, about 180mg / mL to about 200mg / mL, 2mg / mL to about 150mg / mL, about 5mg / mL to about 150mg / mL, about 10mg / mL to about 150mg / mL, about 15mg / mL to about 150mg / mL, about 20mg / mL mL~about 150mg / mL, about 25mg / mL~about 150mg / mL, about 30mg / mL~about 150mg / mL, about 35mg / mL~about 150mg / mL, about 40mg / mL~about 150mg / mL, about 50mg / mL~about 150mg / mL, about 60mg / mL~about 150mg / mL mL, about 80 mg / mL to about 150 mg / mL, about 100 mg / mL to about 150 mg / mL, 2 mg / mL to about 100 mg / mL, about 5 mg / mL to about 100 mg / mL, about 10 mg / mL to about 100 mg / mL, about 15 mg / mL to about 100 mg / mL, about 20 mg / mL ~100mg / mL, 25mg / mL~100mg / mL, 30mg / mL~100mg / mL, 35mg / mL~100mg / mL, 40mg / mL~100mg / mL, 50mg / mL~100mg / mL, 60mg / mL~100mg / mL , about 80 mg / mL to about 100 mg / mL, 2 mg / mL to about 80 mg / mL, about 5 mg / mL to about 80 mg / mL, about 10 mg / mL to about 80 mg / mL, about 15 mg / mL to about 80 mg / mL, about 20 mg / mL to about 80 mg / mL, about 25 mg / mL to about 80 mg / mL , about 30 mg / mL to about 80 mg / mL, about 35 mg / mL to about 80 mg / mL, about 40 mg / mL to about 80 mg / mL, about 60 mg / mL to about 80 mg / mL, 2 mg / mL to about 60 mg / mL, about 5 mg / mL to about 60 mg / mL, about 10 mg / mL to about 60 mg / mL,It is present in the solution at concentrations of approximately 15 mg / mL to 60 mg / mL, 20 mg / mL to 60 mg / mL, 25 mg / mL to 60 mg / mL, 30 mg / mL to 60 mg / mL, 35 mg / mL to 60 mg / mL, 40 mg / mL to 60 mg / mL, 2 mg / mL to 40 mg / mL, 5 mg / mL to 40 mg / mL, 10 mg / mL to 40 mg / mL, 15 mg / mL to 40 mg / mL, 20 mg / mL to 40 mg / mL, or 25 mg / mL to 40 mg / mL.

[0327] In some embodiments, the antisense oligomer is present in the solution at concentrations of approximately 1 mg / mL, approximately 2 mg / mL, approximately 3 mg / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL, approximately 7 mg / mL, approximately 8 mg / mL, approximately 9 mg / mL, approximately 10 mg / mL, approximately 12 mg / mL, approximately 14 mg / mL, approximately 15 mg / mL, approximately 16 mg / mL, approximately 18 mg / mL, approximately 20 mg / mL, approximately 22 mg / mL, approximately 24 mg / mL, approximately 26 mg / mL, approximately 28 mg / mL, approximately 30 mg / mL, approximately 35 mg / mL, approximately 40 mg / mL, approximately 50 mg / mL, approximately 60 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 140 mg / mL, approximately 160 mg / mL, approximately 180 mg / mL, or approximately 200 mg / mL.

[0328] In some embodiments, the pharmaceutical composition is prepared by diluting a concentrate containing an antisense oligomer.

[0329] In some embodiments, the antisense oligomer is approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 200 mg / mL, approximately 100 mg / mL to approximately 200 mg / mL, approximately 150 mg / mL to approximately 200 mg / mL, approximately 180 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 150 mg / mL, approximately 35 mg / mL to approximately 150 mg / mL, approximately 40 mg / mL to approximately 150 mg / mL, approximately 50 mg / mL to approximately 150 mg / mL, approximately 60 mg / mL to approximately 150 mg / mL, approximately It is present in the anonymized material at concentrations of approximately 80 mg / mL to 150 mg / mL, approximately 100 mg / mL to 150 mg / mL, approximately 30 mg / mL to 100 mg / mL, approximately 35 mg / mL to 100 mg / mL, approximately 40 mg / mL to 100 mg / mL, approximately 50 mg / mL to 100 mg / mL, approximately 60 mg / mL to 100 mg / mL, approximately 80 mg / mL to 100 mg / mL, approximately 30 mg / mL to 80 mg / mL, approximately 35 mg / mL to 80 mg / mL, approximately 40 mg / mL to 80 mg / mL, approximately 60 mg / mL to 80 mg / mL, approximately 30 mg / mL to 60 mg / mL, approximately 35 mg / mL to 60 mg / mL, or approximately 40 mg / mL to 60 mg / mL.

[0330] In some embodiments, the antisense oligomer is present in the concentrate at concentrations of approximately 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 120 mg / mL, 140 mg / mL, 160 mg / mL, 180 mg / mL, or 200 mg / mL.

[0331] In some embodiments, the concentrate is phosphate-buffered. In some embodiments, the antisense oligomer is solubilized or diluted in a solution containing one or more of sodium chloride, sodium diphosphate, potassium monophosphate, or water for injection. In some embodiments, the antisense oligomer is solubilized or diluted in a solution containing sodium chloride, sodium diphosphate, potassium monophosphate, and water for injection. In some embodiments, the antisense oligomer is solubilized or diluted in an isotonic solution. In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate buffer at pH 5.8. In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate buffer (pH 6.6-7.6). In some embodiments, the pharmaceutical formulation is preservative-free. In some embodiments, the pharmaceutical formulation is suitable for intravitreal injection. In some embodiments, the pharmaceutical formulation is packaged in single-use vials.

[0332] In some embodiments, the pharmaceutical composition is assembled into a kit comprising: (i) a concentrate containing an antisense oligomer (ASO), wherein the ASO contains a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 6-275 or 280-299; (ii) a diluent, wherein the concentrate is miscible with the diluent; and (iii) instructions for diluting the concentrate with the diluent.

[0333] In some embodiments, the pharmaceutical composition is assembled into a kit comprising: (i) a concentrate containing an antisense oligomer (ASO), wherein the ASO contains a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 6-275 or 280-299; (ii) a diluent, wherein the concentrate is miscible with the diluent; and (iii) instructions for diluting the concentrate with the diluent.

[0334] In some embodiments, the antisense oligomer is approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 200 mg / mL, approximately 100 mg / mL to approximately 200 mg / mL, approximately 150 mg / mL to approximately 200 mg / mL, approximately 180 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 150 mg / mL, approximately 35 mg / mL to approximately 150 mg / mL, approximately 40 mg / mL to approximately 150 mg / mL, approximately 50 mg / mL to approximately 150 mg / mL, approximately 60 mg / mL to approximately 150 mg / mL, approximately It is present in the anonymized material at concentrations of approximately 80 mg / mL to 150 mg / mL, approximately 100 mg / mL to 150 mg / mL, approximately 30 mg / mL to 100 mg / mL, approximately 35 mg / mL to 100 mg / mL, approximately 40 mg / mL to 100 mg / mL, approximately 50 mg / mL to 100 mg / mL, approximately 60 mg / mL to 100 mg / mL, approximately 80 mg / mL to 100 mg / mL, approximately 30 mg / mL to 80 mg / mL, approximately 35 mg / mL to 80 mg / mL, approximately 40 mg / mL to 80 mg / mL, approximately 60 mg / mL to 80 mg / mL, approximately 30 mg / mL to 60 mg / mL, approximately 35 mg / mL to 60 mg / mL, or approximately 40 mg / mL to 60 mg / mL.

[0335] In some embodiments, the antisense oligomer is present in the concentrate at concentrations of approximately 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 120 mg / mL, 140 mg / mL, 160 mg / mL, 180 mg / mL, or 200 mg / mL.

[0336] In some embodiments, the concentrate is phosphate-buffered. In some embodiments, the diluent is a solution containing one or more of sodium chloride, sodium diphosphate, potassium monophosphate, or water for injection. In some embodiments, the diluent is a solution containing sodium chloride, sodium diphosphate, potassium monophosphate, and water for injection. In some embodiments, the diluent contains an isotonic solution. In some embodiments, the diluent contains a phosphate buffer with a pH of at least 5.8. In some embodiments, the diluent contains a phosphate buffer (pH 6.6-7.6). In some embodiments, the concentrate or diluent does not contain a preservative.

[0337] In some embodiments, the diluent is a solution comprising one or more of sodium chloride, sodium diphosphate, potassium monophosphate, or water for injection. In some embodiments, the diluent is a solution comprising sodium chloride, sodium diphosphate, potassium monophosphate, and water for injection. In some embodiments, the diluent is an isotonic solution. In some embodiments, the diluent is a phosphate buffer with a pH of at least 5.8. In some embodiments, the diluent is a phosphate buffer (pH 6.6-7.6). In some embodiments, the concentrate or diluent does not consist of a preservative.

[0338] In some embodiments, instructions for diluting the concentrate with a diluent include instructions for diluting or solubilizing the ASO in the diluent to a concentration of about 2 mg / mL to 200 mg / mL.

[0339] In some embodiments, instructions for diluting the concentrate with a diluent describe diluting or solubilizing the ASO in the diluent to approximately 5 mg / mL to approximately 200 mg / mL, approximately 10 mg / mL to approximately 200 mg / mL, approximately 15 mg / mL to approximately 200 mg / mL, approximately 20 mg / mL to approximately 200 mg / mL, approximately 25 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 2 00mg / mL, about 100mg / mL to about 200mg / mL, about 150mg / mL to about 200mg / mL, about 180mg / mL to about 200mg / mL, 2mg / mL to about 150mg / mL, about 5mg / mL to about 150mg / mL, about 10mg / mL to about 150mg / mL, about 1 5mg / mL to about 150mg / mL, about 20mg / mL to about 150mg / mL, about 25mg / mL to about 150mg / mL, about 30mg / mL to about 150mg / mL, about 35mg / mL to about 150mg / mL, about 40mg / mL to about 150mg / mL, about 50mg / mL to about 150 mg / mL, about 60 mg / mL to about 150 mg / mL, about 80 mg / mL to about 150 mg / mL, about 100 mg / mL to about 150 mg / mL, 2 mg / mL to about 100 mg / mL, about 5 mg / mL to about 100 mg / mL, about 10 mg / mL to about 100 mg / mL, about 15 mg / mL mL~about 100mg / mL, about 20mg / mL~about 100mg / mL, about 25mg / mL~about 100mg / mL, about 30mg / mL~about 100mg / mL, about 35mg / mL~about 100mg / mL, about 40mg / mL~about 100mg / mL, about 50mg / mL~about 100mg / m L, about 60 mg / mL to about 100 mg / mL, about 80 mg / mL to about 100 mg / mL, 2 mg / mL to about 80 mg / mL, about 5 mg / mL to about 80 mg / mL, about 10 mg / mL to about 80 mg / mL, about 15 mg / mL to about 80 mg / mL, about 20 mg / mL to about 80 mg / m L, about 25 mg / mL to about 80 mg / mL, about 30 mg / mL to about 80 mg / mL, about 35 mg / mL to about 80 mg / mL, about 40 mg / mL to about 80 mg / mL, about 60 mg / mL to about 80 mg / mL, 2 mg / mL to about 60 mg / mL, about 5 mg / mL to about 60 mg / mL,Includes instructions for adjusting concentrations to approximately 10 mg / mL to 60 mg / mL, 15 mg / mL to 60 mg / mL, 20 mg / mL to 60 mg / mL, 25 mg / mL to 60 mg / mL, 30 mg / mL to 60 mg / mL, 35 mg / mL to 60 mg / mL, 40 mg / mL to 60 mg / mL, 2 mg / mL to 40 mg / mL, 5 mg / mL to 40 mg / mL, 10 mg / mL to 40 mg / mL, 15 mg / mL to 40 mg / mL, 20 mg / mL to 40 mg / mL, or 25 mg / mL to 40 mg / mL.

[0340] In some embodiments, instructions for diluting the concentrate with a diluent describe diluting or solubilizing the antisense oligomer in the diluent to approximately 2 mg / mL, approximately 3 mg / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL, approximately 7 mg / mL, approximately 8 mg / mL, approximately 9 mg / mL, approximately 10 mg / mL, approximately 12 mg / mL, approximately 14 mg / mL, approximately 15 mg / mL, approximately 16 mg / mL, approximately 18 mg / mL, and approximately Includes instructions for adjusting concentrations to 20 mg / mL, approximately 22 mg / mL, approximately 24 mg / mL, approximately 26 mg / mL, approximately 28 mg / mL, approximately 30 mg / mL, approximately 35 mg / mL, approximately 40 mg / mL, approximately 50 mg / mL, approximately 60 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 140 mg / mL, approximately 160 mg / mL, approximately 180 mg / mL, or approximately 200 mg / mL.

[0341] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 90% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 6-275 or 280-299.

[0342] In some embodiments, the antisense oligomer consists of a nucleotide sequence having at least 90% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 6-275 or 280-299.

[0343] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 36, 236, 242, 250, 280-283, 288, and 290-292.

[0344] In some embodiments, the antisense oligomer consists of a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 36, 236, 242, 250, 280-283, 288, and 290-292.

[0345] In some embodiments, the antisense oligomer includes a modification of the backbone, including a phosphorothioate bond or a phosphorodiamidate bond. In some embodiments, the antisense oligomer includes a phosphorodiamidate morpholino, roq nucleic acid, peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-ON-methylacetamide (2'-NMA), or a 2'-O-methoxyethyl moiety. In some embodiments, the antisense oligomer includes at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomer includes 5'-methylcytosine (5'-MeC). In some embodiments, each cytosine in the antisense oligomer is 5'-methylcytosine (5'-MeC). In some embodiments, the antisense oligomer includes 5'-methyluracil (5'-MeU).

[0346] In some embodiments, the antisense oligomer consists of a modified skeleton including a phosphorothioate bond or a phosphorodiamidate bond. In some embodiments, the antisense oligomer consists of a phosphorodiamidate morpholino, roq nucleic acid, peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-ON-methylacetamide (2'-NMA), or a 2'-O-methoxyethyl moiety. In some embodiments, the antisense oligomer consists of at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomer consists of 5'-methylcytosine (5'-MeC). In some embodiments, each cytosine in the antisense oligomer is 5'-methylcytosine (5'-MeC). In some embodiments, the antisense oligomer consists of 5'-methyluracil (5'-MeU).

[0347] In some embodiments, each cytosine or thymidine in the antisense oligomer is 5'-methyluracil (5'-MeU). In some embodiments, the antisense oligomer consists of phosphorothioate bonds. In some embodiments, each nucleoside bond in the ASO is a phosphorothioate bond. In some embodiments, the antisense oligomer consists of loc nucleic acid (LNA).

[0348] In some embodiments, the antisense oligomer is composed of 8-50 nucleic acid bases, 8-40 nucleic acid bases, 8-35 nucleic acid bases, 8-30 nucleic acid bases, 8-25 nucleic acid bases, 8-20 nucleic acid bases, 8-15 nucleic acid bases, 9-50 nucleic acid bases, 9-40 nucleic acid bases, 9-35 nucleic acid bases, 9-30 nucleic acid bases, 9-25 nucleic acid bases, 9-20 nucleic acid bases, 9-15 nucleic acid bases, 10-50 nucleic acid bases, 10-40 nucleic acid bases, 10-35 nucleic acid bases, and 10-30 It consists of 10 nucleic acid bases, 10-25 nucleic acid bases, 10-20 nucleic acid bases, 10-15 nucleic acid bases, 11-50 nucleic acid bases, 11-40 nucleic acid bases, 11-35 nucleic acid bases, 11-30 nucleic acid bases, 11-25 nucleic acid bases, 11-20 nucleic acid bases, 11-15 nucleic acid bases, 12-50 nucleic acid bases, 12-40 nucleic acid bases, 12-35 nucleic acid bases, 12-30 nucleic acid bases, 12-25 nucleic acid bases, 12-20 nucleic acid bases, or 12-15 nucleic acid bases.

[0349] In some embodiments, the antisense oligomer is composed of 8-50 nucleic acid bases, 8-40 nucleic acid bases, 8-35 nucleic acid bases, 8-30 nucleic acid bases, 8-25 nucleic acid bases, 8-20 nucleic acid bases, 8-15 nucleic acid bases, 9-50 nucleic acid bases, 9-40 nucleic acid bases, 9-35 nucleic acid bases, 9-30 nucleic acid bases, 9-25 nucleic acid bases, 9-20 nucleic acid bases, 9-15 nucleic acid bases, 10-50 nucleic acid bases, 10-40 nucleic acid bases, 10-35 nucleic acid bases, and 10-30 It consists of 10 nucleic acid bases, 10-25 nucleic acid bases, 10-20 nucleic acid bases, 10-15 nucleic acid bases, 11-50 nucleic acid bases, 11-40 nucleic acid bases, 11-35 nucleic acid bases, 11-30 nucleic acid bases, 11-25 nucleic acid bases, 11-20 nucleic acid bases, 11-15 nucleic acid bases, 12-50 nucleic acid bases, 12-40 nucleic acid bases, 12-35 nucleic acid bases, 12-30 nucleic acid bases, 12-25 nucleic acid bases, 12-20 nucleic acid bases, or 12-15 nucleic acid bases.

[0350] In some embodiments, the antisense oligomer has the following chemical structure:

[0351] [ka] [ka] [ka] It has one of the following.

[0352] In some embodiments, the antisense oligomer has the following chemical structure:

[0353] [ka] [ka] [ka] It contains one of the following, or a pharmaceutically acceptable salt thereof.

[0354] This specification describes the use of antisense oligomers for manufacturing a pharmaceutical to treat a disease or condition characterized by reduced OPA1 protein expression or function in a human subject requiring such treatment, or to reduce the likelihood of the subject developing a disease or condition, wherein the pharmaceutical is administered to one eye of the subject in a dose of about 0.005 mg to about 20 mg, and the antisense oligomer comprises a sequence having at least 80% sequence identity with one of SEQ ID NOs: 6-275 or 280-299.

[0355] This specification includes, in some embodiments, the use of antisense oligomers to manufacture a pharmacopoeia for treating a disease or condition characterized by reduced OPA1 protein expression or function in a human subject requiring such treatment, or for reducing the likelihood of the subject developing the disease or condition, wherein the pharmacopoeia is administered to one eye of the subject in a dose of about 0.005 mg to about 20 mg, and the antisense oligomer consists of a sequence having at least 80% sequence identity with one of SEQ ID NOs: 6-275 or 280-299.

[0356] In some embodiments, the pharmaceutical is an antisense oligomer in the following concentrations: approximately 0.005 mg to approximately 15 mg, approximately 0.005 mg to approximately 10 mg, approximately 0.005 mg to approximately 5 mg, approximately 0.005 mg to approximately 1 mg, approximately 0.01 mg to approximately 15 mg, approximately 0.01 mg to approximately 10 mg, approximately 0.01 mg to approximately 5 mg, approximately 0.01 mg to approximately 2.5 mg, approximately 0.01 mg to approximately 1.0 mg, approximately 0.01 mg to approximately 0.5 mg, approximately 0.01 mg to approximately 0.25 mg, approximately 0.01 mg to approximately 0.1 mg, and approximately 0.01 mg to approximately 0.05 mg. This includes administering the drug to one eye of the subject in doses of mg, approximately 0.05 mg to 10 mg, approximately 0.05 mg to 5 mg, approximately 0.05 mg to 2.5 mg, approximately 0.05 mg to 1.0 mg, approximately 0.05 mg to 0.5 mg, approximately 0.05 mg to 0.25 mg, approximately 0.05 mg to 0.1 mg, approximately 0.1 mg to 5 mg, approximately 0.1 mg to 2.5 mg, approximately 0.1 mg to 1.5 mg, approximately 0.1 mg to 1.0 mg, approximately 0.1 mg to 0.5 mg, or approximately 0.1 mg to 0.25 mg.

[0357] In some embodiments, the pharmaceutical product is an antisense oligomer in the following concentrations: approximately 0.005 mg, approximately 0.01 mg, approximately 0.05 mg, approximately 0.1 mg, approximately 0.2 mg, approximately 0.3 mg, 0.4 mg, approximately 0.5 mg, approximately 0.6 mg, approximately 0.7 mg, approximately 0.8 mg, approximately 0.9 mg, approximately 1.0 mg, approximately 1.1 mg, approximately 1.2 mg, approximately 1.3 mg, approximately 1.4 mg, approximately 1.5 mg, and approximately 1. The drug is administered to one eye of the patient in doses of 75 mg, approximately 2.0 mg, approximately 2.25 mg, approximately 2.5 mg, approximately 2.75 mg, approximately 3 mg, approximately 3.5 mg, approximately 4.0 mg, approximately 4.5 mg, approximately 5.0 mg, approximately 5.5 mg, approximately 6.0 mg, approximately 7.0 mg, approximately 8.0 mg, approximately 9.0 mg, approximately 10 mg, approximately 12.5 mg, approximately 15 mg, approximately 17.5 mg, or approximately 20 mg.

[0358] In some embodiments, the pharmaceutical is an antisense oligomer in an amount of approximately 0.1 mg to 1.5 mg, approximately 0.1 mg to 1.4 mg, approximately 0.1 mg to 1.2 mg, approximately 0.1 mg to 1.0 mg, approximately 0.1 mg to 0.8 mg, approximately 0.1 mg to 0.7 mg, approximately 0.1 mg to 0.5 mg, approximately 0.1 mg to 0.3 mg, approximately 0.2 mg to 1.5 mg, and approximately 0.2 mg ~ about 1.4mg, about 0.2mg - about 1.2mg, about 0.2mg - about 1.0mg, about 0.2mg - about 0.8mg, about 0.2mg - about 0.7mg, about 0.2mg - about 0.5 mg, about 0.3mg to about 1.5mg, about 0.3mg to about 1.4mg, about 0.3mg to about 1.2mg, about 0.3mg to about 1.0mg, about 0.3mg to about 0.8mg, about 0.3m g ~ about 0.7mg, about 0.3mg - about 0.5mg, about 0.5mg - about 1.5mg, about 0.5mg - about 1.4mg, about 0.5mg - about 1.2mg, about 0.5mg - about 1.0m g, about 0.5mg to about 0.8mg, about 0.5mg to about 0.7mg, about 0.7mg to about 1.5mg, about 0.7mg to about 1.4mg, about 0.7mg to about 1.2mg, about 0.7mg The drug is administered to one eye of the patient in the following doses: approximately 1.0 mg, 0.8 mg to 1.5 mg, 0.8 mg to 1.4 mg, 0.8 mg to 1.2 mg, 0.8 mg to 1.0 mg, 1.0 mg to 1.5 mg, 1.0 mg to 1.4 mg, 1.0 mg to 1.2 mg, 1.2 mg to 1.5 mg, or 1.2 mg to 1.4 mg.

[0359] In some embodiments, the pharmaceutical is administered to one eye of the subject in doses of approximately 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, or 1.5 mg of the antisense oligomer. In some cases, the method involves administering the pharmaceutical composition to one eye of the subject in doses of approximately 0.1 mg to 1.2 mg.

[0360] Combination therapy In some embodiments, the ASOs disclosed herein may be used in combination with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents may include small molecules. For example, one or more additional therapeutic agents may include the small molecules described in WO2016128343A1, WO2017053982A1, WO2016196386A1, WO201428459A1, WO201524876A2, WO2013119916A2, and WO2014209841A2. These documents are incorporated herein by reference in their entirety. In some embodiments, the one or more additional therapeutic agents include ASOs that may be used to correct intron retention.

[0361] Target treatment Any of the compositions provided herein may be administered to an organism. “Organization” may be used interchangeably with “Subject” or “Patient.” An organism may be a mammal, such as a human, or an animal such as a non-human primate, rodent, rabbit, rat, mouse, horse, donkey, goat, cat, dog, cow, pig, or sheep. In some embodiments, the organism is a human. In some embodiments, the organism is a fetus, embryo, or child. In other embodiments, the organism may be another eukaryote, such as a plant. In some embodiments, the compositions provided herein are administered to cells ex vivo.

[0362] In some embodiments, the compositions provided herein are administered to an individual as a method for treating a disease or disorder. In some embodiments, the individual has a genetic disorder, such as one of the diseases described herein. In some embodiments, the individual is at risk of having a disease, such as one of the diseases described herein. In some embodiments, the individual is at high risk of having a disease or disorder caused by a deficiency in the amount or activity of a protein. If the individual is at “high risk” of having a disease or disorder caused by a deficiency in the amount or activity of a protein, the method involves prophylactic or preventive treatment. For example, an individual may be at high risk of having such a disease or disorder because of a family history of the disease. Typically, an individual at high risk of having such a disease or disorder would benefit from preventive treatment (e.g., by preventing or delaying the onset or progression of the disease or disorder). In embodiments, a fetus is treated in utero, for example, by administering the ASO composition directly or indirectly to the fetus (e.g., via the mother).

[0363] In some cases, the pharmaceutical compositions and methods in question are applicable to the treatment of conditions or diseases associated with OPA1 deficiency. In some cases, the pharmaceutical compositions and methods in question are applicable to the treatment of eye diseases or conditions. In some cases, the pharmaceutical compositions and methods in question are applicable to the treatment of type 1 optic atrophy, autosomal dominant optic atrophy (ADOA), ADOA-plus syndrome, mitochondrial dysfunction, glaucoma, normal-tension glaucoma, Charcot-Marie-Tooth disease, mitochondrial dysfunction, diabetic retinopathy, age-related macular degeneration, retinal ganglion cell death, mitochondrial fission-mediated mitochondrial dysfunction, progressive extraocular muscle palsy, hearing loss, ataxia, motor neuropathy, sensory neuropathy, myopathy, Beale syndrome, brain dysfunction, encephalopathy, peripheral neuropathy, fatal infantile mitochondrial encephalomyopathy, hypertrophic cardiomyopathy, spastic ataxia syndrome, sensorimotor peripheral neuropathy, hypotonia, gastrointestinal motility disorders and dysphagia, and optic nerve It is applicable to the treatment of atrophy, optic nerve atrophy plus syndrome, mitochondrial DNA depletion syndrome14, late-onset cardiomyopathy, diabetic cardiomyopathy, Alzheimer's disease, focal segmental glomerulosclerosis, kidney disease, Huntington's disease, cognitive decline associated with healthy aging, prion disease, late-onset dementia and parkinsonism, mitochondrial myopathy, Leigh syndrome, Friedreich's ataxia, Parkinson's disease, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), pyruvate dehydrogenase complex deficiency, chronic kidney disease, Leber's hereditary optic neuropathy, obesity, age-related systemic neurodegeneration, skeletal muscle atrophy, ischemic injury of the heart and brain, or massive hepatocyte apoptosis.

[0364] In some embodiments, the disease or condition includes type 1 optic atrophy. In some embodiments, the disease or condition includes autosomal dominant optic atrophy (ADOA).

[0365] In some embodiments, the subject is characterized by having (i) a heterozygous OPA1 gene variant, (ii) a clear ocular medium to enable proper visualization of the vitreous humor and fundus and to achieve proper quality of all ophthalmic evaluations, (iii) an AREDS clinical lens standard with posterior subcapsular (PSC) opacity <1, (iv) each eye administered with the pharmaceutical composition individually having a BCVA EDTRS letter score of ≥35 and ≤70, or (v) any combination of (i) to (viii).

[0366] In some embodiments, subjects meet the following criteria: (1) do not have a gain-of-function mutation in the OPA1 gene, or a compound heterozygous or homozygous pathogenic or pathogenic mutation; (2) do not have a benign variant or only a potentially benign variant in the OPA1 gene; (3) do not have extraocular phenotypic expression of (symptomatic) ADOA (ADOA-plus); (4) have never been diagnosed with Beer syndrome; (5) do not have a known pathogenic mutation in another gene involved in optic nerve atrophy or retinal disease; (6) do not have diabetic retinopathy that may lead to proliferative diabetic retinopathy, diabetic macular edema, or optic neuropathy; (7) do not have any eye conditions or a history of any eye conditions in either eye; (8) have no history of intraocular surgery, including refractive surgery, or corneal surgery in either eye within 12 weeks prior to administration; (9) have no history of retinal photocoagulation; (10) have no history of retinal vein occlusion. (11) Not considered at risk of uveitis or eye infection due to having had an active relapse of non-infectious uveitis in either eye or an episode of infectious uveitis or other eye infection within 12 months prior to administration, (12) Not having age-related macular degeneration in dry skin in either eye, (13) Not having high myopia (>6 diopters), (14) No history of cancer (excluding a diagnosis of successfully treated basal cell carcinoma, squamous cell carcinoma, or cervical intraepithelial neoplasia), (15) Not having taken or ingested any drug or treatment that may cause or could cause optic neuropathy, (16) No history of nutritional deficiencies (including B12 and / or folic acid deficiency), or no known serum B12 or folic acid deficiency, not having undergone bariatric surgery, or (17) Any combination of (1) to (16).

[0367] In some embodiments, a method for treating a subject having a disease or condition, or for reducing the likelihood of a subject developing a disease or condition, comprises administering a pharmaceutical composition comprising an antisense oligomer to the subject, wherein the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity with a sequence described in any one of SEQ ID NOs. 6-275 or 280-299, and the method comprises administering the pharmaceutical composition to one eye of the subject in a dose of about 0.005-20 mg of the antisense oligomer.

[0368] In some embodiments, a method for treating a subject having a disease or condition, or for reducing the likelihood of a subject developing a disease or condition, comprises administering a pharmaceutical composition comprising an antisense oligomer to the subject, wherein the antisense oligomer comprises a nucleotide sequence having at least 90% sequence identity with a sequence described in any one of SEQ ID NOs. 6-275 or 280-299, and the method comprises administering the pharmaceutical composition to one eye of the subject in a dose of about 0.005-20 mg of the antisense oligomer.

[0369] In some embodiments, a method for treating a subject having a disease or condition, or for reducing the likelihood of a subject developing a disease or condition, comprises administering a pharmaceutical composition comprising an antisense oligomer to the subject, wherein the antisense oligomer comprises a nucleotide sequence having at least 100% sequence identity with any one of the sequences described in SEQ ID NOs. 6-275 or 280-299, and the method comprises administering the pharmaceutical composition to one eye of the subject in a dose of about 0.005-20 mg of the antisense oligomer.

[0370] In some embodiments, the method involves an antisense oligomer of approximately 0.005 mg to 15 mg, approximately 0.005 mg to 10 mg, approximately 0.005 mg to 5 mg, approximately 0.005 mg to 1 mg, approximately 0.01 mg to 15 mg, approximately 0.01 mg to 10 mg, approximately 0.01 mg to 5 mg, approximately 0.01 mg to 2.5 mg, approximately 0.01 mg to 1.0 mg, approximately 0.01 mg to 0.5 mg, approximately 0.01 mg to 0.25 mg, approximately 0.01 mg to 0.1 mg, approximately 0.01 mg to 0.05 mg. This includes administering the pharmaceutical composition to one eye of the subject in doses of approximately 0.05 mg to 10 mg, approximately 0.05 mg to 5 mg, approximately 0.05 mg to 2.5 mg, approximately 0.05 mg to 1.0 mg, approximately 0.05 mg to 0.5 mg, approximately 0.05 mg to 0.25 mg, approximately 0.05 mg to 0.1 mg, approximately 0.1 mg to 5 mg, approximately 0.1 mg to 2.5 mg, approximately 0.1 mg to 1.5 mg, approximately 0.1 mg to 1.0 mg, approximately 0.1 mg to 0.5 mg, or approximately 0.1 mg to 0.25 mg.

[0371] In some embodiments, the method involves administering approximately 0.005 mg, 0.01 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, and 1.75 mg of antisense oligomers. This includes administering the pharmaceutical composition to one eye of the target in doses of approximately 2.0 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 5.5 mg, 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, or 20 mg.

[0372] In some embodiments, the method involves antisense oligomers in amounts of approximately 0.1 mg to 1.5 mg, 0.1 mg to 1.4 mg, 0.1 mg to 1.2 mg, 0.1 mg to 1.0 mg, 0.1 mg to 0.8 mg, 0.1 mg to 0.7 mg, 0.1 mg to 0.5 mg, 0.1 mg to 0.3 mg, 0.2 mg to 1.5 mg, and 0.2 mg. ~1.4mg, 0.2mg~1.2mg, 0.2mg~1.0mg, 0.2mg~0.8mg, 0.2mg~0.7mg, 0.2mg~0.5mg, approx. 0.3mg to about 1.5mg, about 0.3mg to about 1.4mg, about 0.3mg to about 1.2mg, about 0.3mg to about 1.0mg, about 0.3mg to about 0.8mg, about 0.3mg to about 0. 7mg, about 0.3mg to about 0.5mg, about 0.5mg to about 1.5mg, about 0.5mg to about 1.4mg, about 0.5mg to about 1.2mg, about 0.5mg to about 1.0mg, about 0.5 mg ~ about 0.8mg, about 0.5mg - about 0.7mg, about 0.7mg - about 1.5mg, about 0.7mg - about 1.4mg, about 0.7mg - about 1.2mg, about 0.7mg - about 1.0mg This includes administering the pharmaceutical composition to one eye of the subject in doses of approximately 0.8 mg to 1.5 mg, approximately 0.8 mg to 1.4 mg, approximately 0.8 mg to 1.2 mg, approximately 0.8 mg to 1.0 mg, approximately 1.0 mg to 1.5 mg, approximately 1.0 mg to 1.4 mg, approximately 1.0 mg to 1.2 mg, approximately 1.2 mg to 1.5 mg, or approximately 1.2 mg to 1.4 mg.

[0373] In some embodiments, the method involves administering the pharmaceutical composition to one eye of the subject in doses of approximately 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, or 1.5 mg of the antisense oligomer. In some cases, the method involves administering the pharmaceutical composition to one eye of the subject in doses of approximately 0.1 mg to 1.2 mg.

[0374] In some embodiments, the method involves using an antisense oligomer in quantities of approximately 5 μL to 250 μL, 10 μL to 250 μL, 20 μL to 250 μL, 30 μL to 250 μL, 40 μL to 250 μL, 50 μL to 250 μL, 60 μL to 250 μL, 70 μL to 250 μL, 80 μL to 250 μL, 100 μL to 250 μL, 120 μL to 250 μL, 150 μL to 250 μL, 160 μL to 250 μL, 180 μL to 500 μL, 200 μL to 250 μL, 220 μL to 250 μL, and 5 μL to 22 0 μL, approximately 10 μL to approximately 220 μL, approximately 20 μL to approximately 220 μL, approximately 30 μL to approximately 220 μL, approximately 40 μL to approximately 220 μL, approximately 50 μL to approximately 220 μL, approximately 60 μL to approximately 220 μL, approximately 70 μL to approximately 220 μL, approximately 80 μL to approximately 220 μL, approximately 100 μL to approximately 220 μL, approximately 1 20μL to approx. 220μL, approx. 150μL to approx. 220μL, approx. 160μL to approx. 220μL, approx. 180μL to approx. 220μL, approx. 5μL to approx. 20 0μL, about 10μL to about 200μL, about 20μL to about 200μL, about 30μL to about 200μL, about 40μL to about 200μL, about 50μL to Approximately 200μL, approximately 60μL to approximately 200μL, approximately 70μL to approximately 200μL, approximately 80μL to approximately 200μL, approximately 100μL to approximately 200μL, approximately 120μL ~ approx. 200μL, approx. 150μL ~ approx. 200μL, approx. 160μL ~ approx. 200μL, approx. 180μL ~ approx. 200μL, approx. 5μL ~ approx. 1 80 μL, approximately 10 μL to approximately 180 μL, approximately 20 μL to approximately 180 μL, approximately 30 μL to approximately 180 μL, approximately 40 μL to approximately 180 μL, approximately 50 μL to approximately 180 μL, approximately 60 μL to approximately 180 μL, approximately 70 μL to approximately 180 μL, approximately 80 μL to approximately 180 μL, approximately 100 μL to approximately 180 μL, approximately 120μL ~ Approx. 180μL, Approx. 150μL ~ Approx. 180μL, Approx. 5μL ~ Approx. 150μL, Approx. 10μL ~ Approx. 150μL, Approx. 20μL ~ Approx. 150 μL, approximately 30 μL to approximately 150 μL, approximately 40 μL to approximately 150 μL, approximately 50 μL to approximately 150 μL, approximately 60 μL to approximately 150 μL, approximately 70 μL to approximately 150 μL, approximately 80 μL to approximately 150 μL, approximately 100 μL to approximately 150 μL, approximately 120 μL to approximately 150 μL, approximately 5 μL to approximately 150 μL, approximately 10 μL to approximately 120 μL, approximately 20 μL to approximately 120 μL, approximately 30 μL to approximately 120 μL, approximately 40 μL to approximately 120 μL, approximately 50 μL to approximately 120 μL,Approximately 60μL to 120μL, approximately 70μL to 120μL, approximately 80μL to 120μL, approximately 100μL to 120μL, approximately 5μL to 100μL, approximately 10μL to 100μL, approximately 20μL to 100μL, approximately 30μL to 100μL, approximately 40μL to 100μL, approximately 50μL to 100μL, approximately 60μL to 100μL, approximately 70μL to 100μL, approximately 80μL to 100μL, approximately 5μL to 80 The method involves administering a pharmaceutical composition to one eye of the subject in a volume of approximately μL, approximately 10 μL to approximately 80 μL, approximately 20 μL to approximately 80 μL, approximately 30 μL to approximately 80 μL, approximately 40 μL to approximately 80 μL, approximately 50 μL to approximately 80 μL, approximately 60 μL to approximately 80 μL, approximately 5 μL to approximately 60 μL, approximately 10 μL to approximately 60 μL, approximately 20 μL to approximately 60 μL, approximately 30 μL to approximately 60 μL, approximately 40 μL to approximately 60 μL, or approximately 50 μL to approximately 60 μL.

[0375] In some embodiments, the method includes administering the pharmaceutical composition to one eye of the subject in a volume of approximately 5 μL, approximately 8 μL, approximately 10 μL, approximately 12 μL, approximately 15 μL, approximately 18 μL, approximately 20 μL, approximately 25 μL, approximately 28 μL, approximately 30 μL, approximately 35 μL, approximately 40 μL, approximately 45 μL, approximately 48 μL, approximately 50 μL, approximately 55 μL, approximately 60 μL, approximately 65 μL, approximately 70 μL, approximately 75 μL, approximately 80 μL, approximately 90 μL, approximately 100 μL, approximately 120 μL, approximately 150 μL, approximately 160 μL, approximately 180 μL, approximately 200 μL, approximately 220 μL, or approximately 250 μL. In some cases, the method includes administering the pharmaceutical composition to one eye of the subject in a volume of approximately 50 μL.

[0376] In some embodiments, the method includes administering the pharmaceutical composition to both the left and right eyes of the subject. In some embodiments, the method includes administering the same dose of the pharmaceutical composition to both the left and right eyes of the subject. In some embodiments, the method includes administering different doses of the pharmaceutical composition to the left and right eyes of the subject. In some embodiments, the method includes administering the pharmaceutical composition to at least one eye of the subject.

[0377] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 90% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 6-275 or 280-299.

[0378] In some embodiments, the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 36, 236, 242, 250, 280-283, 288, and 290-292.

[0379] In some embodiments, administration includes administering a multi-dose pharmaceutical composition to a human subject. In some embodiments, administration includes administering an initial dose of the pharmaceutical composition to a human subject, and a subsequent dose of the pharmaceutical composition to a human subject. In some embodiments, the subsequent dose is less than the previous dose, in accordance with instructions that the previous dose is not tolerated. In some embodiments, the subsequent dose is the same as the previous dose, in accordance with instructions that the previous dose is tolerated. In some embodiments, the subsequent dose is greater than the previous dose, in accordance with instructions that the previous dose is tolerated. In some embodiments, the subsequent dose is the same as the previous dose, in accordance with instructions that the previous dose is effective. In some embodiments, the subsequent dose is less than the previous dose, in accordance with instructions that the previous dose is effective. In some embodiments, the subsequent dose is greater than the previous dose, in accordance with instructions that the previous dose is not effective.

[0380] In some embodiments, the pharmaceutical composition comprises a liquid composition. In some embodiments, the method comprises administering the pharmaceutical composition as a bolus injection over a period of 1 to 60 minutes, 1 to 50 minutes, 1 to 40 minutes, 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes, 1 to 5 minutes, or 1 to 3 minutes.

[0381] In some embodiments, the method includes administering the pharmaceutical composition as a bolus injection. In some embodiments, the antisense oligomer is solubilized or diluted in a solution containing one or more of sodium chloride, sodium diphosphate, potassium monophosphate, or water for injection. In some embodiments, the antisense oligomer is solubilized or diluted in a solution containing one or more of sodium chloride, sodium diphosphate, potassium monophosphate, and water for injection. In some embodiments, the antisense oligomer described herein is solubilized or diluted in an isotonic solution. In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate buffer at pH 5.8. In some embodiments, the antisense oligomer is solubilized or diluted in a phosphate buffer (pH 6.6-7.6). In some embodiments, the pharmaceutical formulation does not contain preservatives.

[0382] In some embodiments, the antisense oligomer is present in the pharmaceutical composition at a concentration of approximately 2 mg / mL to approximately 200 mg / mL.

[0383] In some embodiments, the antisense oligomer is approximately 2 mg / mL to 200 mg / mL, approximately 5 mg / mL to 200 mg / mL, approximately 10 mg / mL to 200 mg / mL, approximately 15 mg / mL to 200 mg / mL, approximately 20 mg / mL to 200 mg / mL, approximately 25 mg / mL to 200 mg / mL, approximately 30 mg / mL to 200 mg / mL, approximately 35 mg / mL to 200 mg / mL, approximately 40 mg / mL to 200 mg / mL, approximately 50 mg / mL to 200 mg / mL, approximately 60 mg / mL to 200 mg / mL, and approximately 80 mg / mL to 200 mg / mL. L, about 100mg / mL to about 200mg / mL, about 150mg / mL to about 200mg / mL, about 180mg / mL to about 200mg / mL, 2mg / mL to about 150mg / mL, about 5mg / mL to about 150mg / mL, about 10mg / mL to about 150mg / mL, about 15mg / m L ~ about 150mg / mL, about 20mg / mL - about 150mg / mL, about 25mg / mL - about 150mg / mL, about 30mg / mL - about 150mg / mL, about 35mg / mL - about 150mg / mL, about 40mg / mL - about 150mg / mL, about 50mg / mL - about 150mg / m L, about 60 mg / mL to about 150 mg / mL, about 80 mg / mL to about 150 mg / mL, about 100 mg / mL to about 150 mg / mL, 2 mg / mL to about 100 mg / mL, about 5 mg / mL to about 100 mg / mL, about 10 mg / mL to about 100 mg / mL, about 15 mg / mL About 100mg / mL, about 20mg / mL to about 100mg / mL, about 25mg / mL to about 100mg / mL, about 30mg / mL to about 100mg / mL, about 35mg / mL to about 100mg / mL, about 40mg / mL to about 100mg / mL, about 50mg / mL to about 100mg / mL, Approximately 60 mg / mL to approximately 100 mg / mL, approximately 80 mg / mL to approximately 100 mg / mL, 2 mg / mL to approximately 80 mg / mL, approximately 5 mg / mL to approximately 80 mg / mL, approximately 10 mg / mL to approximately 80 mg / mL, approximately 15 mg / mL to approximately 80 mg / mL, approximately 20 mg / mL to approximately 80 mg / mL , about 25 mg / mL to about 80 mg / mL, about 30 mg / mL to about 80 mg / mL, about 35 mg / mL to about 80 mg / mL, about 40 mg / mL to about 80 mg / mL, about 60 mg / mL to about 80 mg / mL, 2 mg / mL to about 60 mg / mL, about 5 mg / mL to about 60 mg / mL,It is present in the pharmaceutical composition at concentrations of approximately 10 mg / mL to 60 mg / mL, 15 mg / mL to 60 mg / mL, 20 mg / mL to 60 mg / mL, 25 mg / mL to 60 mg / mL, 30 mg / mL to 60 mg / mL, 35 mg / mL to 60 mg / mL, 40 mg / mL to 60 mg / mL, 2 mg / mL to 40 mg / mL, 5 mg / mL to 40 mg / mL, 10 mg / mL to 40 mg / mL, 15 mg / mL to 40 mg / mL, 20 mg / mL to 40 mg / mL, or 25 mg / mL to 40 mg / mL.

[0384] In some embodiments, the antisense oligomer is present in the pharmaceutical composition at concentrations of approximately 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 22 mg / mL, 24 mg / mL, 26 mg / mL, 28 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 140 mg / mL, 160 mg / mL, 180 mg / mL, or 200 mg / mL. Autosomal dominant optic atrophy (ADOA) is the most common hereditary optic neuropathy, characterized by the loss of retinal ganglion cells. In some cases, 65–90% of ADOA cases are caused by mutations in one allele of the OPA1 gene. The OPA1 gene encodes the OPA1 protein, a mitochondrial GTPase that can play a vital role in maintaining mitochondrial structure and function. Most OPA1 mutations lead to haploinsufficiency, which can result in a reduction of approximately 50% from normal OPA1 protein levels. Approximately 1 in 30,000 people worldwide are affected, and due to the founder effect, approximately 1 in 10,000 people are affected in Denmark. ADOA can develop within the first 10 years of life. 80% of ADOA patients are symptomatic before the age of 10. The disease can cause progressive and irreversible vision loss, and up to 46% of patients are legally registered as blind.

[0385] In some embodiments, a method for treating a subject having a disease or condition, or for reducing the likelihood of a subject developing a disease or condition, comprises administering a pharmaceutical composition comprising an antisense oligomer to the subject, wherein the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity with any one of the sequences described in SEQ ID NOs: 6-275 or 280-299, the disease or condition comprises autosomal dominant optic atrophy (ADOA), and the subject is characterized by having (i) a heterozygous OPA1 gene variant, (ii) a clear ocular medium for enabling adequate visualization of the vitreous humor and fundus and achieving adequate quality of all ophthalmic evaluations, (iii) an AREDS clinical lens standard with posterior subcapsular (PSC) opacity <1, (iv) each eye administered with the pharmaceutical composition individually having BCVA EDTRS letter scores of ≥35 and ≤70, or (v) any combination of (i) to (viii).

[0386] In some embodiments, a method for treating a subject having a disease or condition, or for reducing the likelihood of a subject developing a disease or condition, comprises administering a pharmaceutical composition to the subject comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 90% sequence identity with any one of the sequences described in SEQ ID NOs: 6-275 or 280-299, the disease or condition comprises autosomal dominant optic atrophy (ADOA), and the subject is characterized by having (i) a heterozygous OPA1 gene variant, (ii) a clear ocular medium for enabling adequate visualization of the vitreous humor and fundus and achieving adequate quality of all ophthalmic evaluations, (iii) an AREDS clinical lens standard with posterior subcapsular (PSC) opacity <1, (iv) each eye administered with the pharmaceutical composition individually having BCVA EDTRS letter scores ≥35 and ≤70, or (v) any combination of (i) to (viii).

[0387] In some embodiments, a method for treating a subject having a disease or condition, or for reducing the likelihood of a subject developing a disease or condition, comprises administering a pharmaceutical composition to the subject comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 100% sequence identity with any one of the sequences described in SEQ ID NOs: 6-275 or 280-299, the disease or condition comprises autosomal dominant optic atrophy (ADOA), and the subject is characterized by having (i) a heterozygous OPA1 gene variant, (ii) a clear ocular medium for enabling proper visualization of the vitreous humor and fundus and achieving proper quality of all ophthalmic evaluations, (iii) an AREDS clinical lens standard with posterior subcapsular (PSC) opacity <1, (iv) each eye administered with the pharmaceutical composition individually having BCVA EDTRS letter scores ≥35 and ≤70, or (v) any combination of (i) to (viii).

[0388] In some embodiments, a method for treating a subject having a disease or condition, or for reducing the likelihood of a subject developing a disease or condition, comprises administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity with any one of the sequences described in SEQ ID NOs: 6-275 or 280-299, the disease or condition comprises autosomal dominant optic atrophy (ADOA), and the subject is (1) a gain-of-function mutation, or compound heterozygous or homozygous disease in the OPA1 gene. (2) No possibility of virulence or pathogenic variant, (3) No possibility of benign variant or only likely benign variant in the OPA1 gene, (4) No extraocular phenotypic expression of (symptomatic) ADOA (ADOA-plus), (5) Never been diagnosed with Beer syndrome, (6) No known pathogenic mutations in other genes involved in optic nerve atrophy or retinal disease, (7) No diabetic retinopathy that may lead to proliferative diabetic retinopathy, diabetic macular edema, or optic neuropathy, (8) No eye conditions or any other eye conditions (8) No history of the eye condition, no history of intraocular surgery, including refractive surgery, or corneal surgery in either eye within 12 weeks prior to administration, (9) No history of retinal photocoagulation, (10) No history of or presence of retinal vein occlusion, (11) Not considered at risk of uveitis or eye infection due to having had an active relapse of non-infectious uveitis in either eye, or an episode of infectious uveitis or another eye infection within 12 months prior to administration, (12) No dry age-related macular degeneration in either eye, (13) Severe myopia (14) No visual impairment (>6 diopters), no history of cancer (excluding a diagnosis of successfully treated basal cell carcinoma, squamous cell carcinoma, or cervical intraepithelial neoplasia), (15) No history of taking or ingesting any drug or treatment that may cause or could cause optic neuropathy, (16) No history of nutritional deficiencies (including B12 and / or folic acid deficiency), no known serum B12 or folic acid deficiency, no history of bariatric surgery, or (17) Any combination of (1) to (16).

[0389] In some embodiments, a method for treating a subject having a disease or condition, or for reducing the likelihood of a subject developing a disease or condition, comprises administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 90% sequence identity with one of SEQ ID NOs: 6-275 or 280-299, the disease or condition comprises autosomal dominant optic atrophy (ADOA), and the subject is (1) a gain-of-function mutant, or compound heterozygous or homozygous pathogenicity or pathogenicity of the OPA1 gene. (1) No possibility of sex variant, (2) No possibility of benign variant or only likely benign variant in the OPA1 gene, (3) No extraocular phenotypic expression of (symptomatic) ADOA (ADOA-plus), (4) Never been diagnosed with Beer syndrome, (5) No known pathogenic mutations in other genes involved in optic nerve atrophy or retinal disease, (6) No diabetic retinopathy that may lead to proliferative diabetic retinopathy, diabetic macular edema, or optic neuropathy, (7) No eye conditions or any eye conditions in either eye. (8) No history of any eye condition, no history of intraocular surgery including refractive surgery or corneal surgery in either eye within 12 weeks prior to administration, (9) No history of retinal photocoagulation, (10) No history or presence of retinal vein occlusion, (11) Not considered at risk of uveitis or eye infection due to having had an active relapse of non-infectious uveitis in either eye, or an episode of infectious uveitis or another eye infection within 12 months prior to administration, (12) No dry age-related macular degeneration in either eye, (13) No high myopia (17) Having any of the following characteristics: (1) No (>6 diopters), (14) No history of cancer (excluding a diagnosis of successfully treated basal cell carcinoma, squamous cell carcinoma, or cervical intraepithelial neoplasia), (15) Never having taken or ingested any drug or treatment that may cause or could cause optic neuropathy, (16) No history of nutritional deficiencies (including B12 and / or folic acid deficiency), or no known serum B12 or folic acid deficiency, not having undergone bariatric surgery, or (17) Any combination of (1) to (16).

[0390] In some embodiments, a method for treating a subject having a disease or condition, or for reducing the likelihood of a subject developing a disease or condition, comprises administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer comprises a nucleotide sequence having at least 100% sequence identity with any of SEQ ID NOs: 6-275 or 280-299, the disease or condition comprises autosomal dominant optic atrophy (ADOA), and the subject is (1) a gain-of-function mutant, or compound heterozygous or homozygous pathogenicity or disease in the OPA1 gene. (1) No possibility of a virulent variant, (2) No possibility of a benign variant or only a likely benign variant in the OPA1 gene, (3) No extraocular phenotypic expression of (symptomatic) ADOA (ADOA-plus), (4) Never been diagnosed with Beer syndrome, (5) No known pathogenic mutations in other genes involved in optic nerve atrophy or retinal disease, (6) No diabetic retinopathy that may lead to proliferative diabetic retinopathy, diabetic macular edema, or optic neuropathy, (7) No eye conditions or any eye conditions in either eye. (8) No history of any eye condition, no history of intraocular surgery including refractive surgery or corneal surgery in either eye within 12 weeks prior to administration, (9) No history of retinal photocoagulation, (10) No history or presence of retinal vein occlusion, (11) Not considered at risk of uveitis or eye infection due to having had an active relapse of non-infectious uveitis in either eye, or an episode of infectious uveitis or another eye infection within 12 months prior to administration, (12) No dry age-related macular degeneration in either eye, (13) No high myopia (17) Having any of the following characteristics: (1) No (>6 diopters), (14) No history of cancer (excluding a diagnosis of successfully treated basal cell carcinoma, squamous cell carcinoma, or cervical intraepithelial neoplasia), (15) Never having taken or ingested any drug or treatment that may cause or could cause optic neuropathy, (16) No history of nutritional deficiencies (including B12 and / or folic acid deficiency), or no known serum B12 or folic acid deficiency, not having undergone bariatric surgery, or (17) Any combination of (1) to (16).

[0391] In some cases, the therapeutic agent includes an oligonucleotide. In some cases, the therapeutic agent includes a vector, e.g., a viral vector, that expresses an oligonucleotide that binds to a target region of premRNA encoding a target peptide sequence. The methods provided herein may be adapted to bring a drug-encoding vector, e.g., an oligonucleotide, into contact with a cell, so that the drug binds to premRNA in the cell and modulates the processing of the premRNA. In some cases, the viral vector includes an adenovirus vector, an adeno-associated virus (AAV) vector, a lentiviral vector, a herpes simplex virus (HSV) virus vector, a retroviral vector, or any applicable viral vector. In some cases, the therapeutic agent includes a gene editing tool configured to modify a gene encoding a target peptide sequence so that a gene region encoding an inefficiently translatable region is deleted. In some cases, the gene editing tool includes a vector for gene editing based on CRISPR-Cas9, TALEN, zinc finger, or other applicable technologies, e.g., a viral vector.

[0392] The preferred route for administering the ASOs of this disclosure may vary depending on the cell type to which the ASOs are to be delivered. Several tissues and organs are affected by ADOAs, with the eye being the most significantly affected. The ASOs of this disclosure may be administered parenterally to patients, for example, by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreous injection, or intravenous injection.

[0393] In some embodiments, the pharmaceutical composition is administered via intravitreal injection. In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a small molecule. In some embodiments, the additional therapeutic agent comprises an antisense oligomer. In some embodiments, the additional therapeutic agent comprises an ophthalmic drug. In some embodiments, the additional therapeutic agent consists of an antisense oligomer. In some embodiments, the additional therapeutic agent consists of an ophthalmic drug.

[0394] In embodiments, the antisense oligonucleotide is administered together with one or more agents capable of facilitating the penetration of the antisense oligonucleotide of the subject across the blood-brain barrier by any method known in the art. For example, the delivery of an agent by administration of an adenovirus vector to motor neurons in muscle tissue is described in U.S. Patent No. 6,632,427, “Adenoviral-vector-mediated gene transfer into medullary motor neurons,” which is incorporated herein by reference. The direct delivery of a vector to the brain, e.g., the striatum, thalamus, hippocampus, or substantia nigra, is described, for example, in U.S. Patent No. 6,756,523, “Adenovirus vectors for the transfer of foreign genes into the cell of the central nervous system particularly in brain,” which is incorporated herein by reference.

[0395] In some embodiments, antisense oligonucleotides are conjugated to or linked to agents that impart desired pharmaceutical or pharmacological properties. In embodiments, antisense oligonucleotides are conjugated to substances known in the art, such as antibodies against transferrin receptors, that facilitate penetration or transport across the blood-brain barrier. In embodiments, antisense oligonucleotides are conjugated to viral vectors that enhance the effect of antisense compounds or increase transport across the blood-brain barrier. In embodiments, permeable blood-brain barrier disruption is achieved by sugars, such as mesoerythritol, xylitol, D(+)galactose, D(+)lactose, D(+)xylose, dulciitol, myo-inositol, L(-)fructose, D(-)mannitol, D(+)glucose, D(+)arabinose, D(-)arabinose, cellobiose, D(+)maltose, D(+)raffinose, L(+)rhamnose, D(+)melibiose, D(-)ribose, adonis It is supplemented by intravenous administration of toll, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-) lyxose, or amino acids such as glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine.Methods and materials for enhancing penetration across the blood-brain barrier are described, for example, in U.S. Patent No. 9,193,969, "Compositions and methods for selective delivery of oligonucleotide molecules to specific neuron types," U.S. Patent No. 4,866,042, "Method for the delivery of genetic material across the blood-brain barrier," U.S. Patent No. 6,294,520, "Material for passage through the blood-brain barrier," and U.S. Patent No. 6,936,589, "Parenteral delivery systems," each of which is incorporated herein by reference.

[0396] In some embodiments, the subject treated with the method and composition is evaluated for improvement in condition using any of the methods known and described in the Art.

[0397] How to identify additional ASOs that induce exon skipping Methods for identifying or determining ASOs that induce exon skipping of OPA1 NMD exon-containing premRNA are also within the scope of this disclosure. For example, the method may include identifying or determining ASOs that induce pseudoexon skipping of OPA1 NMD exon-containing premRNA. ASOs that improve the rate and / or degree of splicing of target introns may be identified or determined by screening for ASOs that specifically hybridize to various nucleotides within the target region of the premRNA. In some embodiments, the ASOs may block or interfere with the binding sites of splicing repressors / silencers. Any method known in the art may be used to identify (determine) ASOs that produce a desired effect (e.g., pseudoexon skipping, protein or functional RNA production) when hybridized to the target region of an exon. These methods may also be used to identify ASOs that induce exon skipping of a containing exon by binding to a target region in an intron adjacent to the containing exon, or to a target region in an uncontaining exon. Examples of possible methods are provided below.

[0398] A screening round referred to as an ASO "walk" may be performed using ASOs designed to hybridize to a target region of premRNA. For example, the ASOs used in an ASO walk may be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 3' splice site of the containing exon (e.g., the sequence portion of the exon located upstream of the target / containing exon) to approximately 100 nucleotides downstream of the 3' splice site of the target / containing exon, and / or approximately 100 nucleotides upstream of the 5' splice site of the containing exon to approximately 100 nucleotides downstream of the 5' site of the target / containing exon (e.g., the sequence portion of the exon located downstream of the target / containing exon). For example, a first ASO of 15 nucleotides in length may be designed to specifically hybridize to +6 to +20 nucleotides relative to the 3' splice site of the target / containing exon. A second ASO may be designed to specifically hybridize to +11 to +25 nucleotides relative to the 3' splice site of the target / contained exon. The ASO is designed to extend to the target region of the premRNA. In embodiments, the ASO may be more finely tiled, for example, every 1, 2, 3, or 4 nucleotides. Furthermore, the ASO may be tiled from 100 nucleotides downstream from the 5' splice site to 100 nucleotides upstream from the 3' splice site. In some embodiments, the ASO may be tiled from about 1,160 nucleotides upstream from the 3' splice site to about 500 nucleotides downstream from the 5' splice site. In some embodiments, the ASO may be tiled from about 500 nucleotides upstream from the 3' splice site to about 1,920 nucleotides downstream from the 3' splice site.

[0399] One or more ASOs, or control ASOs (ASOs with scrambled sequences, sequences not expected to hybridize to the target region), are delivered to disease-associated cell lines expressing the target premRNA (e.g., the NMD exon-containing premRNAs described herein), for example, by transfection. The exon-skipping effect of each ASO can be evaluated by any method known in the art, for example, by reverse transcriptase (RT)-PCR using primers extending to the splice junction, as described in Example 4. If, compared to cells treated with a control ASO, cells treated with an ASO show a decrease or absence of longer products in RT-PCR products produced using primers extending to the region containing the included exons (e.g., adjacent exons of the NMD exon), it is suggested that splicing of the target NMD exon has been enhanced. In some embodiments, the exon skipping efficiency (or splicing efficiency of splicing introns containing NMD exons), the ratio of spliced ​​premRNA to unspliced ​​premRNA, the splicing rate, or the degree of splicing may be improved using the ASOs described herein. The amount of protein or functional RNA encoded by the target premRNA can also be evaluated to determine whether each ASO achieved the desired effect (e.g., improved production of functional protein). Any method known in the art for evaluating and / or quantifying protein production may be used, for example, Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0400] A second round of screening, referred to as ASO "microwalks," may be carried out using ASOs designed to hybridize to a target region of premRNA. The ASOs used in ASO microwalks are tiled nucleotide by nucleotide to further refine the nucleotide acid sequence of the premRNA, resulting in exon skipping (or improved splicing of NMD exons) when hybridized with the ASO.

[0401] Regions defined by ASOs that facilitate target intron splicing are further explored in detail by ASO "microwalks," which include ASOs spaced 1nt apart, as well as longer ASOs, typically 18–25nt long.

[0402] As described above regarding ASO walks, ASO microwalks are performed by delivering one or more ASOs, or control ASOs (ASOs with scrambled sequences, sequences not expected to hybridize to the target region), to disease-associated cell lines expressing the target premRNA, for example, by transfection. The splicing-inducing effect of each ASO can be evaluated by any method known in the art, for example, by reverse transcriptase (RT)-PCR using primers extending to NMD exons, as described herein (see, e.g., Example 4). If longer products are reduced or absent in RT-PCR products produced using primers extending to NMD exons in ASO-treated cells compared to cells treated with a control ASO, it is suggested that exon skipping (or splicing of NMD exon-containing target introns) has been enhanced. In some embodiments, the exon skipping efficiency (or splicing efficiency of splicing introns containing NMD exons), the ratio of spliced ​​premRNA to unspliced ​​premRNA, the splicing rate, or the degree of splicing may be improved using the ASOs described herein. The amount of protein or functional RNA encoded by the target premRNA can also be evaluated to determine whether each ASO achieved the desired effect (e.g., improved production of functional protein). Any method known in the art for evaluating and / or quantifying protein production may be used, for example, Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0403] ASOs, which, when hybridized to the premRNA region, result in exon skipping (or enhanced splicing of introns containing NMD exons) and increased protein production, may be tested in vivo using animal models, such as transgenic mouse models with full-length human genes knocked in, or humanized mouse models of diseases. The preferred route for administering ASOs may vary depending on the disease and / or the cell type to which ASO delivery is desired. ASOs may be administered, for example, by intravitreous, intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous, or intravenous injection. After administration, the effects of ASO treatment may be determined by evaluating the cells, tissues, and / or organs of the model animals to assess splicing (e.g., efficiency, rate, degree) and protein production, for example, by methods known in the art and methods described herein. Animal models may also be any phenotypic or behavioral indicators of the disease or the severity of the disease.

[0317] Furthermore, methods for identifying or validating NMD-inducing exons in the presence of NMD inhibitors, such as cycloheximide, are also within the scope of this disclosure. An exemplary method is provided in Example 2.

[0404] Specific Embodiments Embodiment A1. A method for treating type 1 optic neuropathy in a subject requiring the treatment thereof by increasing the expression of a target protein or functional RNA by cells of the subject, wherein the cells have mRNA containing a nonsense-mediated RNA decay-inducing exon (NMD exon mRNA), the NMD exon mRNA encoding a target protein or functional RNA, and the method comprises contacting the cells of the subject with a therapeutic agent that binds to a target portion of the NMD exon mRNA encoding the target protein or functional RNA, thereby removing the nonsense-mediated RNA decay-inducing exon from the NMD exon mRNA encoding the target protein or functional RNA, thereby increasing the level of the mRNA encoding the target protein or functional RNA and increasing the expression of the target protein or functional RNA in the cells of the subject.

[0405] Embodiment A2. The method according to Embodiment A1, wherein the target protein is OPA1.

[0406] Embodiment A3. A method for increasing OPA1 protein expression in cells having mRNA encoding the OPA1 protein and containing a nonsense-mediated RNA decay-inducing exon (NMD exon mRNA), the method comprising contacting the cells with a drug that binds to a target region of the NMD exon mRNA encoding the OPA1 protein, thereby removing the nonsense-mediated RNA decay-inducing exon from the NMD exon mRNA encoding the OPA1 protein, thereby increasing the level of mRNA encoding the OPA1 protein and increasing OPA1 protein expression in the cells.

[0407] Embodiment A4. The method according to any one of Embodiments A1 to A3, wherein a nonsense-mediated RNA decay-inducing exon is removed by splicing from NMD exon mRNA encoding a target protein or functional RNA.

[0408] Embodiment A5. The method according to any one of Embodiments A1 to A4, wherein the target protein does not contain an amino acid sequence encoded by a nonsense-mediated RNA decay-inducible exon.

[0409] Embodiment A6. The method according to any one of Embodiments A1 to A5, wherein the target protein is a full-length target protein.

[0410] Embodiment A7. The method according to any one of Embodiments A1 to A6, wherein the drug is an antisense oligomer (ASO) complementary to the target portion of NMD exon mRNA.

[0411] Embodiment A8. The method according to any one of Embodiments A1 to A7, wherein the mRNA is premRNA.

[0412] Embodiment A9. The method according to any one of Embodiments A1 to A8, wherein contact comprises bringing a therapeutic agent into contact with mRNA, the mRNA being located in the nucleus of a cell.

[0413] Embodiment A10. The method according to any one of Embodiments A1 to A9, wherein the target protein or functional RNA corrects the deficiency of the target protein or functional RNA.

[0414] Embodiment A11. The method according to any one of Embodiments A1 to A10, wherein the cells are from or within a subject having a condition caused by a deficiency or activity of the OPA1 protein.

[0415] Embodiment A12. The method according to any one of Embodiments A1 to A11, wherein the amount of target protein deficiency is caused by haploinsufficiency of the target protein, the subject has a first allele encoding a functional target protein and a second allele that does not produce the target protein, or a second allele encoding a non-functional target protein, and the antisense oligomer binds to the target portion of NMD exon mRNA transcribed from the first allele.

[0416] Embodiment A13. The subject has a condition caused by a disorder resulting from a deficiency in the amount or function of a target protein, (a) The first mutant allele, (i) The target protein is produced at a lower level compared to production from the wild-type allele, (ii) A less functional target protein is produced compared to an equivalent wild-type protein, or (iii) The first mutant allele in which the target protein is not produced, (b) A second mutant allele, (i) The target protein is produced at a lower level compared to production from the wild-type allele, (ii) A less functional target protein is produced compared to an equivalent wild-type protein, or (iii) Having a second mutant allele in which the target protein is not produced, The method according to any one of Embodiments A1 to A11, wherein if the subject has a first mutant allele (a)(iii), the second mutant allele is (b)(i) or (b)(ii), and if the subject has a second mutant allele (b)(iii), the first mutant allele is (a)(i) or (a)(ii), and the NMD exon mRNA is transcribed from either the first mutant allele being (a)(i) or (a)(ii) and / or the second allele being (b)(i) or (b)(ii).

[0417] Embodiment A14. The method according to Embodiment A13, wherein the target protein is produced in a less functional form compared to an equivalent wild-type protein.

[0418] Embodiment A15. The method according to Embodiment A13, wherein the target protein is produced in a fully functional form compared to an equivalent wild-type protein.

[0419] Embodiment A16. The method according to any one of Embodiments A1 to A15, wherein the target portion of the NMD exon mRNA is located within a nonsense-mediated RNA decay-inducing exon.

[0420] Embodiment A17. The method according to any one of Embodiments A1 to A15, wherein the target portion of the NMD exon mRNA is either upstream or downstream of the nonsense-mediated RNA decay-inducing exon.

[0421] Embodiment A18. The method according to any one of Embodiments A1 to A17, wherein the NMD exon mRNA comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with SEQ ID NO: 2 or 3.

[0422] Embodiment A19. The method according to any one of Embodiments A1 to A17, wherein the NMD exon mRNA is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with SEQ ID NO: 1.

[0423] Embodiment A20. The method according to any one of Embodiments A1 to A17, wherein the target portion of the NMD exon mRNA includes a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to a region containing at least eight consecutive nucleic acids of either SEQ ID NO: 2 or 3.

[0424] Embodiment A21. The method according to any one of Embodiments A1 to A20, wherein the drug is an antisense oligomer (ASO), and the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least eight consecutive nucleic acids of SEQ ID NO: 2 or 3.

[0425] Embodiment A22. The method according to any one of Embodiments A1 to A15, wherein the target portion of the NMD exon mRNA is located within the nonsense-mediated RNA decay-inducing exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1.

[0426] Embodiment A23. The method according to any one of Embodiments A1 to A15, wherein the target portion of the NMD exon mRNA is upstream or downstream of OPA1's nonsense-mediated RNA decay-inducing exon 6x, OPA1's exon 7x, or OPA1's exon 28x.

[0427] Embodiment A24. The method according to any one of Embodiments A1 to A15, wherein the target portion of the NMD exon mRNA includes the exon-intron junction exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1.

[0428] Embodiment A25. The method according to any one of Embodiments A1 to A24, wherein the target protein produced is a full-length protein or a wild-type protein.

[0429] Embodiment A26. The total amount of target protein or functional RNA produced in cells that come into contact with the antisense oligomer is approximately 1.1 to 10 times, approximately 1.5 to 10 times, approximately 2 to 10 times, approximately 3 to 10 times, approximately 4 to 10 times, approximately 1.1 to 5 times, approximately 1.1 to 6 times, approximately 1.1 to 7 times, approximately 1.1 to 8 times, approximately 1.1 to 9 times, approximately 2 to 5 times, approximately The method according to any one of Embodiments A1 to A25, which increases to 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0430] Embodiment A27. The total amount of mRNA encoding the target protein or functional RNA produced in cells in contact with the antisense oligomer is approximately 20% to 300%, 50% to 300%, 100% to 300%, 150% to 300%, 20% to 50%, 20% to 100%, 20% to 150%, 20% to 200%, 20% to 250%, 50% to 100%, and 50% to 150% compared to the total amount of target protein produced in control cells. The method according to any one of Embodiments A1 to A25, increasing by %, approximately 50% to approximately 200%, approximately 50% to approximately 250%, approximately 100% to approximately 150%, approximately 100% to approximately 200%, approximately 100% to approximately 250%, approximately 150% to approximately 200%, approximately 150% to approximately 250%, approximately 200% to approximately 250%, at least approximately 10%, at least approximately 20%, at least approximately 50%, at least approximately 100%, at least approximately 150%, at least approximately 200%, at least approximately 250%, or at least approximately 300%.

[0431] Embodiment A28. The total amount of target protein produced by cells in contact with the antisense oligomer is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, 2 to 7 times, and approximately The method according to any one of Embodiments A1 to A25, which increases to 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0432] Embodiment A29. The total amount of target protein produced by cells in contact with the antisense oligomer is approximately 20% to 300%, 50% to 300%, 100% to 300%, 150% to 300%, 20% to 50%, 20% to 100%, 20% to 150%, 20% to 200%, 20% to 250%, 50% to 100%, 50% to 150%, 50% to 50% The method according to any one of Embodiments A1 to A25, increasing by 200%, approximately 50% to approximately 250%, approximately 100% to approximately 150%, approximately 100% to approximately 200%, approximately 100% to approximately 250%, approximately 150% to approximately 200%, approximately 150% to approximately 250%, approximately 200% to approximately 250%, at least approximately 10%, at least approximately 20%, at least approximately 50%, at least approximately 100%, at least approximately 150%, at least approximately 200%, at least approximately 250%, or at least approximately 300%.

[0433] Embodiment A30. The method according to any one of Embodiments A1 to A29, wherein the drug is an antisense oligomer (ASO), and the antisense oligomer includes a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond.

[0434] Embodiment A31. The method according to any one of Embodiments A1 to A30, wherein the drug is an antisense oligomer (ASO), and the antisense oligomer comprises a phosphorodiamidate morpholino, roq nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

[0435] Embodiment A32. The method according to any one of Embodiments A1 to A31, wherein the drug is an antisense oligomer (ASO), and the antisense oligomer comprises at least one modified sugar moiety.

[0436] Embodiment A33. The method according to Embodiment A32, wherein each sugar portion is a modified sugar portion.

[0437] Embodiment A34. The drug is an antisense oligomer (ASO), and the drug is composed of 8-50 nucleic acid bases, 8-40 nucleic acid bases, 8-35 nucleic acid bases, 8-30 nucleic acid bases, 8-25 nucleic acid bases, 8-20 nucleic acid bases, 8-15 nucleic acid bases, 9-50 nucleic acid bases, 9-40 nucleic acid bases, 9-35 nucleic acid bases, 9-30 nucleic acid bases, 9-25 nucleic acid bases, 9-20 nucleic acid bases, 9-15 nucleic acid bases, 10-50 nucleic acid bases, 10-40 nucleic acid bases, 10-35 nucleic acid bases, and 10-30 nucleic acid salts. The method according to any one of Embodiments A1 to A33, comprising a base, 10 to 25 nucleic acid bases, 10 to 20 nucleic acid bases, 10 to 15 nucleic acid bases, 11 to 50 nucleic acid bases, 11 to 40 nucleic acid bases, 11 to 35 nucleic acid bases, 11 to 30 nucleic acid bases, 11 to 25 nucleic acid bases, 11 to 20 nucleic acid bases, 11 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, 12 to 30 nucleic acid bases, 12 to 25 nucleic acid bases, 12 to 20 nucleic acid bases, or 12 to 15 nucleic acid bases.

[0438] Embodiment A35. The method according to any one of Embodiments A1 to A34, wherein the drug is an antisense oligomer (ASO) and the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the target portion of the protein-coding NMD exon mRNA.

[0439] Embodiment A36. The method according to any one of Embodiments A1 to A35, further comprising evaluating OPA1 mRNA levels or protein expression.

[0440] Embodiment A37. The method according to any one of Embodiments A1 to A36, wherein type optic nerve atrophy is treated, and an antisense oligomer binds to a target region of OPA1 NMD exon mRNA, the target region being located within Sequence ID No. 2 or 3.

[0441] Embodiment A38. The method according to any one of Embodiments A1 to A37, wherein the subject is a human.

[0442] Embodiment A39. The method according to any one of Embodiments A1 to A38, wherein the subject is a non-human animal.

[0443] Embodiment A40. The method according to any one of Embodiments A1 to A39, wherein the subject is a fetus, embryo, or child.

[0444] Embodiment A41. The method according to any one of Embodiments A1 to A40, wherein the cells are ex vivo.

[0445] Embodiment A42. The method according to any one of Embodiments A1 to A41, wherein the therapeutic agent is administered by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the target.

[0446] Embodiment A43. The method according to any one of Embodiments A1 to A42, further comprising administering a second therapeutic agent to a target.

[0447] Embodiment A44. The method according to Embodiment A43, wherein the second therapeutic agent is a small molecule.

[0448] Embodiment A45. The method according to Embodiment A43, wherein the second therapeutic agent is an ASO.

[0449] Embodiment A46. The method according to any one of Embodiments A43 to A45, wherein the second therapeutic agent modifies intron retention.

[0450] Embodiment A47. An antisense oligomer used in the method described in any of Embodiments A1 to A46.

[0451] Embodiment A48. An antisense oligomer comprising a region containing at least eight consecutive nucleic acids of SEQ ID NO: 2 or 3 and a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity.

[0452] Embodiment A49. A pharmaceutical composition comprising the antisense oligomer of Embodiment A47 or A48 and an excipient.

[0453] Embodiment A50. A method for treating a subject in need of the pharmaceutical composition described in Embodiment A49, wherein the administration is carried out by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreous injection, or intravenous injection.

[0454] Embodiment A51. A composition comprising a therapeutic agent for use in a method of increasing the expression of a target protein or functional RNA by cells for the treatment of type 1 optic neuropathy in a subject requiring the treatment thereof, wherein the deficient protein or deficient functional RNA is deficient in amount or activity in the subject, and the target protein is (a) Deficient protein, or (b) Complementary proteins that functionally enhance or replace the deficient protein, or replace it in a target, Furthermore, functional RNA, (c) Deficient RNA, or (d) Complementary functional RNA that functionally enhances or replaces the deficient functional RNA in the subject, A composition in which a therapeutic agent enhances the elimination of nonsense-mediated RNA decay-inducing exons from NMD exon mRNA encoding a target protein or functional RNA, thereby increasing the production or activity of the target protein or functional RNA in the subject.

[0455] Embodiment A52. A composition comprising a therapeutic agent for use in a method for treating a condition associated with the OPA1 protein in a subject requiring such treatment, the method comprising the step of increasing OPA1 protein expression by cells of the subject, wherein the cells contain nonsense-mediated RNA decay-inducible exons (NMD exon mRNA) and have mRNA encoding the OPA1 protein, and the method comprising contacting the cells with the therapeutic agent, thereby removing the nonsense-mediated RNA decay-inducible exons from the NMD exon mRNA encoding the OPA1 protein, thereby increasing the level of mRNA encoding the OPA1 protein and increasing OPA1 protein expression in the cells of the subject.

[0456] Embodiment A53. The composition according to Embodiment A52, wherein the condition is a disease or disorder.

[0457] Embodiment A54. The composition according to Embodiment A53, wherein the disease or disorder is type 1 optic nerve atrophy.

[0458] Embodiment A55. The composition according to any one of Embodiments A52 to A54, wherein the ...

Claims

1. A method for treating a subject having a disease or condition, or a method for reducing the likelihood that the subject will develop the disease or condition, wherein the method comprises administering a pharmaceutical composition containing an antisense oligomer to the subject. The antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity with any one of the sequences described in SEQ ID NOs: 6-275 or 280-299. The method comprises administering a pharmaceutical composition to one eye of the subject in a dose of about 0.005 to about 20 mg of antisense oligomer.

2. A method for treating a subject having a disease or condition, or a method for reducing the likelihood that the subject will develop the disease or condition, wherein the method comprises administering a pharmaceutical composition containing an antisense oligomer to the subject. The antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity with the sequence described in any one of SEQ ID NOs: 6-275 or 280-299. The disease or condition includes autosomal dominant optic atrophy (ADOA), and the subject is (i) Heterozygous OPA1 gene variant, (ii) A transparent ocular medium to enable proper visualization of the vitreous humor and fundus and to achieve proper quality of all ophthalmic evaluations. (iii) Regarding posterior subcapsular (PSC) opacity <1 AREDS clinical lens standard, (iv) Each eye administered with the pharmaceutical composition individually has a BCVA EDTRS letter score of ≥35 and ≤70, or A method characterized by having any combination of (v)(i) to (viiii).

3. A method for treating a patient having a disease or condition, or a method for reducing the likelihood that a patient will develop a disease or condition, wherein the method comprises administering a pharmaceutical composition containing an antisense oligomer to the subject. The antisense oligomer comprises a nucleotide sequence having at least 80% sequence identity with the sequence described in any one of SEQ ID NOs: 6-275 or 280-299. The disease or condition includes autosomal dominant optic atrophy (ADOA), and the subject is (1) The OPA1 gene does not have a gain-of-function mutant, or a compound heterozygous or homozygous pathogenic or pathogenic mutant. (2) The OPA1 gene does not have a benign variant or only the possibility of being a benign variant, (3) Not exhibiting the extraocular phenotype of (symptomatic) ADOA (ADOA plus), (4) Never been diagnosed with Behr syndrome, (5) Not having known pathogenic mutations in other genes involved in optic nerve atrophy or retinal disease, (6) Not having diabetic retinopathy that may lead to proliferative diabetic retinopathy, diabetic macular edema, or optic neuropathy. (7) Neither eye has any eye condition, nor any history of any eye condition. (8) No history of intraocular surgery, including refractive surgery, or corneal surgery in either eye within 12 weeks prior to administration. (9) No history of retinal photocoagulation, (10) No history of or presence of retinal vein occlusion, (11) Having had an active relapse of non-infectious uveitis in either eye, or an episode of infectious uveitis or another eye infection, within 12 months prior to the administration of the above-mentioned treatment, does not mean that the patient is not at risk of uveitis or eye infection. (12) Neither eye has age-related macular degeneration in dry eyes. (13) No high myopia (>6 diopters) (14) No history of cancer (excluding a diagnosis of basal cell carcinoma, squamous cell carcinoma, or cervical intraepithelial neoplasia with successful treatment), (15) You have never taken or ingested any medication or treatment that may cause or could cause optic neuropathy. (16) No history of nutritional deficiencies (including B12 and / or folic acid deficiency), no known serum B12 or folic acid deficiency, no history of bariatric surgery, or (17) A method having the characteristics of any combination of (1) to (16).

4. A method for treating a subject having a disease or condition, or for reducing the likelihood that the subject will develop the disease or condition, wherein the method comprises administering to the subject a pharmaceutical composition comprising an antisense oligomer, wherein the antisense oligomer has the chemical structure: 【Chemistry 1-1】 [Chemistry 1-2] [Chemistry 1-3] A method comprising a compound comprising any one of the following, or a pharmaceutically acceptable salt thereof.

5. The method according to any one of claims 2 to 4, wherein the method comprises administering the pharmaceutical composition to one eye of the subject in a dose of about 0.005 to about 20 mg of the antisense oligomer.

6. The aforementioned method involves the antisense oligomer in amounts of approximately 0.005 mg to approximately 15 mg, approximately 0.005 mg to approximately 10 mg, approximately 0.005 mg to approximately 5 mg, approximately 0.005 mg to approximately 1 mg, approximately 0.01 mg to approximately 15 mg, approximately 0.01 mg to approximately 10 mg, approximately 0.01 mg to approximately 5 mg, approximately 0.01 mg to approximately 2.5 mg, approximately 0.01 mg to approximately 1.0 mg, approximately 0.01 mg to approximately 0.5 mg, approximately 0.01 mg to approximately 0.25 mg, approximately 0.01 mg to approximately 0.1 mg, approximately 0.01 mg to approximately 0.05 mg, approximately 0.05 mg to approximately 10 mg, and approximately 0. The method according to any one of claims 1 to 5, comprising administering the pharmaceutical composition to one eye of the subject in a dose of approximately 0.5 mg to 5 mg, approximately 0.05 mg to 2.5 mg, approximately 0.05 mg to 1.0 mg, approximately 0.05 mg to 0.5 mg, approximately 0.05 mg to 0.25 mg, approximately 0.05 mg to 0.1 mg, approximately 0.1 mg to 5 mg, approximately 0.1 mg to 2.5 mg, approximately 0.1 mg to 1.5 mg, approximately 0.1 mg to 1.0 mg, approximately 0.1 mg to 0.5 mg, or approximately 0.1 mg to 0.25 mg.

7. The method involves approximately 0.005 mg, 0.01 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.75 mg, 2.0 mg, 2.25 mg, and 2 mg of the antisense oligomer. The method according to any one of claims 1 to 5, comprising administering the pharmaceutical composition to one eye of the subject in a dose of 5 mg, approximately 2.75 mg, approximately 3 mg, approximately 3.5 mg, approximately 4.0 mg, approximately 4.5 mg, approximately 5.0 mg, approximately 5.5 mg, approximately 6.0 mg, approximately 7.0 mg, approximately 8.0 mg, approximately 9.0 mg, approximately 10 mg, approximately 12.5 mg, approximately 15 mg, approximately 17.5 mg, or approximately 20 mg.

8. The aforementioned method involves the antisense oligomer in amounts of approximately 0.1 mg to approximately 1.5 mg, approximately 0.1 mg to approximately 1.4 mg, approximately 0.1 mg to approximately 1.2 mg, approximately 0.1 mg to approximately 1.0 mg, approximately 0.1 mg to approximately 0.8 mg, approximately 0.1 mg to approximately 0.7 mg, approximately 0.1 mg to approximately 0.5 mg, approximately 0.1 mg to approximately 0.3 mg, approximately 0.2 mg to approximately 1.5 mg, approximately 0.2 mg to approximately 1.4 mg, and approximately 0. 2 mg to about 1.2 mg, about 0.2 mg to about 1.0 mg, about 0.2 mg to about 0.8 mg, about 0.2 mg to about 0.7 mg, about 0.2 mg to about 0.5 mg, about 0.3 mg to about 1.5 mg, About 0.3 mg to about 1.4 mg, about 0.3 mg to about 1.2 mg, about 0.3 mg to about 1.0 mg, about 0.3 mg to about 0.8 mg, about 0.3 mg to about 0.7 mg, about 0.3 mg to about 0.5 m g, about 0.5 mg to about 1.5 mg, about 0.5 mg to about 1.4 mg, about 0.5 mg to about 1.2 mg, about 0.5 mg to about 1.0 mg, about 0.5 mg to about 0.8 mg, about 0.5 mg to about 0 .7 mg, about 0.7 mg to about 1.5 mg, about 0.7 mg to about 1.4 mg, about 0.7 mg to about 1.2 mg, about 0.7 mg to about 1.0 mg, about 0.8 mg to about 1.5 mg The method according to any one of Embodiments 1 to 5, comprising administering the pharmaceutical composition to one eye of the subject in a dose of approximately 1.4 mg, approximately 0.8 mg to approximately 1.2 mg, approximately 0.8 mg to approximately 1.0 mg, approximately 1.0 mg to approximately 1.5 mg, approximately 1.0 mg to approximately 1.4 mg, approximately 1.0 mg to approximately 1.2 mg, approximately 1.2 mg to approximately 1.5 mg, or approximately 1.2 mg to approximately 1.4 mg.

9. The method according to any one of claims 1 to 5, wherein the method comprises administering the pharmaceutical composition to one eye of the subject in a dose of about 0.1 mg, about 0.2 mg, about 0.3 mg, 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, or about 1.5 mg of the antisense oligomer.

10. The method is from about 5 μL to about 250 μL, from about 10 μL to about 250 μL, from about 20 μL to about 250 μL, from about 30 μL to about 250 μL, from about 40 μL to about 250 μL, from about 50 μL to about 250 μL, from about 60 μL to about 250 μL, from about 70 μL to about 250 μL, from about 80 μL to about 250 μL, from about 100 μL to about 250 μL, from about 120 μL to about 250 μL, from about 150 μL to about 250 μL, from about 160 μL to about 250 μL, from about 180 μL to about 500 μL, from about 200 μL to about 250 μL, from about 220 μL to about 250 μL, from about 5 μL to about 220 μL, from about 10 μL to about 220 μL, from about 20 μL to about 220 μL, from about 30 μL to about 220 μL, from about 40 μL to about 220 μL, from about 50 μL to about 220 μL, from about 60 μL to about 220 μL, from about 70 μL to about 220 μL, from about 80 μL to about 220 μL, from about 100 μL to about 220 μL, from about 120 μL to about 220 μL, from about 150 μL to about 220 μL, from about 160 μL to about 220 μL, from about 180 μL to about 220 μL, from about 5 μL to about 200 μL, from about 10 μL to about 200 μL, from about 20 μL to about 200 μL, from about 30 μL to about 200 μL, from about 40 μL to about 200 μL, from about 50 μL to about 200 μL, from about 60 μL to about 200 μL, from about 70 μL to about 200 μL, from about 80 μL to about 200 μL, from about 100 μL to about 200 μL, from about 120 μL to about 200 μL, from about 150 μL to about 200 μL, from about 160 μL to about 200 μL, from about 180 μL to about 200 μL, from about 5 μL to about 180 μL, from about 10 μL to about 180 μL, from about 20 μL to about 180 μL, from about 30 μL to about 180 μL, from about 40 μL to about 180 μL, from about 50 μL to about 180 μL, from about 60 μL to about 180 μL, from about 70 μL to about 180 μL, from about 80 μL to about 180 μL, from about 100 μL to about 180 μL, from about 120 μL to about 180 μL, from about 150 μL to about 180 μL, from about 5 μL to about 150 μL, from about 10 μL to about 150 μL, from about 20 μL to about 150 μL, from about 30 μL to about 150 μL, from about 40 μL to about 150 μL, from about 50 μL to about 150 μL, from about 60 μL to about 150 μL, from about 70 μL to about 150 μL, from about 80 μL to about 150 μL, from about 100 μL to about 150 μL, from about 120 μL to about 150 μL, from about 5 μL to about 150 μL, from about 10 μL to about 120 μL, from about 20 μL to about 120 μL, from about 30 μL to about 120 μL, from about 40 μL to about 120 μL, from about 50 μL to about 120 μL, from about 60 μL to about 120 μL, from about 70 μL to about 120 μLApproximately 80 μL to 120 μL, approximately 100 μL to 120 μL, approximately 5 μL to 100 μL, approximately 10 μL to 100 μL, approximately 20 μL to 100 μL, approximately 30 μL to 100 μL, approximately 40 μL to 100 μL, approximately 50 μL to 100 μL, approximately 60 μL to 100 μL, approximately 70 μL to 100 μL, approximately 80 μL to 100 μL, approximately 5 μL to 80 μL, approximately 10 μL to 80 μL, approximately 20 μL to 80 μL The method according to any one of claims 1 to 9, comprising administering the pharmaceutical composition to one eye of the subject in a volume of approximately 30 μL to approximately 80 μL, approximately 40 μL to approximately 80 μL, approximately 50 μL to approximately 80 μL, approximately 60 μL to approximately 80 μL, approximately 5 μL to approximately 60 μL, approximately 10 μL to approximately 60 μL, approximately 20 μL to approximately 60 μL, approximately 30 μL to approximately 60 μL, approximately 40 μL to approximately 60 μL, or approximately 50 μL to approximately 60 μL.

11. The method according to any one of claims 1 to 9, wherein the method comprises administering a pharmaceutical composition to one eye of a subject in a volume of approximately 5 μL, approximately 8 μL, approximately 10 μL, approximately 12 μL, approximately 15 μL, approximately 18 μL, approximately 20 μL, approximately 25 μL, approximately 28 μL, approximately 30 μL, approximately 35 μL, approximately 40 μL, approximately 45 μL, approximately 48 μL, approximately 50 μL, approximately 55 μL, approximately 60 μL, approximately 65 μL, approximately 70 μL, approximately 75 μL, approximately 80 μL, approximately 90 μL, approximately 100 μL, approximately 120 μL, approximately 150 μL, approximately 160 μL, approximately 180 μL, approximately 200 μL, approximately 220 μL, or approximately 250 μL.

12. The method according to any one of claims 1 to 11, wherein the method comprises administering the pharmaceutical composition to both the left and right eyes of the subject.

13. The method according to claim 12, wherein the method comprises administering the same dose of the pharmaceutical composition to both the left and right eyes of the subject.

14. The method according to claim 12, wherein the method comprises administering the pharmaceutical composition to the left and right eyes of the subject in different doses.

15. The method according to any one of claims 1 to 3 or 5 to 14, wherein the antisense oligomer comprises a nucleotide sequence having at least 90% or 100% sequence identity with the sequence described in any one of SEQ ID NOs: 6 to 275 or 280 to 299.

16. The method according to any one of claims 1 to 3 or 5 to 14, wherein the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity with the sequence described in any one of SEQ ID NOs: 36, 236, 242, 250, 280-283, 288, or 290-292.

17. The method according to any one of claims 1 to 3 or 5 to 14, wherein the antisense oligomer modulates the splicing of nonsense-mediated RNA decay-inducing exons (NMD exons) from premRNA in the target cells, and the premRNA encodes the OPA1 protein and contains the NMD exons, thereby modulating the level of treated mRNA processed from the premRNA and regulating the expression of the OPA1 protein in the cells.

18. The aforementioned antisense oligomer (a) Binding to the target region of the premRNA, (b) modulate the binding of factors involved in the splicing of the NMD exons, or (c) The method according to claim 17, which is a combination of (a) and (b).

19. The method according to claim 18, wherein the target portion of the premRNA is located proximal to the NMD exon.

20. The method according to claim 18, wherein the target portion of the premRNA is up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides upstream of the 5' end of the NMD exon.

21. The method according to claim 18, wherein the target portion of the premRNA is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, and about 1 nucleotide upstream of the 5' end of the NMD exon.

22. The method according to claim 18, wherein the target portion of the premRNA is approximately 1,500 nucleotides, 1,000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides downstream of the 3' end of the NMD exon.

23. The method according to claim 18, wherein the target portion of the premRNA is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, and about 1 nucleotide downstream of the 3' end of the NMD exon.

24. The method according to claim 18, wherein the target portion of the premRNA is approximately 1,500 nucleotides, 1,000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides upstream of genomic site GRCh38 / hg38:chr3 193628509.

25. The method according to claim 18, wherein the target portion of the premRNA is approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides upstream of the genomic site GRCh38 / hg38:chr3 193628509.

26. The method according to claim 18, wherein the target portion of the premRNA is approximately 1,500 nucleotides, 1,000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, and 50 nucleotides downstream of the genomic site GRCh38 / hg38:chr3 193628616.

27. The method according to claim 18, wherein the target portion of the premRNA is approximately 1,500 nucleotides, approximately 1,000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, and approximately 50 nucleotides downstream of the genomic site GRCh38 / hg38:chr3 193628616.

28. The method according to claim 18, wherein the target portion of the premRNA is located within an intron region between two standard exon regions of the premRNA, and the intron region contains the NMD exon.

29. The method according to claim 18, wherein the target portion of the premRNA at least partially overlaps with the NMD exon.

30. The method according to claim 18, wherein the target portion of the premRNA at least partially overlaps with an intron upstream or downstream of the NMD exon.

31. The method according to claim 18, wherein the target portion of the premRNA includes a 5'NMD exon-intron junction or a 3'NMD exon-intron junction.

32. The method according to claim 18, wherein the target portion of the premRNA is located within the NMD exon.

33. The method according to claim 18, wherein the target portion of the premRNA comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides of the NMD exon.

34. The method according to any one of claims 17 to 33, wherein the NMD exon comprises a sequence having at least 80%, at least 90%, or 100% sequence identity with sequence number 279.

35. The method according to any one of claims 17 to 33, wherein the NMD exon comprises the sequence of sequence number 279.

36. The method according to claim 18, wherein the target portion of the premRNA is within the range of the nonsense-mediated RNA decay-inducing exon GRCh38 / hg38:chr3 193628509 to 193628616.

37. The method according to claim 18, wherein the target portion of the premRNA is upstream or downstream of the nonsense-mediated RNA decay-inducing exons GRCh38 / hg38:chr3 193628509 to 193628616.

38. The method according to claim 18, wherein the target portion of the premRNA includes the exon-intron junction of exon GRCh38 / hg38:chr3 193628509 to 193628616.

39. The method according to any one of claims 17 to 38, wherein the OPA1 protein expressed from the treated mRNA is a full-length OPA1 protein or a wild-type OPA1 protein.

40. The method according to any one of claims 17 to 38, wherein the OPA1 protein expressed from the processed mRNA is a functional OPA1 protein.

41. The method according to any one of claims 17 to 38, wherein the OPA1 protein expressed from the treated mRNA is at least partially functional compared to the wild-type OPA1 protein.

42. The method according to any one of claims 17 to 38, wherein the OPA1 protein expressed from the treated mRNA is at least partially functional compared to the full-length wild-type OPA1 protein.

43. The method according to any one of claims 17 to 42, wherein the antisense oligomer promotes the exclusion of the NMD exon from the premRNA.

44. The elimination of the NMD exon from the intracellular premRNA in contact with the antisense oligomer is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, and 2 to 6 times compared to the absence of the antisense oligomer. The method according to claim 43, which increases by a factor of two, approximately two to seven times, approximately two to eight times, approximately two to nine times, approximately three to six times, approximately three to seven times, approximately three to eight times, approximately three to nine times, approximately four to seven times, approximately four to eight times, approximately four to nine times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times.

45. The method according to any one of claims 17 to 44, wherein the method results in an increase in the level of the treated mRNA in the cells.

46. The levels of the treated mRNA in the cells that come into contact with the antisense oligomer are approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, and 2 to 7 times higher compared to the case where the antisense oligomer is absent. The method according to claim 45, which increases by a factor of two, approximately two to eight times, approximately two to nine times, approximately three to six times, approximately three to seven times, approximately three to eight times, approximately three to nine times, approximately four to seven times, approximately four to eight times, approximately four to nine times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times.

47. The method according to any one of claims 17 to 46, wherein the method results in an increase in the expression of the OPA1 protein in the cells.

48. The level of OPA1 protein expressed from the treated mRNA in the cell that comes into contact with the antisense oligomer is approximately 1.1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times, 1.1 to 6 times, 1.1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, and 2 to 6 times higher compared to the case where the antisense oligomer is absent. The method according to claim 47, which increases by a factor of two, approximately two to seven times, approximately two to eight times, approximately two to nine times, approximately three to six times, approximately three to seven times, approximately three to eight times, approximately three to nine times, approximately four to seven times, approximately four to eight times, approximately four to nine times, at least approximately 1.1 times, at least approximately 1.5 times, at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 10 times.

49. The method according to any one of claims 1 to 3 or 5 to 48, wherein the antisense oligomer comprises modification of the skeleton including a phosphorothioate bond or a phosphorodiamidate bond.

50. The method according to any one of claims 1 to 3 or 5 to 48, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a loc nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-O-N-methylacetamide (2'-NMA), or a 2'-O-methoxyethyl moiety.

51. The method according to any one of claims 1 to 3 or 5 to 48, wherein the antisense oligomer comprises at least one modified sugar moiety.

52. The method according to claim 51, wherein each sugar portion is a modified sugar portion.

53. The method according to any one of claims 1 to 3 or 5 to 52, wherein the antisense oligomer comprises 5'-methylcytosine (5'-MeC).

54. The method according to any one of claims 1 to 3 or 5 to 53, wherein each cytosine in the antisense oligomer is 5'-methylcytosine (5'-MeC).

55. The method according to any one of claims 1 to 3 or 5 to 54, wherein the antisense oligomer comprises 5'-methyluracil (5'-MeU).

56. The method according to any one of claims 1 to 3 or 5 to 55, wherein the antisense oligomer contains a phosphorothioate bond.

57. The method according to any one of claims 1 to 3 or 5 to 56, wherein the internucleoside bonds of the ASO are phosphorothioate bonds.

58. The method according to any one of claims 1 to 3 or 5 to 57, wherein the antisense oligomer comprises Loc nucleic acid (LNA).

59. The antisense oligomer has a length of 8 to 50 nucleic acid bases, 8 to 40 nucleic acid bases, 8 to 35 nucleic acid bases, 8 to 30 nucleic acid bases, 8 to 25 nucleic acid bases, 8 to 20 nucleic acid bases, 8 to 15 nucleic acid bases, 9 to 50 nucleic acid bases, 9 to 40 nucleic acid bases, 9 to 35 nucleic acid bases, 9 to 30 nucleic acid bases, 9 to 25 nucleic acid bases, 9 to 20 nucleic acid bases, 9 to 15 nucleic acid bases, 10 to 50 nucleic acid bases, 10 to 40 nucleic acid bases, 10 to 35 nucleic acid bases, 10 to 30 nucleic acid bases, and 10 to 25 nucleic acid bases. The method according to any one of claims 1 to 3 or 5 to 58, comprising a base, 10 to 20 nucleic acid bases, 10 to 15 nucleic acid bases, 11 to 50 nucleic acid bases, 11 to 40 nucleic acid bases, 11 to 35 nucleic acid bases, 11 to 30 nucleic acid bases, 11 to 25 nucleic acid bases, 11 to 20 nucleic acid bases, 11 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, 12 to 30 nucleic acid bases, 12 to 25 nucleic acid bases, 12 to 20 nucleic acid bases, or 12 to 15 nucleic acid bases.

60. The aforementioned antisense oligomer has the following chemical structure: 【Chemistry 2-1】 【Chemistry 2-2】 [Chemistry 2-3] The method according to any one of claims 1 to 3 or 5 to 59, comprising any one of the compounds or a pharmaceutically acceptable salt thereof.

61. The aforementioned antisense oligomer has the following structure: 【Chemistry 3-1】 【Chemistry 3-2】 【Chemistry 3-3】 The method according to claim 4 or 60, wherein the compound conforms to any of the following.

62. The method according to any one of claims 1 to 61, wherein the pharmaceutical composition is a liquid composition.

63. The method according to any one of claims 1 to 62, wherein the method comprises administering the pharmaceutical composition as a bolus injection.

64. The method according to any one of claims 1 to 63, wherein the method comprises administering the pharmaceutical composition as a bolus injection over a period of 1 to 60 minutes, 1 to 50 minutes, 1 to 40 minutes, 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes, 1 to 5 minutes, or 1 to 3 minutes.

65. The method according to any one of claims 1 to 64, wherein the antisense oligomer is solubilized or diluted in a solution containing one or more of sodium chloride, sodium diphosphate, potassium monophosphate, or water for injection.

66. The method according to any one of claims 1 to 64, wherein the antisense oligomer is solubilized or diluted in a solution containing one or more of sodium chloride, sodium diphosphate, potassium monophosphate, and water for injection.

67. The method according to any one of claims 1 to 66, wherein the antisense oligomer is solubilized or diluted in an isotonic solution.

68. The method according to any one of claims 1 to 67, wherein the antisense oligomer is solubilized or diluted in a phosphate buffer at pH 5.

8.

69. The method according to any one of claims 1 to 67, wherein the antisense oligomer is solubilized or diluted in a phosphate buffer (pH 6.6 to 7.6).

70. The method according to any one of claims 1 to 69, wherein the pharmaceutical preparation does not contain a preservative.

71. The method according to any one of claims 1 to 70, wherein the antisense oligomer is present in the pharmaceutical composition at a concentration of about 2 mg / mL to about 200 mg / mL.

72. The aforementioned antisense oligomer is present in concentrations of approximately 2 mg / mL to approximately 200 mg / mL, approximately 5 mg / mL to approximately 200 mg / mL, approximately 10 mg / mL to approximately 200 mg / mL, approximately 15 mg / mL to approximately 200 mg / mL, approximately 20 mg / mL to approximately 200 mg / mL, approximately 25 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 200 mg / mL, and approximately 100 mg / mL. L to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, 2 mg / mL to about 15 0 mg / mL, about 5 mg / mL to about 150 mg / mL, about 10 mg / mL to about 150 mg / mL, about 15 mg / mL to about 150 mg / mL, About 20 mg / mL to about 150 mg / mL, about 25 mg / mL to about 150 mg / mL, about 30 mg / mL to about 150 mg / mL, about 35 mg / mL to about 150 mg / mL, about 40 mg / mL to about 150 mg / mL, about 50 mg / mL to about 150 mg / mL, about 60 mg / mL to about 1 50mg / mL, about 80mg / mL to about 150mg / mL, about 100mg / mL to about 150mg / mL, 2mg / mL to about 100mg / m L, about 5 mg / mL to about 100 mg / mL, about 10 mg / mL to about 100 mg / mL, about 15 mg / mL to about 100 mg / mL, about 20 mg / mL to about 100mg / mL, about 25mg / mL to about 100mg / mL, about 30mg / mL to about 100mg / mL, about 35mg / mL to about 100mg / mL, about 40mg / mL to about 100mg / mL, about 50mg / mL to about 100mg / mL, about 60mg / mL to about 100mg / mL mL, about 80 mg / mL to about 100 mg / mL, 2 mg / mL to about 80 mg / mL, about 5 mg / mL to about 80 mg / mL, about 10 mg / m L to about 80 mg / mL, about 15 mg / mL to about 80 mg / mL, about 20 mg / mL to about 80 mg / mL, about 25 mg / mL to about 80 mg / m L, about 30 mg / mL to about 80 mg / mL, about 35 mg / mL to about 80 mg / mL, about 40 mg / mL to about 80 mg / mL, about 60 mg / mL mL to about 80 mg / mL, 2 mg / mL to about 60 mg / mL, about 5 mg / mL to about 60 mg / mL, about 10 mg / mL to about 60 mg / mL,The method according to any one of claims 1 to 70, wherein the pharmaceutical composition contains the following at concentrations: approximately 15 mg / mL to approximately 60 mg / mL, approximately 20 mg / mL to approximately 60 mg / mL, approximately 25 mg / mL to approximately 60 mg / mL, approximately 30 mg / mL to approximately 60 mg / mL, approximately 35 mg / mL to approximately 60 mg / mL, approximately 40 mg / mL to approximately 60 mg / mL, 2 mg / mL to approximately 40 mg / mL, approximately 5 mg / mL to approximately 40 mg / mL, approximately 10 mg / mL to approximately 40 mg / mL, approximately 15 mg / mL to approximately 40 mg / mL, approximately 20 mg / mL to approximately 40 mg / mL, or approximately 25 mg / mL to approximately 40 mg / mL.

73. The method according to any one of claims 1 to 70, wherein the antisense oligomer is present in the pharmaceutical composition at a concentration of approximately 2 mg / mL, approximately 3 mg / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL, approximately 7 mg / mL, approximately 8 mg / mL, approximately 9 mg / mL, approximately 10 mg / mL, approximately 12 mg / mL, approximately 14 mg / mL, approximately 15 mg / mL, approximately 16 mg / mL, approximately 18 mg / mL, approximately 20 mg / mL, approximately 22 mg / mL, approximately 24 mg / mL, approximately 26 mg / mL, approximately 28 mg / mL, approximately 30 mg / mL, approximately 35 mg / mL, approximately 40 mg / mL, approximately 50 mg / mL, approximately 60 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 140 mg / mL, approximately 160 mg / mL, approximately 180 mg / mL, or approximately 200 mg / mL.

74. The method according to any one of claims 1 to 73, wherein the pharmaceutical composition is prepared by diluting a concentrate containing the antisense oligomer.

75. The aforementioned antisense oligomer is present in concentrations of approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 200 mg / mL, approximately 100 mg / mL to approximately 200 mg / mL, approximately 150 mg / mL to approximately 200 mg / mL, approximately 180 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 150 mg / mL, approximately 35 mg / mL to approximately 150 mg / mL, approximately 40 mg / mL to approximately 150 mg / mL, approximately 50 mg / mL to approximately 150 mg / mL, approximately 60 mg / mL to approximately 150 mg / mL, and approximately 80 mg / mL to approximately 15 The method according to claim 74, wherein the concentrate contains the following concentrations: 0 mg / mL, approximately 100 mg / mL to approximately 150 mg / mL, approximately 30 mg / mL to approximately 100 mg / mL, approximately 35 mg / mL to approximately 100 mg / mL, approximately 40 mg / mL to approximately 100 mg / mL, approximately 50 mg / mL to approximately 100 mg / mL, approximately 60 mg / mL to approximately 100 mg / mL, approximately 80 mg / mL to approximately 100 mg / mL, approximately 30 mg / mL to approximately 80 mg / mL, approximately 35 mg / mL to approximately 80 mg / mL, approximately 40 mg / mL to approximately 80 mg / mL, approximately 60 mg / mL to approximately 80 mg / mL, approximately 30 mg / mL to approximately 60 mg / mL, approximately 35 mg / mL to approximately 60 mg / mL, or approximately 40 mg / mL to approximately 60 mg / mL.

76. The method according to claim 74, wherein the antisense oligomer is present in the concentrate at a concentration of approximately 40 mg / mL, approximately 45 mg / mL, approximately 50 mg / mL, approximately 55 mg / mL, approximately 60 mg / mL, approximately 65 mg / mL, approximately 70 mg / mL, approximately 80 mg / mL, approximately 90 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 140 mg / mL, approximately 160 mg / mL, approximately 180 mg / mL, or approximately 200 mg / mL.

77. The method according to any one of claims 74 to 76, wherein the concentrate is a phosphate buffer.

78. The method according to any one of claims 1 to 77, wherein the subject is a human subject.

79. The method according to any one of claims 1 or 4 to 78, wherein the disease or condition is caused by an insufficient amount or activity of the OPA1 protein.

80. The method according to any one of claims 1 or 4 to 79, wherein the disease or condition includes a disease or condition of the eye.

81. The method according to any one of claims 1 or 4 to 79, wherein the disease or condition includes a cardiovascular disease or condition.

82. The method according to any one of claims 1 or 4 to 79, wherein the disease or condition includes a neurological disease or condition.

83. The aforementioned diseases or conditions include ADOA-plus syndrome, mitochondrial disorders, glaucoma, normal-tension glaucoma, Charcot-Marie-Tooth disease, mitochondrial dysfunction, diabetic retinopathy, age-related macular degeneration, retinal ganglion cell death, mitochondrial fission-mediated mitochondrial dysfunction, progressive extraocular muscle palsy, hearing loss, ataxia, motor neuropathy, sensory neuropathy, myopathy, Baer's syndrome, brain dysfunction, encephalopathy, peripheral neuropathy, fatal infantile mitochondrial encephalomyopathy, hypertrophic cardiomyopathy, spastic ataxia syndrome, sensorimotor peripheral neuropathy, hypotonia, gastrointestinal motility disorders and dysphagia, optic nerve atrophy, optic nerve atrophy plus syndrome, and mitochondrial DNA depletion. The method according to any one of claims 1 or 4 to 79, comprising syndrome 14, late-onset cardiomyopathy, diabetic cardiomyopathy, Alzheimer's disease, focal segmental glomerulosclerosis, kidney disease, Huntington's disease, cognitive decline associated with healthy aging, prion disease, late-onset dementia and parkinsonism, mitochondrial myopathy, Leigh syndrome, Friedreich's ataxia, Parkinson's disease, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), pyruvate dehydrogenase complex deficiency, chronic kidney disease, Leber's hereditary optic neuropathy, obesity, age-related systemic neurodegeneration, skeletal muscle atrophy, ischemic injury of the heart and brain, or massive hepatocyte apoptosis.

84. The method according to any one of claims 1 or 4 to 79, wherein the disease or condition includes type 1 optic nerve atrophy.

85. The method according to any one of claims 1 or 4 to 79, wherein the disease or condition includes autosomal dominant optic atrophy (ADOA).

86. The aforementioned subject is, (i) Heterozygous OPA1 gene variant, (ii) A transparent ocular medium to enable proper visualization of the vitreous humor and fundus and to achieve proper quality of all ophthalmic evaluations. (iii) Regarding posterior subcapsular (PSC) opacity <1 AREDS clinical lens standard, (iv) Each eye administered with the pharmaceutical composition individually has a BCVA EDTRS letter score of ≥35 and ≤70, or The method according to claim 85, characterized by having any combination of (v)(i) to (viiii).

87. The aforementioned subject is, (1) The OPA1 gene does not have a gain-of-function mutant, or a compound heterozygous or homozygous pathogenic or pathogenic mutant. (2) The OPA1 gene does not have a benign variant or only the possibility of being a benign variant, (3) Not exhibiting the extraocular phenotype of (symptomatic) ADOA (ADOA plus), (4) Never been diagnosed with Behr syndrome, (5) Not having known pathogenic mutations in other genes involved in optic nerve atrophy or retinal disease, (6) Not having diabetic retinopathy that may lead to proliferative diabetic retinopathy, diabetic macular edema, or optic neuropathy. (7) Neither eye has any eye condition, nor any history of any eye condition. (8) No history of intraocular surgery, including refractive surgery, or corneal surgery in either eye within 12 weeks prior to administration. (9) No history of retinal photocoagulation, (10) No history of or presence of retinal vein occlusion, (11) Having had an active relapse of non-infectious uveitis in either eye, or an episode of infectious uveitis or another eye infection, within 12 months prior to the administration of the above-mentioned treatment, does not mean that the patient is not at risk of uveitis or eye infection. (12) Neither eye has age-related macular degeneration in dry eyes. (13) No high myopia (>6 diopters) (14) No history of cancer (excluding a diagnosis of basal cell carcinoma, squamous cell carcinoma, or cervical intraepithelial neoplasia with successful treatment), (15) You have never taken or ingested any medication or treatment that may cause or could cause optic neuropathy. (16) No history of nutritional deficiencies (including B12 and / or folic acid deficiency), no known serum B12 or folic acid deficiency, and no bariatric surgery, The method according to claim 86, further characterized by having any combination of (1) to (16).

88. The method according to any one of claims 1 to 87, wherein administration includes administering the pharmaceutical composition in multiple doses to the human subject.

89. The method according to claim 88, wherein administration includes administering an initial dose of the pharmaceutical composition to the human subject and a subsequent dose of the pharmaceutical composition to the human subject.

90. The method according to claim 89, wherein the subsequent dose is less than the previous dose, in accordance with instructions that the administration of the previous dose was not tolerable.

91. The method according to claim 89, wherein the subsequent dose is the same as the previous dose, in accordance with instructions that the administration of the previous dose is tolerable.

92. The method according to claim 89, wherein the subsequent dose is greater than the previous dose, in accordance with instructions that the administration of the previous dose is tolerable.

93. The method according to claim 89, wherein the subsequent dose is the same as the previous dose, in accordance with instructions that the administration of the previous dose is effective.

94. The method according to claim 89, wherein the subsequent dose is less than the previous dose, in accordance with instructions that the administration of the previous dose is effective.

95. The method according to claim 89, wherein the subsequent dose is greater than the previous dose, in accordance with instructions that the administration of the previous dose was ineffective.

96. The method according to any one of claims 1 to 95, wherein the pharmaceutical composition is administered by intraventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreous administration, subretinal injection, topical application, implantation, or intravenous injection.

97. The method according to any one of claims 1 to 87, wherein the pharmaceutical composition is administered by intravitreal injection.

98. The method according to any one of claims 1 to 97, further comprising administering an additional therapeutic agent.

99. The method according to claim 98, wherein the additional therapeutic agent comprises small molecules.

100. The method according to claim 98, wherein the additional therapeutic agent comprises an antisense oligomer.

101. The method according to claim 98, wherein the additional therapeutic agent includes an ophthalmic drug.

102. structure: 【Chemistry 4-1】 【Chemistry 4-2】 【Chemistry 4-3】 An antisense oligomer, which is a compound comprising any one of the following, or a pharmaceutically acceptable salt thereof.

103. structure: 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 An antisense oligomer having any of the following properties.

104. It is a pharmaceutical preparation, (a) an antisense oligomer, wherein the antisense oligomer contains a sequence having at least 80% sequence identity with any one of sequence numbers 6-275 or 280-299, (b) comprising a pharmaceutically acceptable diluent, A pharmaceutical formulation in which the antisense oligomer is dissolved or suspended in a solution at a concentration of approximately 1 mg / mL to approximately 200 mg / mL.

105. It is a pharmaceutical preparation, (a) Antisense oligomers (ASOs), and (b) comprising a pharmaceutically acceptable diluent, The antisense oligomer is dissolved or suspended in a solution. The aforementioned antisense oligomer has the following chemical structure: 【Chemistry 6-1】 【Chemistry 6-2】 【Transformation 6-3】 A pharmaceutical preparation comprising a compound or a pharmaceutically acceptable salt thereof, comprising any one of the following.

106. It is a pharmaceutical preparation, (a) Antisense oligomers (ASOs), and (b) comprising a pharmaceutically acceptable diluent, The antisense oligomer is dissolved or suspended in a solution. The aforementioned antisense oligomer has the following chemical structure: 【Chemistry 7-1】 【Chemistry 7-2】 【Transformation 7-3】 A pharmaceutical preparation having one of the following.

107. The pharmaceutical formulation according to claim 105 or 106, wherein the antisense oligomer is present in the solution at a concentration of about 1 mg / mL to about 200 mg / mL.

108. The aforementioned antisense oligomer is present in concentrations of approximately 5 mg / mL to approximately 200 mg / mL, approximately 10 mg / mL to approximately 200 mg / mL, approximately 15 mg / mL to approximately 200 mg / mL, approximately 20 mg / mL to approximately 200 mg / mL, approximately 25 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 200 mg / mL, approximately 100 mg / mL to approximately 200 mg / mL, and approximately 150 mg / mL to about 200 mg / mL, about 180 mg / mL to about 200 mg / mL, 2 mg / mL to about 150 mg / mL, about 5 mg / mL to about 15 0 mg / mL, about 10 mg / mL to about 150 mg / mL, about 15 mg / mL to about 150 mg / mL, about 20 mg / mL to about 150 mg / mL , about 25 mg / mL to about 150 mg / mL, about 30 mg / mL to about 150 mg / mL, about 35 mg / mL to about 150 mg / mL, about 40 m g / mL to about 150 mg / mL, about 50 mg / mL to about 150 mg / mL, about 60 mg / mL to about 150 mg / mL, about 80 mg / mL to about 150mg / mL, about 100mg / mL to about 150mg / mL, 2mg / mL to about 100mg / mL, about 5mg / mL to about 100mg / m L, about 10 mg / mL to about 100 mg / mL, about 15 mg / mL to about 100 mg / mL, about 20 mg / mL to about 100 mg / mL, about 25 m g / mL to about 100 mg / mL, about 30 mg / mL to about 100 mg / mL, about 35 mg / mL to about 100 mg / mL, about 40 mg / mL to About 100 mg / mL, about 50 mg / mL to about 100 mg / mL, about 60 mg / mL to about 100 mg / mL, about 80 mg / mL to about 100 mg / mL, 2 mg / mL to about 80 mg / mL, about 5 mg / mL to about 80 mg / mL, about 10 mg / mL to about 80 mg / mL, about 15 mg / m L to about 80 mg / mL, about 20 mg / mL to about 80 mg / mL, about 25 mg / mL to about 80 mg / mL, about 30 mg / mL to about 80 mg / m L, about 35 mg / mL to about 80 mg / mL, about 40 mg / mL to about 80 mg / mL, about 60 mg / mL to about 80 mg / mL, 2 mg / mL ~about 60mg / mL, about 5mg / mL to about 60mg / mL, about 10mg / mL to about 60mg / mL, about 15mg / mL to about 60mg / mL,A pharmaceutical preparation according to any one of claims 104 to 107, which is present in the solution at a concentration of approximately 20 mg / mL to approximately 60 mg / mL, approximately 25 mg / mL to approximately 60 mg / mL, approximately 30 mg / mL to approximately 60 mg / mL, approximately 35 mg / mL to approximately 60 mg / mL, approximately 40 mg / mL to approximately 60 mg / mL, 2 mg / mL to approximately 40 mg / mL, approximately 5 mg / mL to approximately 40 mg / mL, approximately 10 mg / mL to approximately 40 mg / mL, approximately 15 mg / mL to approximately 40 mg / mL, approximately 20 mg / mL to approximately 40 mg / mL, or approximately 25 mg / mL to approximately 40 mg / mL.

109. The pharmaceutical formulation according to claim 104, wherein the antisense oligomer is present in the solution at a concentration of approximately 1 mg / mL, approximately 2 mg / mL, approximately 3 mg / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL, approximately 7 mg / mL, approximately 8 mg / mL, approximately 9 mg / mL, approximately 10 mg / mL, approximately 12 mg / mL, approximately 14 mg / mL, approximately 15 mg / mL, approximately 16 mg / mL, approximately 18 mg / mL, approximately 20 mg / mL, approximately 22 mg / mL, approximately 24 mg / mL, approximately 26 mg / mL, approximately 28 mg / mL, approximately 30 mg / mL, approximately 35 mg / mL, approximately 40 mg / mL, approximately 50 mg / mL, approximately 60 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 140 mg / mL, approximately 160 mg / mL, approximately 180 mg / mL, or approximately 200 mg / mL.

110. The pharmaceutical formulation according to any one of claims 104 to 109, wherein the pharmaceutical composition is prepared by diluting a concentrate containing the antisense oligomer.

111. The aforementioned antisense oligomer is present in concentrations of approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 200 mg / mL, approximately 100 mg / mL to approximately 200 mg / mL, approximately 150 mg / mL to approximately 200 mg / mL, approximately 180 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 150 mg / mL, approximately 35 mg / mL to approximately 150 mg / mL, approximately 40 mg / mL to approximately 150 mg / mL, approximately 50 mg / mL to approximately 150 mg / mL, approximately 60 mg / mL to approximately 150 mg / mL, and approximately 80 mg / mL to approximately 150 mg / mL. The pharmaceutical preparation according to claim 110, wherein the concentrate contains the following concentrations: g / mL, approximately 100 mg / mL to approximately 150 mg / mL, approximately 30 mg / mL to approximately 100 mg / mL, approximately 35 mg / mL to approximately 100 mg / mL, approximately 40 mg / mL to approximately 100 mg / mL, approximately 50 mg / mL to approximately 100 mg / mL, approximately 60 mg / mL to approximately 100 mg / mL, approximately 80 mg / mL to approximately 100 mg / mL, approximately 30 mg / mL to approximately 80 mg / mL, approximately 35 mg / mL to approximately 80 mg / mL, approximately 40 mg / mL to approximately 80 mg / mL, approximately 60 mg / mL to approximately 80 mg / mL, approximately 30 mg / mL to approximately 60 mg / mL, approximately 35 mg / mL to approximately 60 mg / mL, or approximately 40 mg / mL to approximately 60 mg / mL.

112. The pharmaceutical composition according to claim 110, wherein the antisense oligomer is present in the concentrate at a concentration of approximately 40 mg / mL, approximately 45 mg / mL, approximately 50 mg / mL, approximately 55 mg / mL, approximately 60 mg / mL, approximately 65 mg / mL, approximately 70 mg / mL, approximately 80 mg / mL, approximately 90 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 140 mg / mL, approximately 160 mg / mL, approximately 180 mg / mL, or approximately 200 mg / mL.

113. The pharmaceutical preparation according to any one of claims 104 to 112, wherein the concentrate is phosphate-buffered.

114. The pharmaceutical formulation according to any one of claims 104 to 113, wherein the antisense oligomer is solubilized or diluted in a solution containing one or more of sodium chloride, sodium diphosphate, potassium monophosphate, or water for injection.

115. The pharmaceutical formulation according to any one of claims 104 to 113, wherein the antisense oligomer is solubilized or diluted in a solution comprising sodium chloride, sodium diphosphate, potassium monophosphate, and water for injection.

116. The pharmaceutical formulation according to any one of claims 104 to 113, wherein the antisense oligomer is solubilized or diluted in an isotonic solution.

117. The pharmaceutical formulation according to any one of claims 104 to 116, wherein the antisense oligomer is solubilized or diluted in a phosphate buffer at least pH 5.

8.

118. The pharmaceutical formulation according to any one of claims 104 to 116, wherein the antisense oligomer is solubilized or diluted in a phosphate buffer (pH 6.6 to 7.6).

119. The pharmaceutical preparation according to any one of claims 104 to 118, wherein the pharmaceutical preparation does not contain a preservative.

120. The pharmaceutical preparation according to any one of claims 104 to 119, wherein the pharmaceutical preparation is suitable for intravitreal injection.

121. The pharmaceutical preparation according to any one of claims 104 to 120, wherein the pharmaceutical preparation is packaged in a single-use vial.

122. It's a kit, (i) A concentrate comprising an antisense oligomer (ASO), wherein the ASO comprises a sequence having at least 80% sequence identity with any one of sequence numbers 6-275 or 280-299, (ii) A diluent wherein the concentrate is miscible with the diluent, (iii) A kit comprising instructions for diluting the concentrate with the diluent.

123. The aforementioned antisense oligomer is present in concentrations of approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, approximately 80 mg / mL to approximately 200 mg / mL, approximately 100 mg / mL to approximately 200 mg / mL, approximately 150 mg / mL to approximately 200 mg / mL, approximately 180 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 150 mg / mL, approximately 35 mg / mL to approximately 150 mg / mL, approximately 40 mg / mL to approximately 150 mg / mL, approximately 50 mg / mL to approximately 150 mg / mL, approximately 60 mg / mL to approximately 150 mg / mL, and approximately 80 mg / mL to approximately 150 mg / mL. The kit according to claim 122, wherein the concentrate contains the following concentrations: mg / mL, approximately 100 mg / mL to approximately 150 mg / mL, approximately 30 mg / mL to approximately 100 mg / mL, approximately 35 mg / mL to approximately 100 mg / mL, approximately 40 mg / mL to approximately 100 mg / mL, approximately 50 mg / mL to approximately 100 mg / mL, approximately 60 mg / mL to approximately 100 mg / mL, approximately 80 mg / mL to approximately 100 mg / mL, approximately 30 mg / mL to approximately 80 mg / mL, approximately 35 mg / mL to approximately 80 mg / mL, approximately 40 mg / mL to approximately 80 mg / mL, approximately 60 mg / mL to approximately 80 mg / mL, approximately 30 mg / mL to approximately 60 mg / mL, approximately 35 mg / mL to approximately 60 mg / mL, or approximately 40 mg / mL to approximately 60 mg / mL.

124. The kit according to claim 122, wherein the antisense oligomer is present in the concentrate at a concentration of approximately 40 mg / mL, approximately 45 mg / mL, approximately 50 mg / mL, approximately 55 mg / mL, approximately 60 mg / mL, approximately 65 mg / mL, approximately 70 mg / mL, approximately 80 mg / mL, approximately 90 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 140 mg / mL, approximately 160 mg / mL, approximately 180 mg / mL, or approximately 200 mg / mL.

125. The kit according to any one of claims 122 to 124, wherein the concentrate is phosphate-buffered.

126. The kit according to any one of claims 122 to 125, wherein the diluent is a solution containing one or more of sodium chloride, sodium diphosphate, potassium monophosphate, or water for injection.

127. The kit according to any one of claims 122 to 125, wherein the diluent is a solution comprising sodium chloride, sodium diphosphate, potassium monophosphate, and water for injection.

128. The kit according to any one of claims 122 to 127, wherein the diluent comprises an isotonic solution.

129. The kit according to any one of claims 122 to 128, wherein the diluent comprises a phosphate buffer with a pH of at least 5.

8.

130. The kit according to any one of claims 122 to 128, wherein the diluent comprises a phosphate buffer (pH 6.6 to 7.6).

131. The kit according to any one of claims 122 to 130, wherein the concentrate or diluent does not contain a preservative.

132. The kit according to any one of claims 122 to 131, wherein the instructions for diluting the concentrate with the diluent include instructions for diluting or solubilizing the ASO in the diluent to a concentration of about 2 mg / mL to 200 mg / mL.

133. The instructions for diluting the concentrate with the diluent describe diluting or solubilizing the ASO in the diluent at concentrations of approximately 5 mg / mL to approximately 200 mg / mL, approximately 10 mg / mL to approximately 200 mg / mL, approximately 15 mg / mL to approximately 200 mg / mL, approximately 20 mg / mL to approximately 200 mg / mL, approximately 25 mg / mL to approximately 200 mg / mL, approximately 30 mg / mL to approximately 200 mg / mL, approximately 35 mg / mL to approximately 200 mg / mL, approximately 40 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 60 mg / mL to approximately 200 mg / mL, and approximately 80 mg / mL to approximately 2 00mg / mL, about 100mg / mL to about 200mg / mL, about 150mg / mL to about 200mg / mL, about 180mg / mL to about 200 mg / mL, 2 mg / mL to about 150 mg / mL, about 5 mg / mL to about 150 mg / mL, about 10 mg / mL to about 150 mg / mL, about 1 5 mg / mL to about 150 mg / mL, about 20 mg / mL to about 150 mg / mL, about 25 mg / mL to about 150 mg / mL, about 30 mg / m L to about 150 mg / mL, about 35 mg / mL to about 150 mg / mL, about 40 mg / mL to about 150 mg / mL, about 50 mg / mL to about 150 mg / mL, about 60 mg / mL to about 150 mg / mL, about 80 mg / mL to about 150 mg / mL, about 100 mg / mL to about 150 mg / m L, 2 mg / mL to about 100 mg / mL, about 5 mg / mL to about 100 mg / mL, about 10 mg / mL to about 100 mg / mL, about 15 mg / mL mL to about 100 mg / mL, about 20 mg / mL to about 100 mg / mL, about 25 mg / mL to about 100 mg / mL, about 30 mg / mL to about 1 00mg / mL, about 35mg / mL to about 100mg / mL, about 40mg / mL to about 100mg / mL, about 50mg / mL to about 100mg / m L, about 60 mg / mL to about 100 mg / mL, about 80 mg / mL to about 100 mg / mL, 2 mg / mL to about 80 mg / mL, about 5 mg / m L to about 80 mg / mL, about 10 mg / mL to about 80 mg / mL, about 15 mg / mL to about 80 mg / mL, about 20 mg / mL to about 80 mg / m L, about 25 mg / mL to about 80 mg / mL, about 30 mg / mL to about 80 mg / mL, about 35 mg / mL to about 80 mg / mL, about 40 mg / mL mL to about 80 mg / mL, about 60 mg / mL to about 80 mg / mL, 2 mg / mL to about 60 mg / mL, about 5 mg / mL to about 60 mg / mL,A kit according to any one of claims 122 to 132, including instructions for adjusting the concentration to approximately 10 mg / mL to approximately 60 mg / mL, approximately 15 mg / mL to approximately 60 mg / mL, approximately 20 mg / mL to approximately 60 mg / mL, approximately 25 mg / mL to approximately 60 mg / mL, approximately 30 mg / mL to approximately 60 mg / mL, approximately 35 mg / mL to approximately 60 mg / mL, approximately 40 mg / mL to approximately 60 mg / mL, 2 mg / mL to approximately 40 mg / mL, approximately 5 mg / mL to approximately 40 mg / mL, approximately 10 mg / mL to approximately 40 mg / mL, approximately 15 mg / mL to approximately 40 mg / mL, approximately 20 mg / mL to approximately 40 mg / mL, or approximately 25 mg / mL to approximately 40 mg / mL.

134. The instructions for diluting the concentrate with the diluent describe diluting or solubilizing the antisense oligomer in the diluent in concentrations of approximately 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, and 22 mg. A kit according to any one of claims 122 to 132, comprising instructions for adjusting the concentration to approximately 1 / mL, approximately 24 mg / mL, approximately 26 mg / mL, approximately 28 mg / mL, approximately 30 mg / mL, approximately 35 mg / mL, approximately 40 mg / mL, approximately 50 mg / mL, approximately 60 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 140 mg / mL, approximately 160 mg / mL, approximately 180 mg / mL, or approximately 200 mg / mL.

135. The pharmaceutical composition or kit according to any one of claims 104 or 108-134, wherein the antisense oligomer comprises a nucleotide sequence having at least 90% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 6-275 or 280-299.

136. The pharmaceutical composition or kit according to any one of claims 104 or 108-134, wherein the antisense oligomer comprises a nucleotide sequence having at least 80%, at least 90%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 36, 236, 242, 250, 280-283, 288, and 290-292.

137. The pharmaceutical composition or kit according to any one of claims 104 or 108 to 136, wherein the antisense oligomer comprises a modification of its skeleton including a phosphorothioate bond or a phosphorodiamidate bond.

138. The pharmaceutical composition or kit according to any one of claims 104 or 108 to 137, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a loc nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-O-N-methylacetamide (2'-NMA), or a 2'-O-methoxyethyl moiety.

139. The pharmaceutical composition or kit according to any one of claims 104 or 108 to 138, wherein the antisense oligomer comprises at least one modified sugar moiety.

140. The pharmaceutical composition or kit according to claim 139, wherein each sugar portion is a modified sugar portion.

141. The pharmaceutical composition or kit according to any one of claims 104 or 108 to 140, wherein the antisense oligomer comprises 5'-methylcytosine (5'-MeC).

142. The pharmaceutical composition or kit according to any one of claims 104 or 108 to 141, wherein each cytosine in the antisense oligomer is 5'-methylcytosine (5'-MeC).

143. The pharmaceutical composition or kit according to any one of claims 104 or 108 to 142, wherein the antisense oligomer comprises 5'-methyluracil (5'-MeU).

144. The pharmaceutical composition or kit according to any one of claims 104 or 108 to 143, wherein the antisense oligomer comprises a phosphorothioate bond.

145. A pharmaceutical composition or kit according to any one of claims 104 or 108 to 144, wherein the internucleoside bonds of the ASO are phosphorothioate bonds.

146. The pharmaceutical composition or kit according to any one of claims 104 or 108 to 145, wherein the antisense oligomer comprises Loc nucleic acid (LNA).

147. The antisense oligomers are composed of nucleic acid bases of 8 to 50 lengths, 8 to 40 lengths, 8 to 35 lengths, 8 to 30 lengths, 8 to 25 lengths, 8 to 20 lengths, 8 to 15 lengths, 9 to 50 lengths, 9 to 40 lengths, 9 to 35 lengths, 9 to 30 lengths, 9 to 25 lengths, 9 to 20 lengths, 9 to 15 lengths, 10 to 50 lengths, 10 to 40 lengths, 10 to 35 lengths, 10 to 30 lengths, 10 to 25 lengths, and 10 to A pharmaceutical composition or kit according to any one of claims 104 or 108 to 146, comprising 20 nucleic acid bases, 10 to 15 nucleic acid bases, 11 to 50 nucleic acid bases, 11 to 40 nucleic acid bases, 11 to 35 nucleic acid bases, 11 to 30 nucleic acid bases, 11 to 25 nucleic acid bases, 11 to 20 nucleic acid bases, 11 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, 12 to 30 nucleic acid bases, 12 to 25 nucleic acid bases, 12 to 20 nucleic acid bases, or 12 to 15 nucleic acid bases.

148. The aforementioned antisense oligomer has the following chemical structure: 【Chemistry 8-1】 【Chemistry 8-2】 【Chemistry 8-3】 A pharmaceutical composition or kit according to any one of claims 104 or 108-134, comprising a compound of any one of the pharmaceutically acceptable salts thereof.

149. The antisense oligomer has the following chemical structure: 【Chemistry 9-1】 【Chemistry 9-2】 【Chemistry 9-3】 A pharmaceutical composition or kit according to any one of claims 104 or 108 to 134, having one of the above.

150. Use of an antisense oligomer for the treatment of a disease or condition characterized by reduced OPA1 protein expression or function in a human subject requiring such treatment, or for the manufacture of a pharmacopoeia for reducing the likelihood of the subject progressing to the disease or condition, wherein the pharmacopoeia is administered to one eye of the subject in a dose of about 0.005 mg to about 20 mg, and the antisense oligomer comprises a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 6-275 or 280-299.

151. The aforementioned pharmaceutical is the antisense oligomer in amounts of approximately 0.005 mg to 15 mg, approximately 0.005 mg to 10 mg, approximately 0.005 mg to 5 mg, approximately 0.005 mg to 1 mg, approximately 0.01 mg to 15 mg, approximately 0.01 mg to 10 mg, approximately 0.01 mg to 5 mg, approximately 0.01 mg to 2.5 mg, approximately 0.01 mg to 1.0 mg, approximately 0.01 mg to 0.5 mg, approximately 0.01 mg to 0.25 mg, approximately 0.01 mg to 0.1 mg, approximately 0.01 mg to 0.05 mg, approximately 0.05 mg to 0. The use according to claim 150, comprising administering to one eye of the subject in a dose of 10 mg, approximately 0.05 mg to approximately 5 mg, approximately 0.05 mg to approximately 2.5 mg, approximately 0.05 mg to approximately 1.0 mg, approximately 0.05 mg to approximately 0.5 mg, approximately 0.05 mg to approximately 0.25 mg, approximately 0.05 mg to approximately 0.1 mg, approximately 0.1 mg to approximately 5 mg, approximately 0.1 mg to approximately 2.5 mg, approximately 0.1 mg to approximately 1.5 mg, approximately 0.1 mg to approximately 1.0 mg, approximately 0.1 mg to approximately 0.5 mg, or approximately 0.1 mg to approximately 0.25 mg.

152. The aforementioned pharmaceutical is approximately 0.005 mg, approximately 0.01 mg, approximately 0.05 mg, approximately 0.1 mg, approximately 0.2 mg, approximately 0.3 mg, 0.4 mg, approximately 0.5 mg, approximately 0.6 mg, approximately 0.7 mg, approximately 0.8 mg, approximately 0.9 mg, approximately 1.0 mg, approximately 1.1 mg, approximately 1.2 mg, approximately 1.3 mg, approximately 1.4 mg, approximately 1.5 mg, approximately 1.75 mg, approximately 2.0 mg of the aforementioned antisense oligomer. The use according to claim 150, administered to one eye of the subject in doses of approximately 2.25 mg, approximately 2.5 mg, approximately 2.75 mg, approximately 3 mg, approximately 3.5 mg, approximately 4.0 mg, approximately 4.5 mg, approximately 5.0 mg, approximately 5.5 mg, approximately 6.0 mg, approximately 7.0 mg, approximately 8.0 mg, approximately 9.0 mg, approximately 10 mg, approximately 12.5 mg, approximately 15 mg, approximately 17.5 mg, or approximately 20 mg.

153. The aforementioned pharmaceutical is present in the following concentrations of antisense oligomer: approximately 0.1 mg to approximately 1.5 mg, approximately 0.1 mg to approximately 1.4 mg, approximately 0.1 mg to approximately 1.2 mg, approximately 0.1 mg to approximately 1.0 mg, approximately 0.1 mg to approximately 0.8 mg, approximately 0.1 mg to approximately 0.7 mg, approximately 0.1 mg to approximately 0.5 mg, approximately 0.1 mg to approximately 0.3 mg, approximately 0.2 mg to approximately 1.5 mg, and approximately 0.2 mg to approximately 1.4 mg. g, about 0.2 mg to about 1.2 mg, about 0.2 mg to about 1.0 mg, about 0.2 mg to about 0.8 mg, about 0.2 mg to about 0.7 mg, about 0.2 mg to about 0.5 mg, about 0.3 mg to about about 1.5 mg, about 0.3 mg to about 1.4 mg, about 0.3 mg to about 1.2 mg, about 0.3 mg to about 1.0 mg, about 0.3 mg to about 0.8 mg, about 0.3 mg to about 0.7 mg, about 0 .. 3 mg to about 0.5 mg, about 0.5 mg to about 1.5 mg, about 0.5 mg to about 1.4 mg, about 0.5 mg to about 1.2 mg, about 0.5 mg to about 1.0 mg, about 0.5 mg to about 0.8 mg, about 0.5 mg to about 0.7 mg, about 0.7 mg to about 1.5 mg, about 0.7 mg to about 1.4 mg, about 0.7 mg to about 1.2 mg, about 0.7 mg to about 1.0 mg, about 0.8 mg The use according to claim 150, administered to one eye of the subject in doses of approximately 1.5 mg, approximately 0.8 mg to approximately 1.4 mg, approximately 0.8 mg to approximately 1.2 mg, approximately 0.8 mg to approximately 1.0 mg, approximately 1.0 mg to approximately 1.5 mg, approximately 1.0 mg to approximately 1.4 mg, approximately 1.0 mg to approximately 1.2 mg, approximately 1.2 mg to approximately 1.5 mg, or approximately 1.2 mg to approximately 1.4 mg.

154. The use according to claim 150, wherein the pharmaceutical is administered to one eye of the subject in a dose of the antisense oligomer of approximately 0.1 mg, approximately 0.2 mg, approximately 0.3 mg, 0.4 mg, approximately 0.5 mg, approximately 0.6 mg, approximately 0.7 mg, approximately 0.8 mg, approximately 0.9 mg, approximately 1.0 mg, approximately 1.1 mg, approximately 1.2 mg, approximately 1.3 mg, approximately 1.4 mg, or approximately 1.5 mg.

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