OPA1 ANTISENSE OLIGOMERS FOR THE TREATMENT OF PATIENT CONDITIONS AND DISEASES - Patent application

Abstract

Agents that target processed mRNA, for example, the 5'UTR of processed mRNA, can regulate protein expression, for example, through regulating translation of processed mRNA. Alternative splicing events in genes can result in non-productive mRNA transcripts that can result in abnormal protein expression. Agents that target alternative splicing events in genes can regulate protein expression levels. Therapeutic agents that can regulate protein expression by targeting processed mRNA and / or alternative splicing events can promote functional protein expression and / or inhibit abnormal protein expression in patients. Such therapeutic agents can be used to treat pathologies or diseases associated with protein deficiency and / or mitochondrial dysfunction.

Description

[Technical field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 277,405, filed November 9, 2021, and U.S. Provisional Patent Application No. 63 / 277,767, filed November 10, 2021, the contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002]

[0002] Alternative splicing events in genes can result in non-productive mRNA transcripts that can lead to aberrant or reduced protein expression, and therapeutic agents that can target alternative splicing events in genes can regulate the expression levels of functional proteins and / or inhibit aberrant protein expression in patients. Such therapeutic agents can be used to treat pathologies or diseases caused by protein deficiencies.

[0003]

[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 disorders that lead to degeneration of retinal ganglion cells and progressive irreversible vision loss. The majority of ADOA patients have mutations in OPA1, with most mutations leading to haploinsufficiency (Lenaers G. et al., Orphanet J Rare Dis 2012). OPA1 encodes a mitochondrial GTPase that plays a pivotal role in mitochondrial fusion, ATP synthesis, and apoptosis. Currently, there are no approved disease-modifying treatments for ADOA patients, and there is a need for such treatments. Summary of the Invention

[0004]

[0004] In some aspects, provided herein is a method for increasing expression of OPA1 protein in a cell having processed mRNA that includes a translational control element that encodes the OPA1 protein and inhibits translation of the processed mRNA, the method comprising contacting the cell with an agent or a vector encoding the agent, wherein the agent modulates the structure of the translational control element, thereby increasing expression of OPA1 protein in the cell.

[0005]

[0005] In some aspects, provided herein is a method for increasing expression of OPA1 protein in a cell having processed mRNA that includes a translational control element that encodes the OPA1 protein and inhibits translation of the processed mRNA, comprising the step of contacting the cell with an agent or a vector encoding an agent, wherein the agent (a) binds to a targeted portion of the processed mRNA, (b) modulates the interaction between the translational control element and a factor involved in translation of the processed mRNA, or (c) is a combination of (a) and (b), thereby increasing expression of OPA1 protein in the cell.

[0006]

[0006] In some aspects, provided herein is a method for regulating expression of an OPA1 protein in a cell, comprising the step of contacting a cell with an agent or a vector encoding the agent, wherein the agent comprises an antisense oligomer having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 608-1253.

[0007]

[0007] In some aspects, provided herein is a composition comprising a drug or a vector encoding a drug, wherein the drug comprises an antisense oligomer having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 608-1253.

[0008]

[0008] In some aspects, provided herein is a composition comprising a vector encoding an agent, wherein the agent comprises a polynucleic acid comprising a sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 608-1253.

[0009]

[0009] In some aspects, provided herein is a composition comprising an agent, wherein the agent comprises an antisense oligomer that binds to a targeted portion of a processed mRNA encoding an OPA1 protein, wherein the targeted portion of the processed mRNA includes at least one nucleotide of the primary start codon of the processed mRNA or is within the 5'UTR of the processed mRNA.

[0010]

[0010] In some aspects, provided herein is a composition comprising a vector encoding an agent, wherein the agent comprises a polynucleic acid comprising a sequence that binds to a targeting portion of a processed mRNA encoding an OPA1 protein, wherein the targeting portion of the processed mRNA comprises at least one nucleotide of the primary start codon of the processed mRNA or is within the 5'UTR of the processed mRNA.

[0011]

[0011] In some aspects, provided herein is a composition comprising an agent, wherein the agent regulates the structure of a translational control element of a processed mRNA encoding OPA1 protein, thereby increasing expression of the OPA1 protein, and the translational control element inhibits translation of the processed mRNA.

[0012]

[0012] In some aspects, provided herein is a composition comprising a vector encoding an agent, wherein the agent regulates the structure of a translational control element of a processed mRNA encoding OPA1 protein, thereby increasing expression of the OPA1 protein, and the translational control element inhibits translation of the processed mRNA.

[0013]

[0013] In some aspects, provided herein is a composition comprising an agent that increases translation of processed mRNA in a cell, the processed mRNA encoding an OPA1 protein and comprising a translation control element that inhibits translation of the processed mRNA, and the agent modulates the structure of the translation control element, thereby increasing the translation efficiency and / or rate of translation of the processed mRNA, and the agent (a) binds to a targeted portion of the processed mRNA, (b) modulates the interaction of the translation control element with a factor involved in translation of the processed mRNA, or (c) a combination of (a) and (b).

[0014]

[0014] In some aspects, provided herein is a composition comprising a vector encoding an agent, wherein the agent increases translation of processed mRNA in a cell, the processed mRNA encoding an OPA1 protein and comprising a translation control element that inhibits translation of the processed mRNA, and the agent modulates the structure of the translation control element, thereby increasing the translation efficiency and / or rate of translation of the processed mRNA, and the agent (a) binds to a targeted portion of the processed mRNA, (b) modulates the interaction of the translation control element with a factor involved in translation of the processed mRNA, or (c) a combination of (a) and (b).

[0015]

[0015] In some aspects, provided herein is a method for increasing expression of a target protein in a cell having a processed mRNA that includes a translational control element that encodes the target protein and inhibits translation of the processed mRNA, the method comprising the steps of delivering to the cell (1) a first agent or a first nucleic acid sequence encoding the first agent, and (2) a second agent or a second nucleic acid sequence encoding the second agent, wherein the first agent regulates splicing of a pre-mRNA transcribed from a target gene that encodes the target protein, and the second agent regulates the structure of the translational control element of the processed mRNA that encodes the target protein, thereby increasing expression of the target protein in the cell.

[0016]

[0016] In some aspects, provided herein is a method for increasing expression of a target protein in a cell having a processed mRNA that includes a translational control element that encodes the target protein and inhibits translation of the processed mRNA, the method comprising the step of delivering to the cell (1) a first agent or a first nucleic acid sequence encoding the first agent, and (2) a second agent or a second nucleic acid sequence encoding the second agent, wherein the first agent modulates splicing of a pre-mRNA transcribed from a target gene that encodes the target protein, and the second agent (a) binds to a targeted portion of the processed mRNA, (b) modulates the interaction of the translational control element with a factor involved in translation of the processed mRNA, or (c) a combination of (a) and (b), thereby increasing expression of the target protein in the cell.

[0017]

[0017] In some aspects, provided herein is a method for regulating expression of a target protein in a cell, comprising the step of contacting a cell with (1) a first agent or a first nucleic acid sequence encoding the first agent, and (2) a second agent or a second nucleic acid sequence encoding the second agent, wherein the first agent comprises a first antisense oligomer having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6-275 and 280-299, the second agent comprises a second antisense oligomer that binds to the 5'UTR of a processed mRNA encoding the target protein, and the target protein is OPA1 protein.

[0018]

[0018] In some aspects, provided herein is a pharmaceutical composition comprising: (1) a first therapeutic agent or a first nucleic acid sequence encoding the first therapeutic agent; and (2) a second therapeutic agent or a second nucleic acid sequence encoding the second therapeutic agent, wherein the first therapeutic agent comprises a first antisense oligomer having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6-275 and 280-299, and the second therapeutic agent comprises a second antisense oligomer that binds to the 5'UTR of a processed mRNA encoding the OPA1 protein.

[0019]

[0019] In some aspects, provided herein is a composition comprising an antisense oligomer having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 608-1253, wherein the antisense oligomer comprises a backbone modification, a sugar moiety modification, or a combination thereof.

[0020]

[0020] In some aspects, provided herein is a pharmaceutical composition comprising a composition disclosed herein and a pharma- ceutically acceptable excipient.

[0021]

[0021] In some aspects, provided herein is a pharmaceutical composition comprising a therapeutic agent or a vector encoding a therapeutic agent and a pharma- ceutically acceptable carrier or excipient, wherein the therapeutic agent modulates the structure of a translational control element of a processed mRNA encoding OPA1 protein, thereby increasing expression of OPA1 protein, and the translational control element inhibits translation of the processed mRNA.

[0022]

[0022] In some aspects, provided herein is a pharmaceutical composition comprising a therapeutic agent or a vector encoding a therapeutic agent and a pharma- ceutically acceptable carrier or excipient, wherein the therapeutic agent (a) binds to a targeted portion of a processed mRNA that encodes OPA1 protein and contains a translational control element, (b) modulates the interaction of the translational control element with a factor involved in translation of the processed mRNA, or (c) is a combination of (a) and (b), thereby increasing expression of OPA1 protein in the cell, and wherein the translational control element inhibits translation of the processed mRNA.

[0023]

[0023] In some aspects, provided herein is a pharmaceutical composition comprising a first therapeutic agent of the method disclosed herein, a second therapeutic agent, and a pharma- ceutically acceptable excipient.

[0024]

[0024] In some aspects, provided herein is a kit comprising a first therapeutic agent of the methods disclosed herein in a first container and a second therapeutic agent of the methods disclosed herein in a second container.

[0025]

[0025] In some aspects, provided herein is a pharmaceutical composition comprising a first vector encoding a first therapeutic agent of the methods disclosed herein, a second vector encoding a second therapeutic agent, and a pharma- ceutically acceptable excipient.

[0026]

[0026] In some aspects, provided herein is a kit comprising a first vector of the method disclosed herein, a first therapeutic agent in a first container, and a first vector of the method disclosed herein, a second therapeutic agent in a second container.

[0027]

[0027] In some aspects, provided herein is a pharmaceutical composition comprising a vector encoding a first therapeutic agent and a second therapeutic agent of the methods disclosed herein and a pharma- ceutically acceptable excipient.

[0028]

[0028] In some aspects, provided herein is a pharmaceutical composition comprising: (1) a first therapeutic agent or a first nucleic acid sequence encoding the first therapeutic agent; (2) a second therapeutic agent or a second nucleic acid sequence encoding the second therapeutic agent; and (3) a pharma- ceutically acceptable carrier or excipient, wherein the first therapeutic agent modulates splicing of a pre-mRNA transcribed from a target gene encoding a target protein; and the second therapeutic agent (a) binds to a targeted portion of the processed mRNA that encodes the OPA1 protein and contains a translational control element; (b) modulates the interaction of the translational control element with a factor involved in translation of the processed mRNA; or (c) a combination of (a) and (b), wherein the translational control element inhibits translation of the processed mRNA.

[0029]

[0029] In some aspects, provided herein is a pharmaceutical composition comprising: (1) a first therapeutic agent or a first nucleic acid sequence encoding the first therapeutic agent; (2) a second therapeutic agent or a second nucleic acid sequence encoding the second therapeutic agent; and (3) a pharma- ceutically acceptable carrier or excipient, wherein the first therapeutic agent modulates splicing of a pre-mRNA transcribed from a target gene encoding a target protein; and the second therapeutic agent modulates the structure of a translation control element of a processed mRNA encoding the target protein, and the translation control element inhibits translation of the processed mRNA.

[0030]

[0030] In some aspects, provided herein is a method for treating a disease or condition in a subject or reducing the likelihood of developing a disease or condition by regulating expression of OPA1 protein in cells of a subject in need of such treatment, the method comprising contacting cells of the subject with a therapeutic agent disclosed herein, or a first therapeutic agent and a second therapeutic agent disclosed herein. Reference

[0031] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0031]

[0032] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief description of the drawings]

[0032] [Figure 1]

[0033] FIG. 1 is a bar graph showing the fold change in OPA1 protein levels in cells treated with exemplary ASOs according to some embodiments of the present disclosure. [Figure 2A]

[0034] 2A and 2B show the secondary structure of the 5'UTR region and the major start codon of the OPA1 transcript ENST00000361908 based on computational simulations using two different software tools. [Figure 2B] 2A and 2B show the secondary structure of the 5'UTR region and the major start codon of the OPA1 transcript ENST00000361908 based on computational simulations using two different software tools. [Diagram 3]FIG. 3 shows the secondary structure of the 5′UTR region and the major start codon of the OPA1 transcript ENST00000361908 based on computational simulations using two different software tools. [Figure 4]

[0035] Figures 4-5 show the computer predicted secondary structures of the region covering the 5'UTR and major start codon of the OPA1 processed mRNA upon binding to exemplary ASOs ASO-U35 and ASO-U34, respectively. [Diagram 5] Figures 4-5 show the computer predicted secondary structures of the region covering the 5'UTR and major start codon of the OPA1 processed mRNA upon binding to exemplary ASOs ASO-U35 and ASO-U34, respectively. [Figure 6]

[0036] FIG. 6 shows the computer predicted secondary structure of the region covering the 5′UTR and major start codon of the OPA1 processed mRNA upon binding to an exemplary ASO, ASO-U33. [Figure 7]

[0037] Figures 7 to 9 show the computer predicted secondary structures of the region covering the 5'UTR and major start codon of the processed mRNA of OPA1 upon binding with exemplary ASOs ASO-U37, ASO-U35 and ASO-U33, respectively. [Figure 8] Figures 7 to 9 show the computer predicted secondary structures of the region covering the 5'UTR and major start codon of the processed mRNA of OPA1 upon binding with exemplary ASOs ASO-U37, ASO-U35 and ASO-U33, respectively. [Figure 9] Figures 7 to 9 show the computer predicted secondary structures of the region covering the 5'UTR and major start codon of the processed mRNA of OPA1 upon binding with exemplary ASOs ASO-U37, ASO-U35 and ASO-U33, respectively. [Figure 10]

[0038] FIG. 10 shows the computer predicted secondary structure of the region covering the 5′UTR and major start codon of the OPA1 processed mRNA upon binding to an exemplary ASO, ASO-U39. [Figure 11A]

[0039] 11A-11C are schematic diagrams of target mRNAs containing nonsense-mediated mRNA decay-inducing exons (NMD exon mRNAs) and therapeutic agent-mediated elimination of nonsense-mediated mRNA decay-inducing exons, which increase expression of full-length target proteins or functional RNAs. FIG. 11A shows a cell separated into nuclear and cytoplasmic compartments. In the nucleus, a pre-mRNA transcript of a target gene undergoes splicing to generate an mRNA, which is transported to the cytoplasm and translated into a target protein. For this target gene, a certain fraction of the mRNA contains the nonsense-mediated mRNA decay-inducing exon (NMD exon mRNA) that is degraded in the cytoplasm and thus does not result in target protein production. FIG. 11B shows an example of the same cell separated into nuclear and cytoplasmic compartments. Treatment with a therapeutic agent, e.g., an antisense oligomer (ASO), promotes the elimination of the nonsense-mediated mRNA decay-inducing exon, resulting in an increase in the mRNA, which is then translated into higher levels of the target protein. FIG. 11C shows an exemplary schematic of a novel nonsense-mediated decay (NMD) exon inclusion event (exon X) identified in the OPA1 gene, which leads to the introduction of a premature stop codon (PTC) resulting in a nonproductive mRNA transcript that is degraded by NMD. [Figure 11B]11A-11C are schematic diagrams of target mRNAs containing nonsense-mediated mRNA decay-inducing exons (NMD exon mRNAs) and therapeutic agent-mediated elimination of nonsense-mediated mRNA decay-inducing exons, which increase expression of full-length target proteins or functional RNAs. FIG. 11A shows a cell separated into nuclear and cytoplasmic compartments. In the nucleus, a pre-mRNA transcript of a target gene undergoes splicing to generate an mRNA, which is transported to the cytoplasm and translated into a target protein. For this target gene, a certain fraction of the mRNA contains the nonsense-mediated mRNA decay-inducing exon (NMD exon mRNA) that is degraded in the cytoplasm and thus does not result in target protein production. FIG. 11B shows an example of the same cell separated into nuclear and cytoplasmic compartments. Treatment with a therapeutic agent, e.g., an antisense oligomer (ASO), promotes the elimination of the nonsense-mediated mRNA decay-inducing exon, resulting in an increase in the mRNA, which is then translated into higher levels of the target protein. FIG. 11C shows an exemplary schematic of a novel nonsense-mediated decay (NMD) exon inclusion event (exon X) identified in the OPA1 gene, which leads to the introduction of a premature stop codon (PTC) resulting in a nonproductive mRNA transcript that is degraded by NMD. [Figure 11C]11A-11C are schematic diagrams of target mRNAs containing nonsense-mediated mRNA decay-inducing exons (NMD exon mRNAs) and therapeutic agent-mediated elimination of nonsense-mediated mRNA decay-inducing exons, which increase expression of full-length target proteins or functional RNAs. FIG. 11A shows a cell separated into nuclear and cytoplasmic compartments. In the nucleus, a pre-mRNA transcript of a target gene undergoes splicing to generate an mRNA, which is transported to the cytoplasm and translated into a target protein. For this target gene, a certain fraction of the mRNA contains the nonsense-mediated mRNA decay-inducing exon (NMD exon mRNA) that is degraded in the cytoplasm and thus does not result in target protein production. FIG. 11B shows an example of the same cell separated into nuclear and cytoplasmic compartments. Treatment with a therapeutic agent, e.g., an antisense oligomer (ASO), promotes the elimination of the nonsense-mediated mRNA decay-inducing exon, resulting in an increase in the mRNA, which is then translated into higher levels of the target protein. FIG. 11C shows an exemplary schematic of a novel nonsense-mediated decay (NMD) exon inclusion event (exon X) identified in the OPA1 gene, which leads to the introduction of a premature stop codon (PTC) resulting in a nonproductive mRNA transcript that is degraded by NMD. [Figure 12]

[0040] FIG. 12 shows the identification of an exemplary nonsense-mediated mRNA decay (NMD)-induced exon in the OPA1 gene. The identification of an NMD-induced exon in the OPA1 gene using RNA sequencing is shown as visualized in the UCSC genome browser. The top panel shows a graphical representation of the OPA1 gene to scale. Peaks corresponding to RNA sequencing reads were identified in intron GRCh38 / hg38:chr3 193626204-193631611 and are shown in the middle panel. Bioinformatics analysis identified an exon-like sequence (bottom panel, sequence highlighted in capital letters; GRCh38 / hg38:chr3 193628509-193628616) flanked by 3' and 5' splice sites. Inclusion of this exon results in the introduction of a premature stop codon, making the transcript a target for NMD. FIG. 12 discloses SEQ ID NO: 300. [Figure 13]

[0041] FIG. 13 shows the identification of an exemplary nonsense-mediated mRNA decay (NMD)-induced exon in the OPA1 gene. Identification of an NMD-induced exon in the OPA1 gene using RNA sequencing, visualized in the UCSC genome browser. The top panel shows a graphical representation of the OPA1 gene to scale. Peaks corresponding to RNA sequencing reads were identified in intron GRCh38 / hg38:chr3 193593374-193614710 and shown in the middle panel. Bioinformatics analysis identified an exon-like sequence (bottom panel, sequence highlighted in capital letters; GRCh38 / hg38:chr3 193603500-193603557) flanked by 3' and 5' splice sites. Inclusion of this exon results in the introduction of a premature stop codon, making the transcript a target for NMD. FIG. 13 discloses SEQ ID NO: 301. [Figure 14]

[0042] Figure 14 shows confirmation of NMD-induced exons via puromycin or cycloheximide treatment in various cell lines, as well as confirmation of NMD-induced exons in brain and retina samples. RT-PCR analysis using total RNA from water-treated, DMSO-treated, puromycin-treated, or cycloheximide-treated cells confirmed the presence of a band corresponding to the NMD-induced exon 7x (GRCh38 / hg38:chr3 193628509-193628616) of the OPA1 gene. [Figure 15]

[0043] FIG. 15 shows an exemplary ASO walk around the OPA1 exon 7x (GRCh38 / hg38:chr3 193628509 193628616) region. A graphical representation of ASO walks performed around the OPA1 exon 7x (GRCh38 / hg38:chr3 193628509 193628616) region targeting sequences upstream of the 3' splice site, across the 3' splice site, exon 7x, across the 5' splice site, and downstream of the 5' splice site. ASOs were designed to encompass these regions by moving 5 nucleotides at a time, or 3 nucleotides across the splice site region. FIG. 15 discloses SEQ ID NOs: 302-304, respectively, in order of appearance. [Figure 16]

[0044] Figure 16 shows OPA1 exon 7x (GRCh38 / hg38:chr3 193628509 193628616) region ASO walking assessed by Taqman RT-qPCR. A graph of the fold change in OPA1 productive mRNA product compared to sham is plotted. [Figure 17]

[0045] Figure 17 shows OPA1 exon 7x (GRCh38 / hg38:chr3 193628509 193628616) region ASO walking assessed by Taqman RT-qPCR. A graph of the fold change of OPA1 productive mRNA product compared to sham is plotted. [Figure 18]

[0046] FIG. 18 shows expression of OPA1 transcripts containing the NMD exon in HEK293 cells treated with increasing amounts of cycloheximide. [Figure 19A]

[0047] Figure 19A shows RT-PCR data from the posterior eye of Chlorocebus sabaeus (green monkey) at postnatal day P93 (3 months) and postnatal day P942 (2.6 years), confirming expression of OPA1 transcripts containing the NMD exon in these cells. [Figure 19B]

[0048] FIG. 19B shows quantification of NMD exon abundance from FIG. 19A. [Figure 20A]

[0049] FIG. 20A shows RT-PCR of productive and non-productive OPA1 mRNA following treatment of HEK293 cells with various ASOs and cycloheximide. [Figure 20B]

[0050] FIG. 20B shows a quantification of the data in FIG. 20A. [Figure 21]

[0051] FIG. 21 shows productive OPA1 mRNA expression by quantitative PCR in HEK293 cells treated with various ASOs and without cycloheximide. [Figure 22A]

[0052] FIG. 22A shows RT-PCR of non-productive OPA1 mRNA in HEK293 cells after treatment with ASO-14 and cycloheximide. [Figure 22B]

[0053] FIG. 22B shows quantification of productive OPA1 mRNA in HEK293 cells following treatment with ASO-14 in the absence of cycloheximide. [Figure 22C]

[0054] Figure 22C shows OPA1 protein expression in HEK293 cells after treatment with ASO-14 in the absence of cycloheximide. [Figure 23A]

[0055] FIG. 23A shows OPA1 gene mRNA and protein levels in OPA1 haploinsufficient (OPA1+ / −) HEK293 cells. [Figure 23B]

[0056] Figure 23B shows OPA1 protein expression in OPA1 haploinsufficient (OPA1+ / -) HEK293 cells following treatment with ASO-14. [Figure 23C]

[0057] Figure 23C shows quantification of OPA1 protein expression in OPA1 haploinsufficient (OPA1+ / -) HEK293 cells following treatment with ASO-14. [Figure 24A]

[0058] FIG. 24A shows the study design for the in vivo rabbit experiment of Example 2.14. [Figure 24B]

[0059] FIG. 24B shows the levels of productive and non-productive OPA1 mRNA and protein. [Figure 24C]

[0060] FIG. 24C shows quantification of the data from FIG. 24B. [Figure 25-1]

[0061] Figure 25 shows an exemplary OPA1 ASO of the present disclosure. The two right columns in the diagram show the chemical modifications of the exemplary ASOs. Each nucleotide of all ASOs has a 2'-O-methoxyethyl (2'MOE) modification ("MOE") unless otherwise noted, e.g., the larger font size (e.g., G) is a locked nucleic acid ("LNA"), the underlined letter (e.g., C) is a 5' methyl-cytosine with a 2'-MOE moiety ("5MeC-MOE"), and some ASOs are annotated as phosphorodiamidate morpholino oligomers ("PMO"). FIG. 25 shows, in column order, SEQ ID NOs: 6 to 148, 148, 148, 149, 149, 149, 150, 150, 150 to 151, 151, 151, 123, 152, 152, 152 to 153, 153, 153 to 154, 154, 154, 144 to 146, 93, 81 to 82, 36, 155, 155 to 156, 156 to 157, 157 to 161, 125, 162, 126, 163 to 166, 92, 167 to 179, 156, 180, 157, 159, 181, 160, 182, 161, 183 to 275, and 305 to 607, respectively. [Figure 25-2] Same as Figure 25-1. [Figure 25-3] Same as Figure 25-1. [Figure 25-4] Same as Figure 25-1. [Figure 25-5] Same as Figure 25-1. [Figure 25-6] Same as Figure 25-1. [Figure 25-7] Same as Figure 25-1. [Figure 25-8] Same as Figure 25-1. [Figure 25-9] Same as Figure 25-1. [Figure 25-10] Same as Figure 25-1. [Figure 25-11] Same as Figure 25-1. [Figure 25-12] Same as Figure 25-1. [Figure 25-13] Same as Figure 25-1. [Figure 25-14] Same as Figure 25-1. [Figure 25-15] Same as Figure 25-1. [Figure 25-16] Same as Figure 25-1. [Figure 26A]

[0062] Figure 26A shows RT-PCR results for OPA1 mRNA using a probe spanning exon 7 and exon 8 in HEK293 cells after treatment with ASO-14 and cycloheximide. [Figure 26B]

[0063] Figure 26B shows quantification of OPA1 mRNA in HEK293 cells after treatment with ASO-14 in the absence of cycloheximide based on qPCR using a probe spanning exons 6 and 8, a probe spanning exons 7 and 8, or a probe spanning exons 23 and 24. [Figure 26C]

[0064] Figure 26C shows sequencing data regarding the relative amounts of various OPA1 mRNA transcripts in ASO-14-transfected HEK293 cells. [Figure 27A]

[0065] Figure 27A shows RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 in HEK293 cells after treatment with various exemplary OPA1 ASOs. [Figure 27B]

[0066] Figure 27B shows the relative ratio of OPA1 mRNA transcripts with exons 6, 7, and 8 in tandem ("6-7-8") to the total amount of "6-7-8" transcripts and transcripts with exons 6 and 8 in tandem ("6-8") in HEK293 cells after treatment with various exemplary OPA1 ASOs. [Figure 27C]

[0067] Figures 27C and 27D show quantification of OPA1 mRNA using probes spanning exons 6 and 8 and probes spanning exons 7 and 8, respectively, in HEK293 cells after treatment with various exemplary OPA1 ASOs. [Figure 27D]Figures 27C and 27D show quantification of OPA1 mRNA using probes spanning exons 6 and 8 and probes spanning exons 7 and 8, respectively, in HEK293 cells after treatment with various exemplary OPA1 ASOs. [Figure 28A]

[0068] Figure 28A shows RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("Exon 6-8 PCR"), or using a probe spanning exon 7x and exon 8 ("Exon 7x-8 PCR"), in HEK293 cells after treatment with various exemplary OPA1 ASOs and treatment with cycloheximide. [Figure 28B]

[0069] Figure 28B shows OPA1 protein expression levels in HEK293 cells after treatment with various exemplary OPA1 ASOs. [Figure 28C]

[0070] Figure 28C shows dose response in OPA1 mRNA using a probe spanning exon 6 and exon 8 in HEK293 cells after treatment with various exemplary OPA1 ASOs. [Figure 28D]

[0071] Figures 28D and 28E show quantification of dose response in OPA1 mRNA using a probe spanning exons 6 and 8, a probe spanning exons 7 and 8, and a probe spanning exons 23 and 24, respectively, in HEK293 cells following treatment with various exemplary OPA1 ASOs. Figure 28D summarizes the Ct values ​​of the qPCR reactions, and Figure 28E summarizes the relative amounts. [Figure 28E] Figures 28D and 28E show quantification of dose response in OPA1 mRNA using a probe spanning exons 6 and 8, a probe spanning exons 7 and 8, and a probe spanning exons 23 and 24, respectively, in HEK293 cells following treatment with various exemplary OPA1 ASOs. Figure 28D summarizes the Ct values ​​of the qPCR reactions, and Figure 28E summarizes the relative amounts. [Figure 28F]

[0072] Figure 28F shows the dose response in OPA1 protein expression levels in HEK293 cells following treatment with various exemplary OPA1 ASOs. [Figure 29A]

[0073] Figures 29A-29D show RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("exon 6-8") or a probe spanning exon 7x and exon 8 ("exon 7-8") in HEK293 cells after treatment with various exemplary OPA1 ASO 18-mers and with or without cycloheximide. [Figure 29B] Figures 29A-29D show RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("exon 6-8") or a probe spanning exon 7x and exon 8 ("exon 7-8") in HEK293 cells after treatment with various exemplary OPA1 ASO 18-mers and with or without cycloheximide. [Figure 29C] Figures 29A-29D show RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("exon 6-8") or a probe spanning exon 7x and exon 8 ("exon 7-8") in HEK293 cells after treatment with various exemplary OPA1 ASO 18-mers and with or without cycloheximide. [Figure 29D] Figures 29A-29D show RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("exon 6-8") or a probe spanning exon 7x and exon 8 ("exon 7-8") in HEK293 cells after treatment with various exemplary OPA1 ASO 18-mers and with or without cycloheximide. [Diagram 30]

[0074] Figure 30A shows RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("exon 6-8") or a probe spanning exon 7x and exon 8 ("exon 7x-8") in HEK293 cells after treatment with various exemplary OPA1 ASO 18-mers and with or without cycloheximide. Figure 30B shows RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("exon 6-8") or a probe spanning exon 7x and exon 8 ("exon 7x-8") in HEK293 cells after treatment with various exemplary OPA1 ASO 18-mers and with or without cycloheximide. [Figure 31A]

[0075] Figures 31A-31D show RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("exon 6-8") or a probe spanning exon 7x and exon 8 ("exon 7-8") in HEK293 cells after treatment with various exemplary OPA1 ASO 16-mers and with or without cycloheximide. [Figure 31B] Figures 31A-31D show RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("exon 6-8") or a probe spanning exon 7x and exon 8 ("exon 7-8") in HEK293 cells after treatment with various exemplary OPA1 ASO 16-mers and with or without cycloheximide. [Figure 31C] Figures 31A-31D show RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("exon 6-8") or a probe spanning exon 7x and exon 8 ("exon 7-8") in HEK293 cells after treatment with various exemplary OPA1 ASO 16-mers and with or without cycloheximide. [Figure 31D]Figures 31A-31D show RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("exon 6-8") or a probe spanning exon 7x and exon 8 ("exon 7-8") in HEK293 cells after treatment with various exemplary OPA1 ASO 16-mers and with or without cycloheximide. [Figure 32-1]

[0076] Figure 32A shows RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("exon 6-8") or a probe spanning exon 7x and exon 8 ("exon 7x-8") in HEK293 cells after treatment with various exemplary OPA1 ASO 15-mers and with or without cycloheximide. Figure 32B shows RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("exon 6-8") or a probe spanning exon 7x and exon 8 ("exon 7x-8") in HEK293 cells after treatment with various exemplary OPA1 ASO 15-mers and with or without cycloheximide. Figure 30C shows RT-PCR results for OPA1 mRNA using a probe spanning exon 6 and exon 8 ("exon 6-8") or a probe spanning exon 7x and exon 8 ("exon 7x-8") in HEK293 cells after treatment with various exemplary OPA1 ASO 15-mers and with or without cycloheximide. [Figure 32-2] Same as Figure 32-1. [Figure 33A]

[0077] Figures 33A-33B show dose responses in OPA1 mRNA having exon 6 and exon 8 ("6-8"), having exon 7 and exon 8 ("7-8"), or having exon 7x and exon 8 ("7x-8") in HEK293 cells following treatment with various exemplary OPA1 ASOs at different concentrations. [Figure 33B]Figures 33A-33B show dose responses in OPA1 mRNA having exon 6 and exon 8 ("6-8"), having exon 7 and exon 8 ("7-8"), or having exon 7x and exon 8 ("7x-8") in HEK293 cells following treatment with various exemplary OPA1 ASOs at different concentrations. [Figure 34A]

[0078] Figure 34A is a histogram demonstrating that ATP levels were reduced in mock-treated OPA1+ / - HEK293 cells compared to OPA1+ / + HEK293 cells, and that ASO-14 treatment of OPA1+ / - HEK293 cells increased ATP levels in the cells. [Figure 34B]

[0079] Figures 34B-34C demonstrate that OPA1 protein was increased by ASO-14 in OPA1+ / + HEK293 cells. Figure 34B shows immunoblot gel images of OPA1 and β-actin proteins, and Figure 34C is a histogram summarizing the quantification of the immunoblot results. [Figure 34C] Figures 34B-34C demonstrate that OPA1 protein was increased by ASO-14 in OPA1+ / + HEK293 cells. Figure 34B shows immunoblot gel images of OPA1 and β-actin proteins, and Figure 34C is a histogram summarizing the quantification of the immunoblot results. [Figure 35-1]

[0080] Figure 35A shows a histogram demonstrating that the mRNA expression of OPA1 gene is reduced in fibroblasts derived from diagnosed patients with haploinsufficient mutations of OPA1 gene compared to wild-type (WT) fibroblasts. Figure 35B shows a histogram demonstrating that the protein expression of OPA1 gene is reduced in fibroblasts derived from diagnosed patients with haploinsufficient mutations of OPA1 gene compared to wild-type (WT) fibroblasts. Figure 35C shows a representative immunoblot image of OPA protein expression levels in diseased fibroblasts. [Figure 35-2] Same as Figure 35-1. [Figure 36A]

[0081] Figures 36A, 36B, and 36D show histograms demonstrating that the exemplary antisense oligomer ASO-14 reduced OPA1 NMD exon inclusion (Figure 36A) and increased OPA1 total mRNA (Figure 36B) and protein (Figure 36D) levels in wild-type (WT) fibroblasts and fibroblasts derived from a patient diagnosed with a haploinsufficient mutation in the OPA1 gene. Figure 36C shows representative immunoblot images of OPA1 protein and loading control β-tubulin under all types of conditions. [Figure 36B] Figures 36A, 36B, and 36D show histograms demonstrating that the exemplary antisense oligomer ASO-14 reduced OPA1 NMD exon inclusion (Figure 36A) and increased OPA1 total mRNA (Figure 36B) and protein (Figure 36D) levels in wild-type (WT) fibroblasts and fibroblasts derived from a patient diagnosed with a haploinsufficient mutation in the OPA1 gene. Figure 36C shows representative immunoblot images of OPA1 protein and loading control β-tubulin under all types of conditions. [Figure 36C] Figures 36A, 36B, and 36D show histograms demonstrating that the exemplary antisense oligomer ASO-14 reduced OPA1 NMD exon inclusion (Figure 36A) and increased OPA1 total mRNA (Figure 36B) and protein (Figure 36D) levels in wild-type (WT) fibroblasts and fibroblasts derived from a patient diagnosed with a haploinsufficient mutation in the OPA1 gene. Figure 36C shows representative immunoblot images of OPA1 protein and loading control β-tubulin under all types of conditions. [Figure 36D]Figures 36A, 36B, and 36D show histograms demonstrating that the exemplary antisense oligomer ASO-14 reduced OPA1 NMD exon inclusion (Figure 36A) and increased OPA1 total mRNA (Figure 36B) and protein (Figure 36D) levels in wild-type (WT) fibroblasts and fibroblasts derived from a patient diagnosed with a haploinsufficient mutation in the OPA1 gene. Figure 36C shows representative immunoblot images of OPA1 protein and loading control β-tubulin under all types of conditions. [Figure 37-1]

[0082] Figures 37A-37E demonstrate that patient fibroblasts (cell lines F35 and F36) exhibit reduced mitochondrial bioenergetics. Figure 37A shows representative time courses of oxygen consumption rates for WT, F35 and F36 cells at baseline levels and when sequentially exposed to oligomycin, FCCP, rotenone and antimycin A. Figure 37B shows histograms demonstrating that patient fibroblasts F35 and F36 cells had reduced basal oxygen consumption rates compared to WT fibroblasts. Figure 37C shows histograms demonstrating that patient fibroblasts F35 and F36 cells had reduced ATP production-related respiration compared to WT fibroblasts. Figure 37D shows histograms demonstrating that patient fibroblasts F35 and F36 cells had reduced maximal respiration compared to WT fibroblasts. FIG. 37E shows histograms demonstrating that patient fibroblasts, F35 and F36 cells, had reduced spare respiratory capacity compared to WT fibroblasts. [Figure 37-2] Same as Figure 37-1. [Figure 38-1]

[0083] Figure 38A shows histograms demonstrating that treatment with ASO-14 at 20 nM, 40 nM and 60 nM dose-dependently improved the basal oxygen consumption rate of F35 patient cells. Figure 38B shows histograms demonstrating that treatment with ASO-14 at 20 nM, 40 nM and 60 nM dose-dependently improved the ATP production-related respiration of F35 patient cells. Figure 38C shows histograms demonstrating that treatment with ASO-14 at 20 nM, 40 nM and 60 nM dose-dependently improved the maximal respiration of F35 patient cells. Figure 38D shows histograms demonstrating that treatment with ASO-14 at 20 nM, 40 nM and 60 nM dose-dependently improved the spare respiratory capacity of F35 patient cells. [Figure 38-2] Same as Figure 38-1. [Figure 39-1]

[0084] Figure 39A shows histograms demonstrating that treatment with ASO-14 at 20 nM, 40 nM and 60 nM dose-dependently improved the basal oxygen consumption rate of F36 patient cells. Figure 39B shows histograms demonstrating that treatment with ASO-14 at 20 nM, 40 nM and 60 nM dose-dependently improved the ATP production-related respiration of F36 patient cells. Figure 39C shows histograms demonstrating that treatment with ASO-14 at 20 nM, 40 nM and 60 nM dose-dependently improved the maximal respiration of F36 patient cells. Figure 39D shows histograms demonstrating that treatment with ASO-14 at 20 nM, 40 nM and 60 nM dose-dependently improved the spare respiratory capacity of F36 patient cells. [Figure 39-2] Same as Figure 39-1. [Diagram 40]

[0085] FIG. 40 shows a schematic diagram showing the 5'UTR regions of a series of different OPA1 mRNA transcripts, with overlaid gray boxes indicating the location of the start codons of the three upstream open reading frames and the main start codon, with the main start codon shown to have the best Kozak sequence surrounding it. [Diagram 41]

[0086] Figure 41 shows a schematic diagram depicting the 5'UTR region of a series of different OPA1 mRNA transcripts, with (1) overlaid grey boxes indicating the location of the start codons of three upstream open reading frames and the main start codon, and (2) a zoomed-in view of the sequence of a G-quadruplex motif predicted to be present in the 5'UTR according to G4IPDB. See Mishra SK et al., G4IPDB: A database for G-quadruplex structure forming nucleic acid interacting proteins. Sci Rep. 2016 Dec. 1;6:38144. doi:10.1038 / srep38144. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033]

[0087] Agents that modulate transcription of pre-mRNA from a gene, splicing of the pre-mRNA into mature mRNA (or "processed mRNA"), and / or translation of the mature mRNA can modulate expression of the protein encoded by the gene. In some aspects, provided herein are methods, compositions, and kits related to agents that modulate protein expression by modulating mRNA splicing and / or translation. In some aspects, provided herein are methods, compositions, and kits related to vectors that encode agents that modulate protein expression by modulating mRNA splicing and / or translation.

[0034]

[0088] In some aspects, methods, compositions, and kits are provided herein for agents, e.g., therapeutic agents, that can regulate the translational control elements present in the processed mRNA, for example, by regulating the structure of the translational control elements or by regulating the interaction of the translational control elements with factors involved in the translation of the processed mRNA, and thus regulate the translation of the processed mRNA into the protein encoded by the processed mRNA. In some aspects, methods, compositions, and kits are provided herein for agents, e.g., therapeutic agents, that can bind to at least a portion of the 5' untranslated region ("5'UTR") of the processed mRNA. In some cases, the agents disclosed herein regulate the translational control elements present in the 5'UTR of the processed mRNA, thereby regulating the translation of the processed mRNA and regulating the expression of the protein from the processed mRNA. Thus, modulation of one or more translational control elements present in the processed mRNA according to some embodiments of the present disclosure can regulate the expression of a target protein translated from the processed mRNA, thereby providing therapeutic intervention in a disease or condition associated with aberrant levels or activity of the target protein.

[0035]

[0089] In some aspects, methods, compositions and kits are provided herein for agents, such as therapeutic agents, or vectors encoding agents, that can regulate alternative splicing of pre-mRNA.Alternative splicing events in some genes, such as the OPA1 gene, can result in non-productive mRNA transcripts that can lead to abnormal protein expression.Agents that can target alternative splicing events in those genes can regulate the expression level of functional target proteins and / or inhibit abnormal protein expression in patients.Therefore, such agents can be used to treat diseases or pathologies that are related to abnormal levels or activity of target proteins.

[0036]

[0090] One alternative splicing event that can result in a non-productive mRNA transcript is the inclusion of an additional exon in the mRNA transcript, which can induce nonsense-mediated mRNA decay. In some aspects, the present disclosure provides compositions, methods, and kits for modulating the alternative splicing of a target pre-mRNA, such as the OPA1 pre-mRNA, to increase the production of a processed mRNA that codes for a protein, and thus a translated functional target protein.

[0037]

[0091] In some aspects, provided herein are compositions, methods, and kits for combination therapy utilizing an agent that modulates the translational control element of a processed mRNA encoding a target protein, such as the OPA1 protein, and an agent that modulates the splicing of a pre-mRNA transcribed from a gene encoding a target protein, such as the OPA1 gene. In some aspects, provided herein are compositions, methods, and kits for combination therapy utilizing an agent that targets at least a portion of the 5'UTR of a processed mRNA encoding a target protein, such as the OPA1 protein, and an agent that modulates the splicing of a pre-mRNA transcribed from a gene encoding a target protein, such as the OPA1 gene. Translational regulation

[0092] Without wishing to be bound by any particular theory, eukaryotic mRNAs can be translated by a scanning mechanism that begins with the assembly of a 43S preinitiation complex (PIC) containing methionyl-initiator tRNA (Met-tRNAi) in the form of a ternary complex (TC) with eukaryotic initiation factor 2 (eIF2) bound to guanosine triphosphate (GTP). The assembly of the 43S PIC is stimulated by eIFs 1, 1A, 3 and 5. Subsequent binding of the processed mRNA to the m7G-capped 5' end is facilitated by the eIF4F complex (composed of the cap-binding proteins eIF4E, eIF4G, and the RNA helicase eIF4A) and by poly(A)-binding protein (PABP). The PIC can scan the mRNA 5' untranslated region (UTR) for an AUG nucleotide triplet start codon using complementarity with the anticodon of Met-tRNAi. AUG recognition can trigger hydrolysis of eIF2-bound GTP to generate a stable 48S PIC. Release of eIF2-GDP is followed by addition of the large (60S) ribosomal subunit, catalyzed by eIF5B, to generate the 80S initiation complex that is ready to begin protein synthesis.

[0038]

[0093] RNA molecules can fold into complex shapes that can provide additional layers of control of gene expression beyond those of their sequences. The 5'UTR is the region of the processed mRNA that is immediately upstream from the main start codon. The term "main start codon" or "main start codon" as used herein may refer to the start codon that initiates the translation of the main open reading frame of the processed mRNA. The term "open reading frame" as used herein may refer to the sequence of codons that begins with a start codon and ends with a stop codon. The "main open reading frame" of the processed mRNA as used herein is the open reading frame that encodes the protein that the processed mRNA and the gene into which the processed mRNA is transcribed are primarily responsible for expressing. For example, the main open reading frame of the processed mRNA of OPA1 is the open reading frame on the processed mRNA of OPA1 that encodes the OPA1 protein. Similarly, the major open reading frame of the ALB processed mRNA transcribed from the ALB gene (the gene encoding albumin) is the open reading frame on the ALB processed mRNA that encodes albumin.

[0039]

[0094] 5'UTR can regulate the translation of processed mRNA by different mechanisms in viruses, prokaryotes and eukaryotes. In some processed mRNAs, 5'UTR can be highly structured, and some of the secondary or higher order structures formed by 5'UTR can block ribosome entry. For example, some processed mRNAs can have one or more secondary or higher order structures formed within the 5'UTR or formed by a portion of the sequence of the 5'UTR and other parts of the processed mRNA. In some cases, secondary structures such as stems, loops (e.g., hairpin loops), and / or stem-loops can be formed within the 5'UTR. In some cases, G-quadruplex motifs can be present within the 5'UTR. As used herein, the term "G-quadruplex" may refer to an RNA structure formed in a G-rich region by stacking of at least two G-tetrads, each of which forms a quadrangular structure by non-Watson-Crick interactions between two or more layers of paired G-quartets. In some cases, G-quadruplexes may be extremely stable, e.g., due to melting temperatures higher than physiological temperatures in vitro, especially in the presence of potassium ions (K+) that are specifically chelated within the G-quartet. Descriptions of G-quadruplexes can be found in Beaudoin JD and Perreault JP., Nucleic Acids Res., 2010 November;38(20):7022-36.doi:10.1093 / nar / gkq557, Todd AK et al., Nucleic Acids Res., 33, 2901-2907(2005), Huppert JL and Balasubramanian S., Nucleic Acids Res., 2005 May 24;33(9):2908-16.doi:10.1093 / nar / gki609, each of which is incorporated herein by reference in its entirety. In some cases, G-quadruplex formation may be the most stable RNA structure that can block ribosome scanning.In some cases, the G-quadruplex has a sequence according to the formula Gx-N1-7-Gx-N1-7-Gx-N1-7-Gx, where x≧3 and N is A, C, G, or U. In some cases, the G-rich sequence that can form a G-quadruplex includes the sequence GGGAGCCGGGCUGGGGCUCACACGGGGG. In some cases, a portion of the sequence or secondary or higher order structure of the 5′UTR region of the processed mRNA can recruit one or more RNA binding proteins (RBPs) that can be involved in regulating translation of the processed mRNA.

[0040]

[0095] Upstream open reading frames and upstream start codons can also regulate protein expression by suppressing the translation of processed mRNAs that code for proteins. The term "upstream open reading frame" or "uORF" as used herein may refer to an open reading frame located in the 5'UTR of a processed mRNA, e.g., an open reading frame that is upstream of the main start codon of the processed mRNA. The term "upstream start codon" or "upstream start codon" as used herein may refer to a start codon located in the 5'UTR of a processed mRNA, e.g., a start codon upstream of the main start codon. An upstream start codon can suppress the translation of processed mRNAs that start from the main start codon. The nature of ribosome scanning during translation of processed mRNAs, its 5' to 3' directionality, may indicate that the start codon is frequently the AUG triplet closest to the 5' end that the scanning PIC encounters first. In some embodiments, a portion of the first AUG nucleotide triplet may be skipped (a process called "read-through") and the downstream AUG may be used if it is adjacent to an unfavorable sequence. The preferred sequence relationship in mammals is the "Kozak consensus", 5'(A / G)CCAUGG 3'. When an upstream start codon (e.g., an upstream AUG, or uAUG) is present in frame with a downstream AUG without an intervening stop codon, read-through may occur with some frequency, allowing the production of two protein isomers that differ only in their N-terminal extension, with the longer form often being targeted to a specific cellular compartment. In some cases, when a uAUG is followed by a stop codon in the same open reading frame (ORF), translation of the upstream ORF (uORF) may attenuate translation of the downstream main ORF (mORF) because translation reinitiation is generally inefficient. Some uORFs inhibit downstream translation primarily because ribosomes may stall during their translation, impeding the path of scanning PIC that bypasses the uORF start codon."AUG-like" triplets that differ from AUG by a single base, such as a NUG (where N is any nucleotide) triplet or an A(A / G)G triplet, may be selected by the scanning PIC, albeit less frequently due to mismatches with the anticodon of the tRNAi and the concomitant destabilization of the 48S PIC. The start codons disclosed herein can include AUG and AUG-like triplets.

[0041]

[0096] Non-limiting examples of translation control elements disclosed herein can include secondary structures of processed mRNAs, such as secondary structures in the 5'UTR, such as stems, loops, or stem-loops, G-quadruplex motifs, and upstream start codons. In some cases, the translation control elements of processed mRNAs disclosed herein regulate the translation of processed mRNAs by regulating the translation efficiency and / or rate of translation of the processed mRNAs. In some embodiments, the translation control elements disclosed herein inhibit the translation of processed mRNAs. For example, the translation control elements can block ribosome scanning during translation, thus suppressing the translation efficiency and / or rate of translation. In some cases, the upstream start codon can initiate the translation of the upstream open reading frame it is directed to, thereby preventing the scanning ribosome from resuming translation of the main open reading frame, thus suppressing the expression of the protein encoded by the main open reading frame of the processed mRNA. Targeting processed mRNA

[0097] In some embodiments, the disclosed methods and compositions relate to regulating the translation of processed mRNA transcribed from a target gene, such as the OPA1 gene. In some embodiments, the agents provided herein include a translation control element that targets the processed mRNA encoding a target protein (e.g., OPA1 protein) and inhibits the translation of the processed mRNA. In some cases, the agent modulates the structure of the translation control element, thereby increasing the expression of the target protein in the cell. In some cases, the agent (a) binds to a targeting portion of the processed mRNA, (b) modulates the interaction between the translation control element and a factor involved in the translation of the processed mRNA, or (c) is a combination of (a) and (b), thereby increasing the expression of the target protein (e.g., OPA1 protein) in the cell.

[0042]

[0098] In some cases, the resulting increased expression of OPA1 protein induced by an agent targeting the processed mRNA encoding the OPA1 protein is associated with a condition or disease associated with OPA1 deficiency, such as an ocular disease or condition, optic atrophy type 1, autosomal dominant optic atrophy (ADOA), 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 external ophthalmoplegia; hearing loss; ataxia; motor neuropathy; sensory neuropathy; myopathy; Behr's syndrome; brain dysfunction; encephalopathy; peripheral neuropathy; fatal pediatric mitochondrial encephalopathy; hypertrophic cardiomyopathy; spastic ataxia syndrome; sensorimotor peripheral neuropathy; hypotonia; gastrointestinal motility and swallowing disorders; optic nerve dysfunction; Optic atrophy;Optic atrophy plus syndrome;Mitochondrial DNA depletion syndrome 14;Late-onset cardiomyopathy;Diabetic cardiomyopathy;Alzheimer's disease;Focal segmental glomerulosclerosis;Kidney disease;Huntington's disease;Cognitive decline in healthy aging;Prion diseases;Late-onset dementia and Parkinsonism;Mitochondrial myopathy;Leigh syndrome;Friedreich's ataxia;Parkinson's disease;MELAS (Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes): Pyruvate dehydrogenase complex deficiency;Chronic kidney disease;Leber's hereditary optic neuropathy;Obesity;Age-related generalized neurodegeneration;Skeletal muscle atrophy;Cardiac and cerebral ischemic disorders;Widespread liver apoptosis;NARP (Neuropathy, Ataxia, and Retinitis Pigmentosa);MERRF (Myoclonic epilepsy with ragged-red fibers);Pearson / Kerns-Sayre syndrome syndrome); MIDD (maternally inherited diabetes and deafness); mitochondrial trifunctional protein deficiency; Fuchs endothelial dystrophy; macular telangiectasia; retinitis pigmentosa; Leber's congenital amaurosis; hereditary maculopathy; Stargardt disease; or Sohrsby's fundus degeneration.

[0043]

[0099] In some embodiments, the translation control element is in the 5' untranslated region (5'UTR) of the processed mRNA. In some cases, the translation control element comprises at least a portion of the 5'UTR of the processed mRNA. In some cases, the translation control element comprises a secondary mRNA structure that involves base pairing with at least one nucleotide of the primary start codon of the processed mRNA. In some of these cases, the agent provided herein inhibits base pairing with at least one nucleotide of the primary start codon of the processed mRNA. For example, the agent may inhibit at least one, two, or three of the three unstructured nucleotides of the primary start codon that tend to participate in base pairing in a secondary mRNA structure, such as a stem, a loop, or a stem-loop. In some embodiments, the mRNA secondary structure comprises a stem, a stem-loop, a G-quadruplex, or any combination thereof. In some cases, the agent does not bind to the primary start codon. For example, the agent binds to a portion of the processed mRNA that is different from the primary start codon, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, or 230 nucleotides upstream of the primary start codon, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 400, 500, 600, 800, 1000 nucleotides downstream of the primary start codon, or even more downstream. In other cases, the agent binds to at least 1, 2, or 3 nucleotides of the primary start codon. In some cases, the agent inhibits or reduces the formation of a secondary mRNA structure that includes at least one nucleotide of the primary start codon of the processed mRNA. For example, the agent inhibits or reduces base pairing of at least one nucleotide of the primary start codon of the processed mRNA with another nucleotide. In some cases, the other nucleotide is another nucleotide of the 5'UTR of the processed mRNA.

[0044]

[0100] In some cases, the target protein is OPA1 protein, and the agent inhibits base pairing with at least one nucleotide of the main start codon of the processed mRNA. In some of these cases, the agent binds to a targeting portion of the processed mRNA that is at most 60 nucleotides upstream of the main start codon of the processed mRNA. In some of these cases, the agent binds to a targeting portion of the processed mRNA that is at most 42 nucleotides upstream of the main start codon of the processed mRNA. In some of these cases, the agent binds to a targeting portion of the processed mRNA that is at most 116 nucleotides upstream of the main start codon of the processed mRNA. In some of these cases, the agent binds to a targeting portion of the processed mRNA that is at least 108 nucleotides upstream of the main start codon of the processed mRNA. In some of these cases, the primary start codon of the OPA1 mature mRNA is defined by chromosomal coordinates GRCh38 chr3:193,593,378-193,593,380.

[0045]

[0101] In some embodiments, the translation control element comprises at least a portion of an upstream open reading frame (uORF). In some of these cases, the agent provided herein promotes the formation of a secondary mRNA structure involving at least a portion of the uORF. In some of these cases, the translation control element comprises an upstream start codon. In some of these cases, the agent promotes the formation of a secondary mRNA structure involving base pairing with at least one nucleotide of the upstream start codon. In some cases, the agent does not bind to the upstream start codon. In other cases, the agent binds to the upstream start codon. In some of these embodiments, whether or not the agent binds to the upstream start codon, the agent promotes or increases the formation of a secondary mRNA structure including at least one nucleotide of the upstream start codon. In some cases, the agent promotes or increases base pairing of at least one nucleotide of the upstream start codon with another nucleotide of the processed mRNA, optionally, the other nucleotide is another nucleotide of the 5'UTR of the processed mRNA.

[0046]

[0102] In some embodiments, the target protein is an OPA1 protein and the agent promotes the formation of a secondary mRNA structure with at least a portion of the uORF. In some of these embodiments, the agent binds to a targeting portion of the processed mRNA that is up to 60 nucleotides upstream of the primary start codon of the processed mRNA. In some of these embodiments, the agent binds to a targeting portion of the processed mRNA that is at least 52 nucleotides upstream of the primary start codon of the processed mRNA. In some of these embodiments, the upstream start codon is defined by genomic coordinates GRCh38 chr3:193,593,226 to 193,593,228.

[0047]

[0103] In some embodiments, the translational control element comprises a G-quadruplex formed by a G-rich sequence of the processed mRNA. In some of these embodiments, the agent inhibits the formation of a G-quadruplex. In some embodiments, the G-rich sequence comprises at least a portion of the 5' untranslated region (5'UTR) of the processed mRNA. In some embodiments, the G-rich sequence is present in the 5' untranslated region (5'UTR) of the processed mRNA. In some embodiments, the G-rich sequence comprises a sequence according to the formula Gx-N1-7-Gx-N1-7-Gx-N1-7-Gx, where x > 3 and N is A, C, G or U. In some embodiments, the G-rich sequence comprises the sequence GGGAGCCGGGCUGGGGCUCACACGGGGG. In some cases, at least one, two, three, or all four of the Gx sequences in the 5'UTR of the processed mRNA are structured, present in a secondary structure, or base-paired with another nucleotide, optionally the other nucleotide is C or U. In some of these embodiments, the agents provided herein unwind, promote the transformation, or inhibit or reduce the formation of a G-quadruplex. In some of these embodiments, the agents unwind, promote the transformation, or inhibit or reduce the base-pairing or structure of at least one, two, three, or all four of the G-quadruplex Gx sequences.

[0048]

[0104] In some embodiments, the target protein is an OPA1 protein and the agent inhibits the formation of a G-quadruplex and / or unwinds, promotes deformation of, or inhibits or reduces the base pairing or structure of at least one, two, three, or all four of the G-quadruplex Gx sequences. In some of these embodiments, the agent binds to a targeting portion of the processed mRNA that is up to 60 nucleotides upstream of the main start codon of the processed mRNA. In some of these embodiments, the targeting portion of the processed mRNA is up to 35 nucleotides upstream of the main start codon of the processed mRNA. In some of these embodiments, the targeting portion of the processed mRNA is at least 17 nucleotides upstream of the main start codon of the processed mRNA. In some of these embodiments, the targeting portion of the processed mRNA is up to 60 nucleotides upstream of the main start codon of the processed mRNA and at least 17 nucleotides upstream of the main start codon of the processed mRNA.

[0049]

[0105] In some aspects, the agents provided herein target a targeting portion in the 5'UTR of a processed mRNA. In some embodiments, the targeting portion of the processed mRNA comprises at least one nucleotide upstream of the codon immediately downstream from the main start codon of the processed mRNA. In some cases, the targeting portion of the processed mRNA comprises at least one nucleotide that is up to 234 nucleotides upstream of the first nucleotide of the main start codon of the processed mRNA. In some cases, the targeting portion of the processed mRNA comprises at least one nucleotide that is up to 234, 220, 200, 180, 160, 140, 120, 100, 80, 90, 70, 60, 50, 40, 30, 20, or 10 nucleotides upstream of the first nucleotide of the main start codon of the processed mRNA. In some cases, the targeting portion of the processed mRNA is at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 160, 180, or 200 nucleotides upstream of the primary start codon of the processed mRNA. In some cases, the targeting portion of the processed mRNA is about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 160, 180, 200, or 220 nucleotides upstream of the primary start codon. In some cases, the targeting portion of the processed mRNA is about 110 nucleotides upstream of the primary start codon.

[0050]

[0106] In some embodiments, the target protein is OPA1 protein and the processed mRNA is a processed mRNA encoding OPA1 protein. In some of these embodiments, the targeted portion of the processed mRNA has a sequence having at least 80%, 82%, 84%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 9%, 96%, 97%, 98%, or 99%, or 100% sequence identity to any of the sequences listed in Table 1.4. In some of these embodiments, the targeting portion of the processed mRNA has a sequence that has at least 80%, 82%, 84%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 9%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 8 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1263-1271. In some cases, the processed mRNA has a sequence that has at least 80%, 82%, 84%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 9%, 96%, 97%, 98%, or 99%, or 100% sequence identity to any of the sequences listed in Table 1.3. In some cases, the processed mRNA has a sequence having at least 80%, 82%, 84%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 9%, 96%, 97%, 98% or 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1254-1262. In some cases, the agent comprises an antisense oligomer having at least 80%, 82%, 84%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 9%, 96%, 97%, 98% or 99%, or 100% sequence identity to any of the sequences listed in Table 8. In some cases, the agent comprises an antisense oligomer having at least 80%, 82%, 84%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 9%, 96%, 97%, 98% or 99%, or 100% sequence identity to any sequence selected from the group consisting of SEQ ID NOs: 608-1253.In some cases, the agent comprises an antisense oligomer having about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 608-1253. In some cases, the antisense oligomer has at least 80%, 82%, 84%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 9%, 96%, 97%, 98% or 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 827-847, 932-937, 953, 968, 988-1023. In some cases, the antisense oligomer has about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 827-847, 932-937, 953, 968, 988-1023.

[0051]

[0107] In some embodiments, one or more of the translational control elements disclosed herein inhibit translation of the processed mRNA by inhibiting the translation efficiency and / or rate of translation of the processed mRNA. In some embodiments, agents provided herein that target the processed mRNA increase expression of the OPA1 protein in a cell by increasing the translation efficiency and / or rate of translation of the processed mRNA.

[0052]

[0108] In some embodiments, the agents disclosed herein modulate (e.g., promote or inhibit) the binding of factors that control translation. Such factors are known in the art and are also described in Patricia R. Araujo et al., Before It Gets Started: Regulating Translation at the 5'UTR, International Journal of Genomics, Vol. 2012, Article ID 475731, p. 8, 2012, which is incorporated herein by reference in its entirety.

[0053]

[0109] In some embodiments, the translation efficiency and / or rate of processed mRNA encoding the OPA1 protein in cells contacted with an agent or a vector encoding an agent is increased compared to the translation efficiency and / or translation of processed mRNA in control cells not contacted with the agent or a vector encoding an agent.

[0054]

[0110] In some cases, the translation efficiency and / or translation rate of the processed mRNA encoding the OPA1 protein in a cell contacted with an agent or a vector encoding an agent disclosed herein is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1 to about 10 ... an increase of 0.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 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.

[0055]

[0111] In some cases, the translation efficiency and / or rate of the processed mRNA encoding the OPA1 protein in a cell contacted with an agent or a vector encoding an agent disclosed herein is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about An increase of 1.1 to about 9 times, about 2 to about 5 times, about 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.

[0056]

[0112] In some embodiments, the processed mRNA transcript targeted by the disclosed methods, compositions or kits is a mutated processed mRNA transcript. In some embodiments, the processed mRNA transcript is not a mutated processed mRNA transcript. In some embodiments, the processed mRNA is processed from a pre-mRNA that is a mutated pre-mRNA. In some embodiments, the processed mRNA is processed from a pre-mRNA that is not a mutated pre-mRNA. mRNA splicing

[0113] Intervening sequences or introns in RNA sequences are removed by a large and highly dynamic RNA-protein complex called the spliceosome, which coordinates the complex interactions between the primary transcript, small nuclear RNAs (snRNAs), and numerous proteins. The spliceosome assembles in an orderly fashion on each intron, starting with the recognition of the 5' splice site (5'ss) by U1 snRNA or the 3' splice site (3'ss) by the U2 pathway, where U2 auxiliary factor (U2AF) binds to the 3'ss region to facilitate U2 binding to the branch point sequence (BPS). U2AF is a stable heterodimer composed of a 65 kD subunit (U2AF65) encoded by U2AF2 that binds to polypyrimidine tracts (PPTs) and a 35 kD subunit (U2AF35) encoded by U2AF1 that interacts with a highly conserved AG dinucleotide in the 3'ss, stabilizing U2AF65 binding. In addition to the BPS / PPT units and 3'ss / 5'ss, accurate splicing requires auxiliary sequences or structures, known as intronic or exonic splicing enhancers or silencers, that activate or repress splice site recognition. These elements allow true splice sites to be recognized among the vast excess of cryptic or pseudo sites in the genomes of higher eukaryotes that have the same sequence but are 10-fold more numerous than the natural sites. Although the elements often have a regulatory function, the exact mechanism of their activation or repression is not fully understood.

[0057]

[0114] The decision to splice or not can typically be modeled as a stochastic rather than a deterministic process, such that even the most well-defined splicing signals can occasionally be spliced ​​incorrectly. However, under normal conditions, pre-mRNA splicing occurs with a surprisingly high degree of fidelity. This is thought to be due, in part, to the activity of adjacent cis-acting auxiliary exonic and intronic splicing control elements (ESRs or ISRs). Typically, these functional elements are classified as either exonic or intronic splicing enhancers (ESEs or ISEs) or silencers (ESSs or ISSs) based on their ability to stimulate or inhibit splicing, respectively. There is now evidence that some auxiliary cis-acting elements may act by influencing the dynamics of spliceosome assembly, for example, by affecting the positioning of the complex between the U1 snRNP and the 5'ss, but it seems highly likely that many elements function in concert 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 with important roles in defining exons. SR proteins promote exon recognition by recruiting components of the pre-spliceosome to adjacent splice sites or by attenuating the effect of ESS in their vicinity. The repressive effect of ESS can be mediated by members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, which can alter the recruitment of core splicing factors to adjacent splice sites. In addition to their role in splicing control, silencer elements are suggested to have a role in suppressing pseudoexons, which are a set of decoy intron splice sites with the typical spacing of an exon but without a functional open reading frame. ESEs and ESSs, along with their cognate trans-acting RBPs, represent key components in a set of splicing control factors that specify how, where, and when mRNA is assembled from its precursor.

[0058]

[0115] Alternative splicing is a regulated 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 being translated from at least some of the resulting mature mRNA isoforms. During this process, certain exons of a gene may be included or excluded from the final processed mRNA produced from that gene. As a result, proteins translated from alternatively spliced ​​mRNAs may contain differences in their amino acid sequences and, in some cases, their biological functions.

[0059]

[0116] As described herein, an "alternatively spliced ​​exon" may refer to an exon of a gene that is naturally included or excluded from a mature mRNA transcript, and thus may result in different protein products translated from different mature mRNA transcripts. The inclusion or skipping of an alternatively spliced ​​exon may occur naturally in a cell in a random or controlled manner, for example, under the control of external physiological or pathological stimuli, or in response to intracellular signaling. In some cases, the production of an alternatively spliced ​​mRNA, for example, the splicing of an alternatively spliced ​​exon, is controlled by a system of trans-acting proteins that bind to cis-acting sites in the primary transcript itself. In some cases, an alternatively spliced ​​exon is a coding exon, for example, an exon that, when included in a mature mRNA transcript, is translated into an amino acid sequence as part of a protein product translated from the mature mRNA transcript. In some cases, inclusion of the alternatively spliced ​​exon into a mature mRNA transcript will maintain a canonical open reading frame compared to a mature mRNA transcript without the alternatively spliced ​​exon, e.g., the number of nucleotides in the alternatively spliced ​​exon is divisible by 3.

[0060]

[0117] Sequences marking exon-intron boundaries are degenerate signals of various strengths that can occur frequently within human genes. In multi-exon genes, different pairs of splice sites can be linked together in many different combinations to generate a wide variety of transcripts from a single gene. This is commonly referred to as alternative pre-mRNA splicing. Although most mRNA isoforms produced by alternative splicing can be exported from the nucleus and translated into functional polypeptides, different mRNA isoforms derived from a single gene can vary greatly in their translation efficiency. Those mRNA isoforms that have a premature stop codon (PTC) at least 50 bp upstream of the exon junction complex are likely to be targeted for degradation by the nonsense-mediated mRNA decay (NMD) pathway. Mutations in traditional (BPS / PPT / 3'ss / 5'ss) and auxiliary splicing motifs can lead to aberrant splicing, e.g., exon skipping, or cryptic (or spurious) exon inclusion or splice site activation, and can be a significant contributor to human morbidity and mortality. Both aberrant and alternative splicing patterns can be influenced by naturally occurring DNA variants in exons and introns.

[0061]

[0118] Considering that exon-intron boundaries can occur at any of three codon positions, it is clear that only a subset of alternative splicing events can maintain the canonical open reading frame. For example, only exons divisible by 3 can be skipped or included in the mRNA without any change in the reading frame. Splicing events that are not in phase with compatibility can induce frameshifts. Unless reversed by downstream events, frameshifts will certainly result in one or more PTCs, possibly resulting in their subsequent degradation by NMD. NMD is a translation-coupled mechanism that excludes mRNAs that contain PTCs. NMD can function as a surveillance pathway that exists in all eukaryotes. NMD can reduce failures in gene expression by eliminating mRNA transcripts that contain premature stop codons. Translation of these aberrant mRNAs can potentially result in deleterious gain-of-function or dominant-negative activity of the resulting protein. NMD targets not only transcripts with PTCs but also a broad range of mRNA isoforms expressed from many endogenous genes, suggesting that NMD is a master regulator driving both fine and coarse regulation of steady-state RNA levels in cells.

[0062]

[0119] An NMD-inducing exon ("NIE" or "NMD exon") is an exon, or a pseudoexon, that is a region within an intron, that can activate the NMD pathway when included in a mature RNA transcript. In a constitutive splicing event, the intron containing the NMD exon is typically excised, but during an alternative or aberrant splicing event, the intron or a portion thereof (e.g., the NMD exon) may be retained. A mature mRNA transcript containing such an NMD exon may be non-productive due to a frameshift that induces the NMD pathway. Inclusion of the NMD exon in a mature RNA transcript may downregulate gene expression. An mRNA transcript containing an NMD exon may be referred to in the present disclosure as an "NIE-containing mRNA" or an "NMD exon mRNA."

[0063]

[0120] Cryptic (or false splice sites) have the same splicing recognition sequence as true splice sites, but are not used in the splicing reaction. They are 10 times more abundant than true splice sites in the human genome and are usually suppressed by molecular mechanisms that are not yet fully understood. Cryptic 5' splice sites have the consensus NNN / GUNNNN or NNN / GCNNNN, where N is any nucleotide and / N is the exon-intron boundary. Cryptic 3' splice sites have the consensus NAG / N. Their activation is positively influenced by the surrounding nucleotides that make them more similar to the optimal consensus of the original splice sites, i.e., MAG / GURAGU and YAG / G, respectively, where M is C or A, R is G or A, and Y is C or U.

[0064]

[0121] Splice sites and their regulatory sequences can be readily identified by the skilled artisan using suitable publicly available algorithms, e.g. those 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 (www.ncbi.nlm.nih.gov / pmc / articles / PMC2095810 / pdf / gkm680.pdf).

[0065]

[0122] A cryptic splice site or splicing control sequence may compete with a splice site of an NMD exon for an RNA binding protein such as U2AF, in some embodiments, an agent may bind to a cryptic splice site or splicing control sequence and prevent binding of the RNA binding protein, thereby favoring binding of the RNA binding protein to the NMD exon splice site.

[0066]

[0123] In some embodiments, the cryptic splice site may not include the 5' or 3' splice site of the NMD exon. In some embodiments, the cryptic 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 cryptic 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. Target transcript

[0124] In some embodiments, the disclosed methods and compositions exploit the presence of NMD exons in pre-mRNA transcribed from the OPA1 gene. The splicing of the identified OPA1 NMD exon pre-mRNA species that produces functional mature OPA1 mRNA can be induced using agents such as ASOs that stimulate exon skipping of the NMD exon. Induction of exon skipping can result in inhibition of the NMD pathway.The resulting mature OPA1 mRNA is normally translated without activating the NMD pathway, thereby increasing the amount of OPA1 protein in the patient's cells and preventing pathologies or disorders associated with OPA1 deficiency, such as eye diseases or conditions, optic atrophy type 1, autosomal dominant optic atrophy (ADOA), 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 dysfunction. afission-mediated mitochondrial dysfunction;progressive external ophthalmoplegia;hearing loss;ataxia;motor neuropathy;sensory neuropathy;myopathy;Beer's syndrome;cerebral dysfunction;encephalopathy;peripheral neuropathy;fatal childhood mitochondrial encephalopathy;hypertrophic cardiomyopathy;spastic ataxia syndrome;sensory-motor peripheral neuropathy;hypotonia;gastrointestinal motility and swallowing disorders;optic atrophy;optic atrophy plus syndrome;mitochondrial DNA depletion syndrome 14;late-onset cardiomyopathy;diabetic cardiomyopathy;Alzheimer's disease;focal segmental chronic glomerulosclerosis;kidney disease;Huntington's disease;cognitive decline in healthy aging;prion diseases;late-onset dementia and Parkinsonism;mitochondrial myopathy;Leigh syndrome;Friedreich's ataxia;Parkinson's disease;MELAS (mitochondrial encephalopathy, 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 disorders;widespread It can reduce the symptoms of liver apoptosis; NARP (neuropathy, ataxia, retinitis pigmentosa); MERRF (myoclonic epilepsy with ragged red fibers); Pearson / Kerns-Sayre syndrome; MIDD (maternally inherited diabetes and deafness); mitochondrial trifunctional protein deficiency; Fuchs endothelial dystrophy; macular telangiectasia; retinitis pigmentosa; Leber congenital amaurosis; hereditary maculopathy; Stargardt disease; or Sohrsby fundus degeneration.

[0067]

[0125] In some embodiments, the disclosed methods and compositions exploit alternative splicing of pre-mRNA transcribed from the OPA1 gene. In some cases, splicing of a coding exon, e.g., an alternatively spliced ​​exon, e.g., OPA1 exon 7 (an exon encoded by the genomic region spanning GRCh38 / hg38:chr3 193626092-193626202) can regulate the level of OPA1 protein expressed from the OPA1 gene. As used herein, the term "OPA1 exon 7" or its grammatical equivalents are used interchangeably with the terms "exon (GRCh38 / hg38:chr3 193626092-193626202)" or "an exon encoded by the genomic region spanning GRCh38 / hg38:chr3 193626092-193626202." Without wishing to be bound by any particular theory, the presence or absence of exon 7 or the amino acid sequence encoded by exon (GRCh38 / hg38:chr3 193626092-193626202) can modulate the stability of the OPA1 protein. For example, in some cases, an OPA1 protein encoded by a mature mRNA transcript lacking exon 7 may have fewer proteolytic cleavage sites compared to an OPA1 protein encoded by a corresponding mature mRNA transcript having / containing exon 7. In some cases, an OPA1 protein encoded by a corresponding mature mRNA transcript having / containing / encoded by a mature mRNA transcript lacking exon 7 is a functional protein. An OPA1 protein encoded by a mature mRNA transcript lacking exon 7 may be at least partially functional compared to an OPA1 protein encoded by a corresponding mature mRNA transcript having / containing exon 7. In some cases, the OPA1 protein encoded by the 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 a mature mRNA transcript lacking exon 7 in a cell may result in a more functional OPA1 protein in the cell due to the greater stability of the OPA1 protein lacking exon 7 and its at least partial functional equivalence.

[0068]

[0126] In other embodiments, a coding exon of the OPA1 pre-mRNA other than exon 7 is targeted by an agent disclosed herein that promotes the elimination of a coding exon other than exon 7. In these other embodiments, the agent that promotes the elimination of a coding exon other than exon 7 increases expression of the OPA1 protein encoded by the mature mRNA transcript lacking the excluded exon.

[0069]

[0127] Alternative splicing of OPA1 pre-mRNA species to produce a functional mature OPA1 protein, e.g., skipping of a coding exon, e.g., an alternatively spliced ​​exon, e.g., exon 7, can be induced using an agent, such as an ASO, that stimulates exon skipping. Induction of exon skipping can result in modulation of the levels of different alternatively spliced ​​mRNA transcripts.The resulting mature OPA1 mRNA is translated into a distinct OPA1 protein, thereby increasing the amount of OPA1 protein in the patient's cells and preventing pathologies or diseases associated with OPA1 deficiency, such as eye diseases or conditions, optic atrophy type 1, autosomal dominant optic atrophy (ADOA), 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 external ophthalmoplegia;Hearing loss;Ataxia;Motor neuropathy;Sensory neuropathy;Myopathy;Beer syndrome;Cerebral dysfunction;Encephalopathy;Peripheral neuropathy;Fatal childhood mitochondrial encephalopathy;Hypertrophic cardiomyopathy;Spastic ataxia syndrome;Sensorimotor peripheral neuropathy;Hypotonia;Gastrointestinal motility and swallowing disorders;Optic atrophy;Optic atrophy plus syndrome;Mitochondrial DNA depletion syndrome 14;Late-onset cardiomyopathy;Diabetic cardiomyopathy;Alzheimer's disease;Focal segmental glomerulopathy Sclerosis;kidney disease;Huntington's disease;cognitive decline in healthy aging;prion diseases;late-onset dementia and Parkinsonism;mitochondrial myopathy;Leigh syndrome;Friedreich's ataxia;Parkinson's disease;MELAS (mitochondrial encephalopathy, 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 disorders;widespread It can reduce symptoms of liver apoptosis; NARP (neuropathy, ataxia, retinitis pigmentosa); MERRF (myoclonic epilepsy with ragged red fibers); Pearson / Kerns-Sayre syndrome; MIDD (maternally inherited diabetes and deafness); mitochondrial trifunctional protein deficiency; Fuchs endothelial dystrophy; macular telangiectasia; retinitis pigmentosa; Leber congenital amaurosis; hereditary maculopathy; Stargardt disease; or Sohrsby fundus degeneration.

[0070]

[0128] In some embodiments, the disease or condition that can be treated or ameliorated using the methods or compositions disclosed herein is not directly related to the target protein (gene) targeted by the therapeutic agent. In some embodiments, the therapeutic agents provided herein may target a protein (gene) that is not directly related to the disease or condition, but modulation of the expression of the target protein (gene) can treat or ameliorate the disease or condition.

[0071]

[0129] In various embodiments, the present disclosure provides therapeutic agents that can target OPA1 mRNA transcripts to modulate splicing or protein expression levels. The therapeutic agent can be a small molecule, polynucleotide, or polypeptide. In some embodiments, the therapeutic agent is an ASO. Various regions or sequences of OPA1 pre-mRNA can be targeted by therapeutic agents, such as ASOs. In some embodiments, the ASO targets OPA1 pre-mRNA transcripts that contain an NMD exon. In some embodiments, the ASO targets a sequence within the NMD exon of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence upstream (or 5') from the 5' end (3'ss) of the NMD exon of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence downstream (or 3') from the 3' end (5'ss) of the NMD exon of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron adjacent to the 5' end of the NMD exon of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron adjacent to the 3' end of the NMD exon of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence that includes an NMD exon-intron boundary of the OPA1 pre-mRNA transcript. The NMD exon-intron boundary may refer to the junction of an intron sequence with an NMD exon region. The intron sequence may be adjacent to the 5' end of the NMD exon or the 3' end of the NMD exon. In some embodiments, the ASO targets a sequence within an exon of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence that includes both a portion of an intron and a portion of an exon of the OPA1 pre-mRNA transcript.

[0072]

[0130] In some embodiments, the ASO targets a sequence about 4 to about 300 nucleotides upstream (or on the 5' side) from the 5' end of the NMD exon. In some embodiments, the ASO targets a sequence about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides upstream (or on the 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 about 4 to about 300 nucleotides downstream (or on the 3' side) from the 3' end of the NMD exon. In some embodiments, the ASO targets a sequence about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides downstream from the 3' end of the NMD exon. In some embodiments, the ASO targets a sequence more than 300 nucleotides downstream from the 3' end of the NMD exon.

[0073]

[0131] In some embodiments, the OPA1 NMD exon-containing pre-mRNA transcript is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the OPA1 NMD exon pre-mRNA transcript comprises a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 2-5.

[0074]

[0132] In some embodiments, the OPA1 NMD exon-containing pre-mRNA transcript (or NMD exon mRNA) comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 2-5. In some embodiments, the OPA1 NMD exon-containing pre-mRNA 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: 2-5. In some embodiments, the targeting portion of the NMD exon mRNA comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 2-5.

[0075]

[0133] In some embodiments, the ASO targets exon 6x of the OPA1 NMD exon-containing pre-mRNA that contains NIE exon 6, exon 7x of the OPA1 NMD exon-containing pre-mRNA that contains NIE exon 7, or exon 28x of the OPA1 NMD exon-containing pre-mRNA that contains NIE exon 28. In some embodiments, the ASO targets an exon (GRCh38 / hg38:chr3 193628509 193628616) of the OPA1 pre-mRNA, or an exon (GRCh38 / hg38:chr3 193603500 193603557) of OPA1. In some embodiments, the ASO targets an NMD exon of the OPA1 pre-mRNA other than the NMD exon (GRCh38 / hg38:chr3 193628509 193628616).

[0076]

[0134] In some embodiments, the ASO targets a sequence about 1500, about 1000, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 80, about 70, about 60, or about 50 nucleotides upstream (or 5') of the 5' end of exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1. In some embodiments, the ASO targets a sequence about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') of GRCh38 / hg38:chr3 193628509 of OPA1; or GRCh38 / hg38:chr3 193603500 of OPA1.

[0077]

[0135] In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') of the 5' end of exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1. In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') of GRCh38 / hg38:chr3 193628509 of OPA1; or GRCh38 / hg38:chr3 193603500 of OPA1.

[0078]

[0136] In some embodiments, the ASO targets a sequence about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') of the 3' end of exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1. In some embodiments, the ASO targets a sequence about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') of GRCh38 / hg38:chr3 193628616 of OPA1; or GRCh38 / hg38:chr3 193603557 of OPA1.

[0079]

[0137] In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') of the 3' end of exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1. In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') of GRCh38 / hg38:chr3 193628616 of OPA1; or GRCh38 / hg38:chr3 193603557 of OPA1.

[0080]

[0138] In some embodiments, the ASO has a sequence complementary to a targeted portion of an NMD exon mRNA set forth in any one of SEQ ID NOs: 2-5, or 279.

[0081]

[0139] In some embodiments, the ASO targets a sequence upstream from the 5' end of an NMD exon. For example, an ASO targeting a sequence upstream from the 5' end of an NMD exon (OPA1 exon 6x, OPA1 exon 7x, or OPA1 exon 28x) comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO:2 or 3. For example, an ASO targeting a sequence upstream from the 5' end of an NMD exon (e.g., exon of OPA1 (GRCh38 / hg38:chr3 193628509-193628616); or exon of OPA1 (GRCh38 / hg38:chr3 193603500 193603557)) can include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 4 or 5.

[0082]

[0140] In some embodiments, the ASO targets a sequence containing an exon-intron boundary (or junction). For example, an ASO targeting a sequence containing an exon-intron boundary can include a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 2-5. In some embodiments, the ASO targets a sequence downstream from the 3' end of an NMD exon. For example, an ASO targeting a sequence downstream from the 3' end of an NMD exon (e.g., exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1) can include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 2 or 3, or to at least 8 contiguous nucleic acids of SEQ ID NO: 2 or 3. For example, an ASO targeting a sequence downstream from the 3' end of an NMD exon (e.g., exon of OPA1 (GRCh38 / hg38:chr3 193628509-193628616); or exon of OPA1 (GRCh38 / hg38:chr3 193603500-193603557)) can include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 4 or 5, or at least 8 contiguous nucleic acids of SEQ ID NO: 4 or 5. In some embodiments, the ASO targets a sequence within an NMD exon.

[0083]

[0141] In some embodiments, the ASO targets exon 6x of an OPA1 NMD exon-containing pre-mRNA containing NIE exon 6, exon 7x of an OPA1 NMD exon-containing pre-mRNA containing NIE exon 7, or exon 28x of an OPA1 NMD exon-containing pre-mRNA containing NIE exon 28. In some embodiments, the ASO targets a sequence downstream (or 3') from the 5' end of exon 6x, exon 7x, or exon 28x of an OPA1 pre-mRNA. In some embodiments, the ASO targets a sequence upstream (or 5') from the 3' end of exon 6x, exon 7x, or exon 28x of an OPA1 pre-mRNA.

[0084]

[0142] In some embodiments, the targeted portion of the OPA1 NMD exon-containing pre-mRNA is in intron 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 to the targeted portion of the NMD exon pre-mRNA results in exon skipping of at least one NMD exon within intron 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, followed by increased OPA1 protein production. In some embodiments, the targeted portion of the OPA1 NMD exon-containing pre-mRNA is in intron 6 of OPA1 or intron 28 of OPA1. In some embodiments, the targeted portion of the OPA1 NMD exon-containing pre-mRNA is an intron of OPA1 (GRCh38 / hg38:chr3 193626203-193631611; or an intron of OPA1 (GRCh38 / hg38:chr3 193593374-193614710)).

[0085]

[0143] In some embodiments, the disclosed methods and compositions are used to increase expression of OPA1 by inducing exon skipping of a pseudoexon in an OPA1 NMD exon-containing pre-mRNA. In some embodiments, the pseudoexon is a sequence within any of introns 1-50. In some embodiments, the pseudoexon is a sequence 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 can be an OPA1 intron or a portion thereof. In some embodiments, the pseudo-exon is within intron 6 of OPA1, or intron 28 of OPA1. In some embodiments, the pseudo-exon is within intron (GRCh38 / hg38:chr3 193626203-193631611) of OPA1, or intron (GRCh38 / hg38:chr3 193593374-193614710) of OPA1.

[0086]

[0144] In some embodiments, the ASO targets the OPA1 pre-mRNA transcript to induce exon skipping of a coding exon, e.g., an alternatively spliced ​​exon. In some embodiments, the ASO targets a sequence within a coding exon, e.g., an alternatively spliced ​​exon, of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence upstream (or 5') from the 5' end (3'ss) of a coding exon of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence downstream (or 3') from the 3' end (5'ss) of a coding exon of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron adjacent to the 5' end of a coding exon of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron adjacent to the 3' end of a coding exon of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence that includes an exon-intron boundary of the OPA1 pre-mRNA transcript. An exon-intron boundary may refer to the junction of an intron sequence and an exon sequence. The intron sequence may be adjacent to the 5' end of a coding exon or the 3' end of a coding exon. In some embodiments, the ASO targets a sequence within an exon of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron of the OPA1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence that includes both a portion of an intron and a portion of an exon of the OPA1 pre-mRNA transcript.

[0087]

[0145] In some embodiments, the ASO targets a sequence about 4 to about 300 nucleotides upstream (or 5' side) from the 5' end of a coding exon, e.g., an alternatively spliced ​​exon. In some embodiments, the ASO targets a sequence about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides upstream (or 5' side) from the 5' end of the coding exon region. In some embodiments, the ASO may target a sequence more than 300 nucleotides upstream from the 5' end of the coding exon. In some embodiments, the ASO targets a sequence about 4 to about 300 nucleotides upstream (or 3' side) from the 3' end of the coding exon. In some embodiments, the ASO targets a sequence about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides downstream from the 3' end of the coding exon region. In some embodiments, the ASO targets a sequence more than 300 nucleotides downstream from the 3' end of the coding exon.

[0088]

[0146] In some embodiments, the OPA1 pre-mRNA transcript is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the OPA1 pre-mRNA transcript comprises a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 2-5.

[0089]

[0147] In some embodiments, the OPA1 pre-mRNA transcript (or NMD exon mRNA) comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 2-5. In some embodiments, the OPA1 pre-mRNA 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: 2-5. In some embodiments, the targeting portion of the OPA1 pre-mRNA comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 2-5.

[0090]

[0148] In some embodiments, the ASO targets exon 7 of the OPA1 pre-mRNA, i.e., the ASO targets exon (GRCh38 / hg38:chr3 193626092-193626202) of the OPA1 pre-mRNA.

[0091]

[0149] In some embodiments, the ASO targets a coding exon of the OPA1 pre-mRNA other than exon 7, i.e., the ASO targets an exon of the OPA1 pre-mRNA other than the exon defined by (GRCh38 / hg38:chr3 193626092-193626202).

[0092]

[0150] In some embodiments, the ASO targets a sequence about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') from the 5' end of exon 7 of OPA1. In some embodiments, the ASO targets a sequence about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') from GRCh38 / hg38:chr3 193626092 of OPA1.

[0093]

[0151] In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') from the 5' end of exon 7 of OPA1. In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') from GRCh38 / hg38:193626092 of OPA1.

[0094]

[0152] In some embodiments, the ASO targets a sequence about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') from the 3' end of exon 7 of OPA1. In some embodiments, the ASO targets a sequence about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') from GRCh38 / hg38:chr3 193626202 of OPA1.

[0095]

[0153] In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') of the 3' end of exon 7 of OPA1. In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') of GRCh38 / hg38:chr3 193626202 of OPA1.

[0096]

[0154] In some embodiments, the ASO has a sequence complementary to a targeted portion of an NMD exon mRNA set forth in any one of SEQ ID NOs: 2-5, or 277.

[0097]

[0155] In some embodiments, the ASO targets a sequence upstream from the 5' end of a coding exon, e.g., an alternatively spliced ​​exon. For example, an ASO targeting a sequence upstream from the 5' end of a coding exon (e.g., exon 7 of OPA1) can include a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least 8 contiguous nucleic acids of SEQ ID NO: 2 or 3. For example, an ASO targeting a sequence upstream from the 5' end of a coding exon (e.g., exon (GRCh38 / hg38:193626092-193626202) of OPA1) can include a sequence that has at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 4 or 5.

[0098]

[0156] In some embodiments, the ASO targets a sequence containing an exon-intron boundary (or junction). For example, an ASO targeting a sequence containing an exon-intron boundary can include a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 2-5. In some embodiments, the ASO targets a sequence downstream from the 3' end of a coding exon, e.g., an alternatively spliced ​​exon. For example, an ASO targeting a sequence downstream from the 3' end of a coding exon (e.g., exon 7 of OPA1) can include a sequence that has at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 2 or 3 or to at least 8 contiguous nucleic acids of SEQ ID NO: 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) can include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 4 or 5, or to at least 8 contiguous nucleic acids of SEQ ID NO: 4 or 5. In some embodiments, the ASO targets a sequence within a coding exon, e.g., an alternatively spliced ​​exon. Exon inclusion

[0157] As used herein, an "NMD exon-containing pre-mRNA" is a pre-mRNA transcript that contains at least one pseudoexon. Alternative or aberrant splicing can result in the inclusion of at least one pseudoexon in a mature mRNA transcript. The terms "mature mRNA", "processed mRNA" and "fully spliced ​​mRNA" are used interchangeably herein to refer to a fully processed mRNA that has a complete splicing event in a cell. The inclusion of at least one pseudoexon can result in a non-productive mRNA, leading to NMD of the mature mRNA. The NMD exon-containing mature mRNA can sometimes lead to aberrant protein expression.

[0099]

[0158] In some embodiments, the included pseudo-exon is the pseudo-exon that is most abundant 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 pseudo-exon that is most abundant in a population of NMD exon-containing pre-mRNAs transcribed from a gene encoding a target protein in a cell, the population of NMD exon-containing pre-mRNAs comprising more than one included pseudo-exon. In some embodiments, an antisense oligomer that targets the pseudo-exon that is most abundant 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 that the antisense oligomer targets or binds. In some embodiments, the targeted region is in the pseudo-exon that is the pseudo-exon that is most abundant in the NMD exon-containing pre-mRNAs encoding the OPA1 protein.

[0100]

[0159] The degree of exon inclusion can be expressed as the exon inclusion ratio, e.g., the percentage of transcripts that include a given pseudoexon. Briefly, the % exon inclusion can be calculated as the percentage of the amount of RNA transcripts with exon inclusion relative to the sum of the average amount of RNA transcripts with exon inclusion and the average amount of RNA transcripts with exon exclusion.

[0101]

[0160] In some embodiments, an included pseudoexon is an exon identified as an included pseudoexon based on a determination of an inclusion rate of 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%. In an embodiment, the included pseudoexons are from about 5% to about 100%, from about 5% to about 95%, from about 5% to about 90%, from about 5% to about 85%, from about 5% to about 80%, from about 5% to about 75%, from about 5% to about 70%, from about 5% to about 65%, from about 5% to about 60%, from about 5% to about 55%, from about 5% to about 50%, from about 5% to about 45%, from about 5% to about 40%, from about 5% to about 35%, from about 5% to about 30%, from about 5% to about 25%, from about 5% to about 20%, from about 5% to about 15%, from about 10% to about 100%, from about 10% to about 95%, from about 10% to about 90%, About 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 100%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 70%, About 15% to about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 100%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45% , about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 25% to about 100%, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, or about 25% to about 35% inclusion rate.ENCODE data (described, for example, 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.

[0102]

[0161] In some embodiments, contacting a cell with an ASO that is complementary to a targeted portion of the OPA1 pre-mRNA transcript results in an increase in the amount of OPA1 protein produced 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 / treatment of the ASO. In some embodiments, the total amount of OPA1 protein produced by cells contacted with an antisense oligomer is 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%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 5 ... An increase of 0% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%. In some embodiments, the total amount of OPA1 protein produced by cells contacted with an antisense oligomer is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 3 to about 10 times, about 4 to about 10 times, about 5 to about 10 times, about 5 to about 10 times, about 6 to about 10 times, about 7 to about 10 times, about 8 to about 10 times, about 10 ... fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold. The control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the pre-mRNA.

[0103]

[0162] In some embodiments, contacting a cell with an ASO that is complementary to a targeted portion of an OPA1 pre-mRNA transcript results in an increase in the amount of OPA1-encoding mRNA, including mature mRNA encoding a target protein. In some embodiments, the amount of OPA1 protein-encoding mRNA or mature mRNA encoding OPA1 protein is increased by 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 / treatment of the ASO. In some embodiments, the total amount of mRNA encoding the OPA1 protein, or mature mRNA encoding the OPA1 protein produced in a cell contacted with an antisense oligomer, is 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%, about 20% to about 300%, about 30% to about 40%, about 40% to about 50%, about 40% to about 60%, about 50% to about 70%, about 60% to about 80%, about 80% to about 90%, about 90% to about 100%, about 100% to about 150%, about 10% to about 20% an increase of about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%.In some embodiments, the total amount of mRNA encoding the OPA1 protein, or mature mRNA encoding the OPA1 protein produced in cells contacted with an antisense oligomer, is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 ...2 to about 10 times, about 2 to about 10 times, about 2 to about 10 times, about 2 to about 10 times, about 2 to about 10 times, about 2 to about .1 to about 7 fold, about 1.1 to about 8 fold, about 1.1 to about 9 fold, about 2 to about 5 fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold. The control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the OPA1 NMD exon-containing pre-mRNA.

[0104]

[0163] The NMD exon can be any length. In some embodiments, the NMD exon includes the complete sequence of the intron, in which case it can be referred to as intron retention. In some embodiments, the NMD exon can be a portion of an intron. In some embodiments, the NMD exon can be a 5' end portion of an intron that includes a 5'ss sequence. In some embodiments, the NMD exon can be a 3' end portion of an intron that includes a 3'ss sequence. In some embodiments, the NMD exon can be a portion within an intron without the inclusion of a 5'ss sequence. In some embodiments, the NMD exon can be a portion within an intron without the inclusion of a 3'ss sequence. In some embodiments, the NMD exon can be a portion within an intron without the inclusion of either a 5'ss or a 3'ss sequence. In some embodiments, the NMD exon can be from 5 nucleotides to 10 nucleotides in length, from 10 nucleotides to 15 nucleotides in length, from 15 nucleotides to 20 nucleotides in length, from 20 nucleotides to 25 nucleotides in length, from 25 nucleotides to 30 nucleotides in length, from 30 nucleotides to 35 nucleotides in length, from 35 nucleotides to 40 nucleotides in length, from 40 nucleotides to 45 nucleotides in length, from 45 nucleotides to 50 nucleotides in length, from 50 nucleotides to 55 nucleotides in length, from 55 nucleotides to 60 nucleotides in length, from 60 nucleotides to 65 nucleotides in length, from 65 nucleotides to 70 nucleotides in length, from 70 nucleotides to 75 nucleotides in length, from 75 nucleotides to 80 nucleotides in length, from 80 nucleotides to 85 nucleotides in length, from 85 nucleotides to 90 nucleotides in length, from 90 nucleotides to 95 nucleotides in length, or from 95 nucleotides to 100 nucleotides in length. In some embodiments, the NMD exon can 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 in length, at least 90 nucleotides, or at least 100 nucleotides in length.In some embodiments, the NMD exon can be 100-200 nucleotides, 200-300 nucleotides, 300-400 nucleotides, 400-500 nucleotides, 500-600 nucleotides, 600-700 nucleotides, 700-800 nucleotides, 800-900 nucleotides, 900-1,000 nucleotides in length, In some embodiments, the NMD exon can be greater than 1,000 nucleotides in length.

[0105]

[0164] Inclusion of the pseudoexon can result in frameshifting and introduction of a premature stop codon (PIC) into the mature mRNA transcript, making the transcript a target for NMD. The mature mRNA transcript containing the NMD exon can be a non-productive mRNA transcript that does not result in protein expression. The PIC can be present at any position downstream of the NMD exon. In some embodiments, the PIC can be present in any exon downstream of the NMD exon. In some embodiments, the PIC can be present within the NMD exon. For example, inclusion of exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1 into the mRNA transcript encoded by the OPA1 gene can induce PIC into the mRNA transcript. For example, inclusion of an exon of OPA1 (GRCh38 / hg38:chr3 193628509 193628616); or an exon of OPA1 (GRCh38 / hg38:chr3 193603500 193603557)) in the mRNA transcript encoded by OPA1.

[0106]

[0165] In some aspects, a method is provided herein for regulating the expression of OPA1 protein by promoting the inclusion of a coding exon. The method can include contacting a cell having an OPA1 pre-mRNA with an agent, the agent comprising an oligonucleotide that binds to (a) a targeting portion of the pre-mRNA in an intron region immediately upstream of the 5' end of the coding exon of the pre-mRNA, or (b) a targeting portion of the pre-mRNA in an intron region immediately downstream of the 3' end of the coding exon of the pre-mRNA, thereby increasing the level of processed mRNA that is processed from the pre-mRNA in the cell and contains the coding exon. In some cases, the included coding exon is an alternatively spliced ​​exon. In some cases, the method promotes the inclusion of the coding exon into the processed mRNA during splicing of the pre-mRNA in the cell.

[0107]

[0166] In some of these embodiments of inclusion of a coding exon, the target portion of the pre-mRNA is within a region spanning 100-50, 100-60, 100-70, 100-80, or 100-90 nucleotides upstream of the 5' end of the coding exon. In some cases, the target portion of the pre-mRNA is within a region spanning 40-100, 50-100, 60-100, 70-100, 80-100, or 90-100 nucleotides downstream of the 3' end of the coding exon. In some cases, the coding exon is exon 7 of OPA1. In some cases, the coding exon comprises a sequence having at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO:277. In some cases, the coding exon comprises SEQ ID NO:277. The targeting portion of the pre-mRNA can be within a region spanning 100-50, 100-60, 100-70, 100-80, or 100-90 nucleotides upstream of the genomic site GRCh38 / hg38:chr3 193626092. In some cases, the targeting portion of the pre-mRNA is within a region spanning 40-100, 50-100, 60-100, 70-100, 80-100, or 90-100 nucleotides downstream of the genomic site GRCh38 / hg38:chr3 193626202.

[0108]

[0167] In some cases, the inclusion of the coding exon in the processed mRNA in a cell contacted with the agent is about 1.1 to about 10 fold, about 1.5 to about 10 fold, about 2 to about 10 fold, about 3 to about 10 fold, about 4 to about 10 fold, about 1.1 to about 5 fold, about 1.1 to about 6 fold, about 1.1 to about 7 fold, about 1.1 to about 8 fold, about 1.1 to about 9 fold, about 2 to about 5 fold, about 2 to about 6 fold , about 2 to about 7 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. Exclusion of both NMD and coding exons

[0168] In some embodiments, methods are provided herein for regulating expression of a target protein by targeting a pre-mRNA and regulating the elimination of both coding exons and nonsense-mediated RNA decay-induced exons (NMD exons) from the pre-mRNA. In some cases, the method includes contacting a cell with an agent, where the agent promotes the elimination of both coding exons and NMD exons from the pre-mRNA, thereby increasing the level of processed mRNA that is processed from the pre-mRNA and lacks both coding exons and NMD exons. In some cases, the agent binds to a targeted portion of the pre-mRNA or modulates the binding of a factor involved in splicing of the coding exon, the NMD exon, or both. In some cases, the agent interferes with the binding of a factor involved in splicing of the coding exon, the NMD exon, or both to a region of the targeted portion. In some cases, the NMD exon is within an intronic region adjacent to the coding exon. In some cases, the NMD exon is within an intronic region immediately upstream of the coding exon. In some cases, the NMD exon is within an intronic region immediately downstream of a coding exon. In some cases, the coding exon is an alternatively spliced ​​exon.

[0109]

[0169] In some cases, the targeting portion of the pre-mRNA is proximal to the coding exon. The targeting portion of the pre-mRNA can be located in an intron region immediately upstream of the coding exon. The targeting portion of the pre-mRNA can be located in an intron region immediately downstream of the coding exon. In some cases, the targeting portion of the pre-mRNA can be located within the coding exon. In some cases, the targeting portion of the pre-mRNA is within a region spanning 49-1, 39-1, 29-1, or 19-1 nucleotides upstream of the 5' end of the coding exon. In some cases, the targeting portion of the pre-mRNA is within a region spanning 100 nucleotides upstream of the coding exon to 100 nucleotides downstream of the coding exon. In some cases, the targeting portion comprises about 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 contiguous nucleotides of a coding exon.

[0110]

[0170] In some cases, the targeting portion of the pre-mRNA is proximal to the NMD exon. In some cases, the targeting portion of the pre-mRNA is located in the intron region immediately upstream of the NMD exon. In some cases, the targeting portion of the pre-mRNA is located in the intron region immediately downstream of the NMD exon. In some cases, the targeting portion of the pre-mRNA is located within the NMD exon. In some cases, the targeting portion of the pre-mRNA is within the region that spans from 100 nucleotides upstream of the NMD exon to 100 nucleotides downstream of the NMD exon.

[0111]

[0171] In some embodiments, the methods described herein are applicable to modulating expression of OPA1 protein by modulating the exclusion of both exon 7 and an NMD exon (e.g., exon 7x) of an OPA1 pre-mRNA that contains both exon 7 and exon 7x. In some cases, the coding exon comprises a sequence having at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO: 277. In some cases, the coding exon comprises SEQ ID NO: 277. In some cases, the targeting portion of the pre-mRNA is immediately upstream of the coding exon GRCh38 / hg38:chr3 193626092-193626202. In some cases, the targeting portion of the pre-mRNA is immediately downstream of the coding exon GRCh38 / hg38:chr3 193626092-193626202. In some cases, the targeting portion of the pre-mRNA is within a region spanning 49-1, 39-1, 29-1, or 19-1 nucleotides upstream of GRCh38 / hg38:chr3 193626092. In some cases, the targeting portion of the pre-mRNA is within a region spanning 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 targeting portion of the pre-mRNA is within the coding exon GRCh38 / hg38:chr3 193626092-193626202. In some cases, the targeting portion of the pre-mRNA includes an exon-intron junction of coding exon GRCh38 / hg38:chr3 193626092-193626202. In some cases, the NMD exon comprises a sequence having at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO: 279. In some cases, the NMD exon comprises SEQ ID NO: 279. In some cases, the targeting portion of the pre-mRNA is immediately upstream of the NMD exon GRCh38 / hg38:chr3 193628509-193628616. In some cases, the targeting portion of the pre-mRNA is immediately downstream of the NMD exon GRCh38 / hg38:chr3 193628509-193628616.In some cases, the targeted portion of the pre-mRNA is within a region spanning from 100 nucleotides upstream of genomic site GRCh38 / hg38:chr3 193628509 to 100 nucleotides downstream of genomic site GRCh38 / hg38:chr3 193628616.

[0112]

[0172] In some cases, the targeting portion of the pre-mRNA is within the NMD exon GRCh38 / hg38:chr3 193628509-193628616. In some cases, the targeting portion of the pre-mRNA includes an exon-intron junction of the NMD exon GRCh38 / hg38:chr3 193628509-193628616. In some cases, the targeting portion includes about 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 contiguous nucleotides of the NMD exon.

[0113]

[0173] In some cases, the exclusion of coding exons from pre-mRNA in cells contacted with the agent is about 1.1 to about 10 fold, about 1.5 to about 10 fold, about 2 to about 10 fold, about 3 to about 10 fold, about 4 to about 10 fold, about 1.1 to about 5 fold, about 1.1 to about 6 fold, about 1.1 to about 7 fold, about 1.1 to about 8 fold, about 1.1 to about 9 fold, about 2 to about 5 fold, about 2 to about 6 fold, , about 2 to about 7 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. In some cases, the exclusion of NMD exons from pre-mRNA in cells contacted with the agent is about 1.1 to about 10 fold, about 1.5 to about 10 fold, about 2 to about 10 fold, about 3 to about 10 fold, about 4 to about 10 fold, about 1.1 to about 5 fold, about 1.1 to about 6 fold, about 1.1 to about 7 fold, about 1.1 to about 8 fold, about 1.1 to about 9 fold, about 2 to about 5 fold, about 2 to about 6 fold, , about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold. In some cases, the method results in an increase in the level of processed mRNA in the cell.The level of processed mRNA in a cell contacted with a drug is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold.

[0114]

[0174] In some cases, the method results in an increase in expression of OPA1 protein in the cells, such that the level of OPA1 protein expressed from processed mRNA in the cells contacted with the agent is about 1.1 to about 10 fold, about 1.5 to about 10 fold, about 2 to about 10 fold, about 3 to about 10 fold, about 4 to about 10 fold, about 1.1 to about 5 fold, about 1.1 to about 6 fold, about 1.1 to about 7 fold, about 1.1 to about 8 fold, about 1.1 to about 9 fold, about 2 to about 5 fold, about 2 to about 6 fold, or about 3 to about 7 fold, compared to the absence of contact with the agent. fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold.

[0115]

[0175] In some cases, the level of OPA1 protein expressed from the processed mRNA in cells contacted with the agent is increased by at least about 1.5-fold compared to the absence of contact with the agent.

[0116]

[0176] In some cases, the OPA1 protein expressed from a processed mRNA lacking exon 7 and exon 7x is a functional OPA1 protein. The OPA1 protein expressed from a processed mRNA lacking exon 7 and exon 7x can be at least partially functional compared to a wild-type OPA1 protein. The OPA1 protein expressed from a processed mRNA lacking exon 7 and exon 7x can be at least partially functional compared to a full-length wild-type OPA1 protein. Protein expression

[0177] In some embodiments, the methods described herein are used to increase the production of a functional target protein, e.g., OPA1 protein. As used herein, the term "functional" refers to the amount of activity or function of a target protein, e.g., OPA1 protein, required to eliminate any one or more symptoms of the condition or disease being treated, e.g., optic atrophy type 1. In some embodiments, the methods are used to increase the production of a partially functional target protein, e.g., OPA1 protein. As used herein, the term "partially functional" refers to any amount of activity or function of a target protein, e.g., OPA1 protein, that is less than the amount of activity or function required to eliminate or prevent any one or more symptoms of the disease or condition. In some embodiments, a partially functional protein or RNA may have 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% less activity than a fully functional protein or RNA.

[0117]

[0178] In some embodiments, the method is a method of increasing expression of a protein, the target protein, by cells of a subject having a target gene encoding the target protein, the subject having a disease or condition caused by a deficient amount of activity of the target protein, the deficient amount of the target protein being caused by haploinsufficiency of the target protein. In such embodiments, the subject has a first allele that encodes a functional target protein and a second allele in which the target protein is not produced. In another such embodiment, the subject has a first allele that encodes a functional target protein and a second allele that encodes a non-functional target protein. In another such embodiment, the subject has a first allele that encodes a functional target protein and a second allele that encodes a partially functional target protein. In any of these embodiments, the agent binds to a targeting portion of the target pre-mRNA transcribed from the second allele, thereby inducing exon skipping of the pseudoexon from the pre-mRNA, causing an increase in the level of mature mRNA encoding the functional target protein and an increase in expression of the target protein in the cells of the subject. Alternatively or additionally, the agent can bind to a targeted portion of the target processed mRNA that encodes the target protein, thereby increasing translation of the target processed mRNA and increasing expression of the target protein.

[0118]

[0179] In some embodiments, the method is a method of increasing expression of a protein that is OPA1 by cells of a subject having the OPA1 gene, the subject having a disease or condition caused by a defective amount of activity of the OPA1 protein, e.g., optic atrophy type 1, the defective amount of the OPA1 protein being caused by haploinsufficiency of the OPA1 protein. In such embodiments, the subject has a first allele that encodes a functional OPA1 protein and a second allele in which no OPA1 protein is produced. In another such embodiment, the subject has a first allele that encodes a functional OPA1 protein and a second allele that encodes a non-functional OPA1 protein. In another such embodiment, the subject has a first allele that encodes a functional OPA1 protein and a second allele that encodes a partially functional OPA1 protein. In any of these embodiments, the antisense oligomer binds to a targeted portion of the OPA1 pre-mRNA transcribed from the second allele, thereby inducing exon skipping of the pseudoexon from the pre-mRNA, causing increased levels of mature mRNA encoding a functional OPA1 protein and increased expression of the OPA1 protein in the cells of the subject. Alternatively or additionally, the agent can bind to a targeted portion of the OPA1 processed mRNA encoding the OPA1 protein, thereby increasing translation of the OPA1 processed mRNA and increasing expression of the OPA1 protein.

[0119]

[0180] In some embodiments, the method is a method of increasing expression of a target protein by cells of a subject, wherein the subject has a disease or condition caused by a defective amount of activity of the target protein, and the defective amount of the target protein is caused by autosomal recessive inheritance.

[0120]

[0181] In some embodiments, the method is a method of increasing expression of OPA1 protein by cells of a subject, the subject having a disease or condition caused by a defective amount of activity of OPA1 protein, and the defective amount of OPA1 protein is caused by autosomal recessive inheritance.

[0121]

[0182] In some embodiments, the method is a method of increasing expression of a target protein by cells of a subject, wherein the subject has a disease or condition caused by a defective amount of activity of the target protein, and the defective amount of the target protein is caused by autosomal dominant inheritance.

[0122]

[0183] In some embodiments, the method is a method of increasing expression of OPA1 protein by cells of a subject having an OPA1 precursor mRNA, the subject having a disease or condition caused by a defective amount of OPA1, protein activity, e.g., optic atrophy type 1, and the defective amount of OPA1 protein is caused by autosomal dominant inheritance.

[0123]

[0184] In a related embodiment, the method is a method of increasing the expression of a protein or functional RNA using ASO. In some embodiments, the ASO can be used to increase the expression of a target protein in a cell of a subject having a target pre-mRNA, and the subject has a deficiency in the amount or function of the target protein.

[0124]

[0185] In a related embodiment, the method is a method of increasing expression of a protein or functional RNA using ASO. In some embodiments, ASO can be used to increase expression of OPA1 protein in cells of a subject having OPA1 pre-mRNA, and the subject has a deficiency in the amount or function of OPA1 protein, for example, optic atrophy type 1.

[0125]

[0186] In some embodiments, a pre-mRNA transcript that encodes a protein that causes a disease or pathology is targeted by an agent, e.g., an oligonucleotide, described herein. In some cases, it is an NMD exon-containing pre-mRNA transcript that is targeted by an agent, e.g., an oligonucleotide, described herein. In some cases, an agent, e.g., an oligonucleotide, described herein, is designed to target a coding exon of a pre-mRNA. In some cases, an agent, e.g., an oligonucleotide, described herein, can induce skipping of an NMD exon, a coding exon, or both. In some embodiments, an NMD exon-containing pre-mRNA transcript that encodes a protein that does not cause a disease is targeted by an ASO. For example, a disease that is the result of a mutation or deficiency of a first protein in a particular pathway may be ameliorated by targeting a pre-mRNA that encodes a second protein, thereby increasing production of the second protein. In some embodiments, the function of the second protein can compensate for the mutation or deficiency of the first protein, which causes the disease or pathology.

[0126]

[0187] In some embodiments, the subject (a)(i) the OPA1 protein is produced at a reduced level compared to production from a wild-type allele; (ii) the OPA1 protein is produced in a form that has reduced functionality compared to the equivalent wild-type protein, or (iii) OPA1 protein or functional RNA is not produced; the first mutant allele; and (b)(i) the OPA1 protein is produced at a reduced level compared to production from a wild-type allele; (ii) the OPA1 protein is produced in a form that has reduced functionality compared to the equivalent wild-type protein, or (iii) OPA1 protein is not produced; Second mutant allele having The NMD exon-containing pre-mRNA is transcribed from the first allele and / or the second allele. In these embodiments, the ASO binds to the targeting portion of the NMD exon-containing pre-mRNA transcribed from the first allele or the second allele, thereby inducing exon skipping of the pseudoexon from the NMD exon-containing pre-mRNA, causing an increase in the level of the mRNA encoding the OPA1 protein and an increase in the expression of the target protein or functional RNA in the cells of the subject. In these embodiments, the target protein or functional RNA having an increased expression level resulting from exon skipping of the pseudoexon from the NMD exon-containing pre-mRNA can be in a form that has reduced function (partially functional) compared to the equivalent wild-type protein, or has full function (fully functional) compared to the equivalent wild-type protein.

[0127]

[0188] In some embodiments, the subject (a)(i) the OPA1 protein is produced at a reduced level compared to production from a wild-type allele; (ii) the OPA1 protein is produced in a form that has reduced functionality compared to the equivalent wild-type protein, or (iii) OPA1 protein or functional RNA is not produced; the first mutant allele; and (b)(i) the OPA1 protein is produced at a reduced level compared to production from a wild-type allele; (ii) the OPA1 protein is produced in a form that has reduced functionality compared to the equivalent wild-type protein, or (iii) OPA1 protein is not produced; Second mutant allele having The OPA1 pre-mRNA is transcribed from the first allele and / or the second allele. In these embodiments, the ASO binds to the targeting portion of the OPA1 pre-mRNA transcribed from the first allele or the second allele, thereby inducing exon skipping of the coding exon from the OPA1 pre-mRNA, causing increased expression of the target OPA1 protein in the cells of the subject. In these embodiments, the target OPA1 protein having increased expression levels resulting from exon skipping of the coding exon from the OPA1 pre-mRNA can be in a form that has reduced function (partially functional) compared to the equivalent full-length wild-type protein, or has full function (fully functional) compared to the equivalent full-length wild-type protein.

[0128]

[0189] In some embodiments, the level of mRNA encoding the OPA1 protein is increased by 1.1-10 fold when compared to the amount of mRNA encoding the OPA1 protein produced in a control cell, e.g., a cell that is not treated with an antisense oligomer, or a cell that is treated with an antisense oligomer that does not bind to a targeted portion of the OPA1 pre-mRNA.

[0129]

[0190] In some embodiments, the level of OPA1 protein expressed in cells contacted with an agent or a vector encoding an agent disclosed herein is increased compared to the level of OPA1 protein in control cells that were not contacted with the agent or a vector encoding an agent. In some cases, the level of OPA1 protein expressed in cells contacted with an agent or a vector encoding an agent is increased by about 1.1 to about 10 fold, about 1.5 to about 10 fold, about 2 to about 10 fold, about 3 to about 10 fold, about 4 to about 10 fold, about 1.1 to about 5 fold, about 1.1 to about 6 fold, about 1.1 to about 7 fold, about 1.1 to about 8 fold, about An increase of 1.1 to about 9 times, about 2 to about 5 times, about 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.

[0130]

[0191] In some cases, the level of OPA1 protein expressed in cells contacted with an agent or a vector encoding an agent disclosed herein is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, or about 1.5 to about 10 times higher than in the absence of the agent. fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold.

[0131]

[0192] In some embodiments, the OPA1 protein translated from the processed mRNA is a functional OPA1 protein. In some embodiments, the OPA1 protein translated from the processed mRNA is fully functional. In some embodiments, the OPA1 protein translated from the processed mRNA is a wild-type OPA1 protein. In some embodiments, the OPA1 protein translated from the processed mRNA is a full-length OPA1 protein. In some embodiments, the OPA1 protein translated from the processed mRNA has at least 80%, 82%, 84%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 9%, 96%, 97%, 98%, or 99%, or 100% sequence identity to any of the sequences listed in Table 2. In some embodiments, the OPA1 protein translated from the processed mRNA has at least 80%, 82%, 84%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 9%, 96%, 97%, 98%, or 99%, or 100% sequence identity to any sequence selected from the group consisting of SEQ ID NOs: 1272-1280.

[0132]

[0193] In some embodiments, subjects treated using the methods of the present disclosure express a partially functional OPA1 protein from one allele, where the partially functional OPA1 protein may be caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion. In some embodiments, subjects treated using the methods of the present disclosure express a non-functional OPA1 protein from one allele, where the non-functional OPA1 protein may be caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion in one allele. In some embodiments, subjects treated using the methods of the present disclosure have an OPA1 full gene deletion in one allele. Therapeutic Agents

[0194] In various embodiments of the present disclosure, compositions and methods are provided that include therapeutic agents that modulate the expression level of a target protein, for example, OPA1 protein. In some embodiments, compositions and methods are provided herein that modulate the translation of a target processed mRNA, for example, OPA1 processed mRNA. In some embodiments, compositions and methods are provided herein that modulate the alternative splicing of a target pre-mRNA, for example, OPA1 pre-mRNA. In some embodiments, compositions and methods are provided herein that induce exon skipping in the splicing of a target pre-mRNA, for example, OPA1 pre-mRNA, for example, that induce pseudoexon skipping during the splicing of a target pre-mRNA, for example, OPA1 pre-mRNA.

[0133]

[0195] The therapeutic agent disclosed herein may be a translation regulator, for example, an agent disclosed herein that regulates the translation of processed mRNA encoding a target protein. The therapeutic agent disclosed herein may be an NIE inhibitor. The therapeutic agent may include a polynucleic acid polymer.

[0134]

[0196] According to one aspect of the present disclosure, provided herein is a method for treating or preventing a condition or disease associated with a functional OPA1 protein deficiency, comprising administering an NIE inhibitor to a subject to increase the level of functional OPA1 protein, wherein the agent binds to a region of a pre-mRNA transcript to reduce the inclusion of an NMD exon in the mature transcript. For example, provided herein is a method for treating or preventing a condition associated with a functional OPA1 protein deficiency, comprising administering an NIE inhibitor to a subject to increase the level of functional OPA1 protein, wherein the agent binds to a region of an intron containing an NMD exon (e.g., exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1) of a pre-mRNA transcript, or an NMD exon activation control sequence in the same intron. For example, provided herein is a method of treating or preventing a condition associated with a deficiency of functional OPA1 protein, comprising administering to a subject an NIE inhibitor to increase the level of functional OPA1 protein, where the agent binds to a region of an intron containing an NMD exon of a pre-mRNA transcript (e.g., exon (GRCh38 / hg38:chr3 193628509 193628616); or exon (GRCh38 / hg38:chr3 193603500 193603557) of OPA1), or to an NMD exon activating regulatory sequence in the same intron. In some embodiments, the method includes administering to the subject an NIE inhibitor to increase the level of functional OPA1 protein, where the agent binds to a region of an intron that contains an NMD exon of a pre-mRNA transcript (e.g., exon 7x defined by (GRCh38 / hg38:chr3 193628509 193628616) or an exon of OPA1 other than the exon defined by (GRCh38 / hg38:chr3 193603500 193603557)), or to an NMD exon activating control sequence in the same intron.In some embodiments, a therapeutic agent promotes the elimination of an NMD exon in an OPA1 pre-mRNA other than exon 7x defined by (GRCh38 / hg38:chr3 193628509 193628616) or an exon defined by GRCh38 / hg38:chr3 193603500 193603557). In some embodiments, a composition disclosed herein comprises an agent that promotes the elimination of an NMD exon in an OPA1 pre-mRNA other than exon 7x defined by (GRCh38 / hg38:chr3 193628509 193628616) or an exon defined by GRCh38 / hg38:chr3 193603500 193603557).

[0135]

[0197] When referring to reducing NMD exon inclusion in mature mRNA, the reduction may be complete, e.g., 100%, or partial. The reduction may be clinically significant. The reduction / correction may be compared to the level of NMD exon inclusion in subjects without treatment, or compared to the amount of NMD exon inclusion in a population of similar subjects. The reduction / correction may be at least 10% less NMD exon inclusion compared to the average subject, or subject before treatment. The reduction may be at least 20% less NMD exon inclusion compared to the average subject, or subject before treatment. The reduction may be at least 40% less NMD exon inclusion compared to the average subject, or subject before treatment. The reduction may be at least 50% less NMD exon inclusion compared to the average subject, or subject before treatment. The reduction may be at least 60% less NMD exon inclusion compared to the average subject, or subject before treatment. The reduction may be at least 80% less NMD exon inclusion compared to the average subject, or subject before treatment. The reduction can be at least 90% less NMD exon inclusion compared to the average subject, or compared to a subject prior to treatment.

[0136]

[0198] According to one aspect of the present disclosure, provided herein is a method for treating or preventing a condition or disease associated with a functional OPA1 protein deficiency, comprising administering an agent to a subject to increase the level of functional OPA1 protein, wherein the agent binds to a region of a pre-mRNA transcript to reduce the inclusion of a coding exon (e.g., exon 7) in the mature transcript. For example, provided herein is a method for treating or preventing a condition associated with a functional OPA1 protein deficiency, comprising administering an agent to a subject to increase the level of functional OPA1 protein, wherein the agent binds to a region of a pre-mRNA transcript that contains a coding exon (e.g., exon 7 of OPA1). For example, provided herein is a method for treating or preventing a condition associated with a functional OPA1 protein deficiency, comprising administering an agent to a subject to increase the level of functional OPA1 protein, wherein the agent binds to a region of a pre-mRNA transcript that contains a coding exon (e.g., exon (GRCh38 / hg38:chr3 193626092-193626202)). In some embodiments, the method includes administering to the subject an agent to increase the level of functional OPA1 protein, where the agent binds to a region of a pre-mRNA transcript that contains a coding exon (e.g., an exon of OPA1 other than exon 7 defined by (GRCh38 / hg38:chr3 193626092-193626202). In some embodiments, the therapeutic agent promotes the elimination of a coding exon of an OPA1 pre-mRNA other than exon 7 defined by (GRCh38 / hg38:chr3 193626092-193626202). In some embodiments, the compositions disclosed herein include an agent that promotes the elimination of a coding exon of an OPA1 pre-mRNA other than exon 7 defined by (GRCh38 / hg38:chr3 193626092-193626202).

[0137]

[0199] When referring to increasing the level of active OPA1 protein, the increase may be clinically significant. The increase may be compared to the level of active OPA1 protein in a subject without treatment, or compared to the amount of active OPA1 protein in a population of similar subjects. The increase may be at least 10% more active PHIP protein compared to the average subject, or subject before treatment. The increase may be at least 20% more active OPA1 protein compared to the average subject, or subject before treatment. The increase may be at least 40% more active OPA1 protein compared to the average subject, or subject before treatment. The increase may be at least 50% more active OPA1 protein compared to the average subject, or subject before treatment. The increase may be at least 80% more active OPA1 protein compared to the average subject, or subject before treatment. The increase may be at least 100% more active OPA1 protein compared to the average subject, or subject before treatment. The increase may be at least 200% more active OPA1 protein compared to the average subject, or subject before treatment. The increase can be at least 500% more active OPA1 protein compared to the average subject, or compared to the subject before treatment.

[0138]

[0200] In embodiments where the NIE inhibitor comprises a polynucleic acid polymer, the polynucleic acid polymer can be about 50 nucleotides in length. The polynucleic acid polymer can be about 45 nucleotides in length. The polynucleic acid polymer can be about 40 nucleotides in length. The polynucleic acid polymer can be about 35 nucleotides in length. The polynucleic acid polymer can be about 30 nucleotides in length. The polynucleic acid polymer can be about 24 nucleotides in length. The polynucleic acid polymer can be about 25 nucleotides in length. The polynucleic acid polymer can be about 20 nucleotides in length. The polynucleic acid polymer can be about 19 nucleotides in length. The polynucleic acid polymer can be about 18 nucleotides in length. The polynucleic acid polymer can be about 17 nucleotides in length. The polynucleic acid polymer can be about 16 nucleotides in length. The polynucleic acid polymer can be about 15 nucleotides in length. The polynucleic acid polymer can be about 14 nucleotides in length. The polynucleic acid polymer can be about 13 nucleotides in length. The polynucleic acid polymer can be about 12 nucleotides in length. The polynucleic acid polymer can be about 11 nucleotides in length. The polynucleic acid polymer can be about 10 nucleotides in length. The polynucleic acid polymer can be about 10 to about 50 nucleotides in length. The polynucleic acid polymer can be about 10 to about 45 nucleotides in length. The polynucleic acid polymer can be about 10 to about 40 nucleotides in length. The polynucleic acid polymer can be about 10 to about 35 nucleotides in length. The polynucleic acid polymer can be about 10 to about 30 nucleotides in length. The polynucleic acid polymer can be about 10 to about 25 nucleotides in length. The polynucleic acid polymer can be about 10 to about 20 nucleotides in length. The polynucleic acid polymer can be about 15 to about 25 nucleotides in length. The polynucleic acid polymer can be about 15 to about 30 nucleotides in length. The polynucleic acid polymer can be about 12 to about 30 nucleotides in length.

[0139]

[0201] The sequence of the polynucleic acid polymer can 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 an mRNA transcript, such as a precursor mRNA transcript or a processed mRNA transcript. The sequence of the polynucleic acid polymer can be 100% complementary to the target sequence of an mRNA transcript (e.g., a precursor mRNA transcript or a processed mRNA transcript).

[0140]

[0202] The sequence of the polynucleic acid polymer may have four or less mismatches to the target sequence of the mRNA transcript. The sequence of the polynucleic acid polymer may have three or less mismatches to the target sequence of the mRNA transcript. The sequence of the polynucleic acid polymer may have two or less mismatches to the target sequence of the mRNA transcript. The sequence of the polynucleic acid polymer may have one or less mismatch to the target sequence of the mRNA transcript. The sequence of the polynucleic acid polymer may have no mismatches to the target sequence of the mRNA transcript.

[0141]

[0203] The polynucleic acid polymer can specifically hybridize to the target sequence of the mRNA transcript.For example, the polynucleic acid polymer can have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence complementarity to the target sequence of the mRNA transcript.Hybridization can be performed under highly stringent hybridization conditions.

[0142]

[0204] The polynucleic acid polymer comprises 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 to a sequence selected from the group consisting of SEQ ID NOs: 2-5. The polynucleic acid polymer may comprise a sequence having about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2-5.

[0143]

[0205] When referring to polynucleic acid polymer sequences, one skilled in the art will understand that one or more substitutions may be tolerated, optionally two substitutions may be tolerated in the sequence, such that the polynucleic acid polymer sequence maintains the ability to hybridize to a target sequence, or to be recognized as a target sequence if the substitution is in the target sequence. The sequence identity criteria may be determined by BLAST sequence alignment using standard / default parameters. For example, a sequence can have 99% identity and still function according to the present disclosure. In other embodiments, a sequence can have 98% identity and still function according to the present disclosure. In another embodiment, a sequence can have 95% identity and still function according to the present disclosure. In another embodiment, a sequence can have 90% identity and still function according to the present disclosure.

[0144]

[0206] In some cases, the agents, e.g., therapeutic agents, disclosed herein, include modified snRNAs, e.g., modified human or mouse snRNAs. In some cases, the agents, e.g., therapeutic agents, include vectors, e.g., viral vectors, that encode the modified snRNAs. In some embodiments, the modified snRNA is a modified U1 snRNA (see, e.g., Alanis et al., Human Molecular Genetics, 2012, Vol. 21, No. 11, pp. 2389-2398). In some embodiments, the modified snRNA is a modified U7 snRNA (see, e.g., Gadgil et al., J Gene Med. 2021;23:e3321). Modified U7 snRNA can be made by any method known in the art, including those described in Meyer, K.; Schumperli, Daniel (2012), Antisense Derivatives of U7 Small Nuclear RNA as Modulators of Pre-mRNA Splicing. In: Stamm, Stefan; Smith, Christopher WJ; Luhrmann, Reinhard (eds.), Alternative pre-mRNA Splicing: Theory and Protocols (pages 481-494), Chichester: John Wiley & Sons 10.1002 / 9783527636778.ch45, which is incorporated herein by reference in its entirety. In some embodiments, modified U7 (smOPT) does not compete with WT U7 (Stefanovic et al., 1995).

[0145]

[0207] In some embodiments, the modified snRNA comprises a smOPT modification. For example, the modified snRNA can comprise the sequence AAUUUUUGGAG. For example, the sequence AAUUUUUGGAG can replace the sequence AAUUUGUCUAG in wild-type U7 snRNA to generate modified U&snRNA (smOPT). In some embodiments, the smOPT modification of U7 snRNA renders the particle functionally inactive in histone pre-mRNA processing (Stefanovic et al., 1995). In some embodiments, modified U7 (smOPT) is stably expressed in the nucleus at higher levels than WT U7 (Stefanovic et al., 1995). In some embodiments, the snRNA comprises a U1 snRNP targeting sequence. In some embodiments, the snRNA comprises a U7 snRNP targeting sequence. In some embodiments, the snRNA comprises a modified U7 snRNP targeting sequence, and the modified U7 snRNP targeting sequence comprises smOPT. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that hybridizes to a pre-mRNA, such as an ASCE-containing pre-mRNA. For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to an OPA1 pre-mRNA. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that hybridizes to a processed mRNA of OPA1, such as the 5'UTR of a processed mRNA of OPA1. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that includes one or more sequences of an ASO disclosed herein. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that hybridizes to a targeting portion of a processed mRNA of OPA1 or a targeting portion of an OPA1 pre-mRNA. In some embodiments, the modified snRNA is modified to contain a single-stranded nucleotide sequence that contains two or more sequences that hybridize to two or more target regions of the OPA1 processed mRNA or two or more target regions of the OPA1 pre-mRNA.For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to at least eight consecutive nucleic acids of the OPA1 processed mRNA or OPA1 pre-mRNA. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that hybridizes to any of the target regions of the OPA1 processed mRNA or OPA1 pre-mRNA disclosed herein. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that includes two or more sequences that hybridize to two or more target regions of the OPA1 processed mRNA or OPA1 pre-mRNA. Antisense oligomers

[0208] Provided herein are compositions comprising antisense oligomers that induce exon skipping by binding to a targeted portion of OPA1 processed mRNA or OPA1 pre-mRNA, e.g., OPA1 NMD exon-containing pre-mRNA. As used herein, the terms "ASO" and "antisense oligomer" are used interchangeably and refer to an oligomer, such as a polynucleotide, that includes nucleobases that hybridize to a target nucleic acid (e.g., OPA1 processed mRNA or OPA1 pre-mRNA, e.g., OPA1 NMD exon-containing pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (GU). ASOs can have exact sequence complementarity to the target sequence or near complementarity (e.g., sufficient complementarity to bind to the target sequence and enhance splicing at the splice site). ASOs are designed to bind (hybridize) to a target nucleic acid (e.g., a targeted portion of an mRNA transcript) and remain hybridized under physiological conditions. Typically, when ASO hybridizes to sites other than the intended (targeted) nucleic acid sequence, it hybridizes to a limited number of sequences that are not the target nucleic acid (a small number of sites other than the target nucleic acid). The design of ASO can take into account the presence of the nucleic acid sequence of the targeted portion of the mRNA transcript (processed mRNA or pre-mRNA) or a sufficiently similar nucleic acid sequence in the genome or elsewhere in the mRNA or transcriptome of the cell, so that the possibility of the ASO binding to other sites and causing "off-target" effects is limited. Any antisense oligomer known in the art (e.g., in PCT / US2014 / 054151, published as WO2015 / 035091, entitled "Reducing Nonsense-Mediated mRNA Decay", incorporated herein by reference) can be used to carry out the methods described herein.

[0146]

[0209] In some embodiments, the ASO "specifically hybridizes" or is "specific" to a target nucleic acid or to a targeted portion of an OPA1-processed mRNA or OPA1 pre-mRNA, e.g., an NMD exon-containing pre-mRNA. Typically, such hybridization occurs at a T substantially greater than 37° C., preferably at least 50° C., typically between 60° C. and approximately 90° C. m Such hybridization preferably corresponds to stringent hybridization conditions. At a given ionic strength and pH, T m is the temperature at which 50% of the target sequence hybridizes to a complementary oligonucleotide.

[0147]

[0210] Oligomers, e.g., oligonucleotides, are "complementary" to each other when hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration. A double-stranded polynucleotide can be "complementary" to another polynucleotide when hybridization can occur between one of the strands of a first polynucleotide and a 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) need not be 100% complementary to the sequence of the target nucleic acid to which it hybridizes. In certain embodiments, the ASO can include 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 to a target region within the target nucleic acid sequence to which it is targeted. For example, an ASO in which 18 of 20 nucleobases of an oligomeric compound are complementary to a target region and therefore can specifically hybridize represents 90% complementarity. In this example, the remaining non-complementary nucleobases can be clustered together or interspersed with complementary nucleobases, and do not need to be contiguous to each other or to complementary nucleobases. The complementarity percentage of an ASO with a region of a target nucleic acid can be routinely determined using BLAST (basic local alignment search tool) and PowerBLAST programs known in the art (Altschul et al., J.Mol.Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0148]

[0211] ASO does not need to hybridize to all nucleobases in the target sequence, and the nucleobases that ASO hybridizes can be continuous or non-contiguous. ASO may hybridize over one or more segments of a pre-mRNA transcript, such that intervening or adjacent segments are not involved in the hybridization event (e.g., loop or hairpin structures may be formed). In certain embodiments, ASO hybridizes to non-contiguous nucleobases in the target pre-mRNA transcript. For example, ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobases that ASO does not hybridize to.

[0149]

[0212] The ASO described herein comprises a nucleobase that is complementary to a nucleobase present in a target portion of an OPA1-processed mRNA or an OPA1 pre-mRNA, e.g., an NMD exon-containing pre-mRNA. The term ASO includes oligonucleotides and any other oligomeric molecules that contain a nucleobase that can hybridize to a complementary nucleobase of a target mRNA, but do not contain a sugar moiety, e.g., a peptide nucleic acid (PNA). The ASO may comprise naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and / or modified backbones. In some embodiments, all nucleotides of the ASO are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described herein will be apparent to one of skill in the art and can be found, for example, in U.S. Pat. No. 6,147,200, U.S. Pat. No. 8,258,109, U.S. Pat. No. 5,656,612, U.S. Patent Publication No. 2012 / 0190728, and Dias and Stein, Mol. Cancer Ther. 2002, pp. 347-355, which are incorporated herein by reference in their entireties.

[0150]

[0213] The one or more nucleobases of the ASO may be any naturally occurring unmodified nucleobase, such as adenine, guanine, cytosine, thymine, and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase to be capable of hydrogen bonding with a nucleobase present in the target pre-mRNA. Examples of modified nucleobases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethoylcytosine.

[0151]

[0214] The ASOs described herein also include backbone structures that connect the components of the oligomer. The terms "backbone structure" and "oligomer linkage" can be used interchangeably and refer to the linkage between the monomers of the ASO. In naturally occurring oligonucleotides, the backbone includes 3'-5' phosphodiester linkages that connect the sugar moieties of the oligomer. The backbone structures or oligomer linkages of the ASOs described herein include, but are not limited to, phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroaniladates, and phosphoroamidates. See, e.g., 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., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990). In some embodiments, the backbone structure of the ASO does not contain phosphorus, but rather contains peptide bonds, such as peptide nucleic acids (PNAs) or linking groups including carbamates, amides, and linear and cyclic hydrocarbon groups. In some embodiments, the backbone modification is a phosphorothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.

[0152]

[0215] In some embodiments, the stereochemistry at each phosphorus internucleotide linkage of the ASO backbone is random. In some embodiments, the stereochemistry at each phosphorus internucleotide linkage of the ASO backbone is controlled and not random. For example, U.S. Patent Application Publication No. 2014 / 0194610, "Methods for the Synthesis of Functionalized Nucleic Acids," incorporated herein by reference, describes methods for independently selecting the handedness of chirality at each phosphorus atom in a nucleic acid oligomer. In some embodiments, the ASOs used in the methods of the present disclosure include ASOs with non-random phosphorus internucleotide linkages, including but not limited to any ASOs listed in Tables 5 and 6 herein. In some embodiments, the compositions used in the methods of the present disclosure include pure diastereomeric ASOs. In some embodiments, a composition used in a method of the disclosure comprises an ASO having a diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

[0153]

[0216] In some embodiments, the ASO has a non-random mixture of Rp and Sp configurations in its phosphointernucleotide linkages. For example, it has been suggested that a mixture of Rp and Sp is required in antisense oligonucleotides to achieve a balance between good 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, the ASOs used in the methods of the disclosure, including but not limited to any of the ASOs set forth herein in SEQ ID NOs: 2-5, comprise 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 and the remaining Sp, or about 100% Rp.In some embodiments, the ASO used in the methods of the present disclosure, including but not limited to any ASO described herein, comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 consecutive nucleic acids of any one of SEQ ID NOs: 2-5, and has an Rp of about 10% to about 100%, about 15% to about 100%, about 20% to about 100%, about 25% to about 100%, about 30% to about 100%, about 35% to about 100%, about 40% to about 40%. 00% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about 60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, or about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp and the remainder Sp.

[0154]

[0217] In some embodiments, the ASO used in the methods of the disclosure, including but not limited to any ASO described herein, comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 consecutive nucleic acids of any one of SEQ ID NOs: 2-5, and has about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about The composition may comprise at least about 25% Sp, 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 Rp, or about 100% Sp. In an embodiment, an ASO used in the methods of the present disclosure, including but not limited to any ASO described herein, comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region containing at least 8 consecutive nucleic acids of any one of SEQ ID NOs: 2 to 5, and has 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, about 4 ... % Sp, about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55% Sp, with the remainder Rp.

[0155]

[0218] Any of the ASOs described herein may contain modified sugar moieties or sugar analogs that contain ribose or deoxyribose, or morpholine rings, as found in naturally occurring nucleotides. Non-limiting examples of modified sugar moieties include 2'-substituents, such as 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'-ON-methyl-acetamide (2'-NMA); 2'F; N3'→P5' phosphoramidate, 2' dimethylaminooxyethoxy, 2' dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidiniumethyl, carbamate modified sugars, and bicyclic modified sugars. In some embodiments, the sugar moiety modification is selected from 2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety modification is an additional crosslink, such as in locked nucleic acids (LNAs). In some embodiments, the sugar analog contains a morpholine ring, e.g., phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises a ribofuransyl or 2' deoxyribofuransyl modification. In some embodiments, the sugar moiety comprises a 2'4'-constrained 2'O-methyloxyethyl (cMOE) modification. In some embodiments, the sugar moiety comprises a cEt 2',4' constrained 2'-O-ethyl BNA modification. In some embodiments, the sugar moiety comprises a tricycloDNA (tcDNA) modification. In some embodiments, the sugar moiety comprises an ethylene nucleic acid (ENA) modification. In some embodiments, the sugar moiety comprises an MCE modification. Modifications are known in the art and described in the literature, e.g., 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 herein by reference for this purpose.

[0156]

[0219] In some embodiments, each monomer of the ASO is modified in the same way, for example, each link in the backbone of the ASO contains a phosphorothioate linkage, or each ribose sugar moiety contains a 2'-O-methyl modification. Such modifications present in each of the monomer components of the ASO are referred to as "uniform modifications". In some cases, a combination of different modifications may be desired, for example, an ASO may contain a combination of phosphorodiamidate linkages and sugar moieties containing morpholine rings (morpholinos). A combination of different modifications to an ASO is referred to as "mixed modifications" or "mixed chemistry".

[0157]

[0220] In some embodiments, the ASO comprises one or more backbone modifications. In some embodiments, the ASO comprises one or more sugar moiety modifications. In some embodiments, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs described herein, or any component of the ASO (e.g., nucleobase, sugar moiety, backbone) may be modified to achieve a desired property or activity of the ASO or to reduce an undesirable property or activity of the ASO. For example, the ASO, or one or more components of any ASO, may be modified to enhance binding affinity for the target sequence of a pre-mRNA transcript, to reduce binding to any non-target sequences, to reduce degradation by cellular nucleases (i.e., RNase H), to improve uptake of the ASO into cells and / or into the nucleus of a cell, to alter the pharmacokinetics or pharmacodynamics of the ASO, and / or to modulate the half-life of the ASO.

[0158]

[0221] In some embodiments, the ASO is comprised of 2'-O-(2-methoxyethyl) (MOE) phosphorothioate modified nucleotides. In some embodiments, the ASO is comprised of 2'NMA phosphorothioate modified nucleotides. ASOs comprised of such nucleotides are particularly well suited for the methods disclosed herein, as oligomers with such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable for oral delivery, for example, in some embodiments described herein. See, e.g., Geary et al., J Pharmacol Exp Ther. 2001;296(3):890-7; Geary et al., J Pharmacol Exp Ther. 2001;296(3):898-904.

[0159]

[0222] Methods for synthesizing ASOs are known to those of skill in the art. Alternatively, or additionally, ASOs may be obtained from commercial suppliers.

[0160]

[0223] Unless otherwise specified, the left-hand end of a single-stranded nucleic acid (e.g., a pre-mRNA transcript, an oligonucleotide, an ASO, etc.) sequence is the 5' end, and the left-hand direction of a single-stranded or double-stranded nucleic acid sequence is referred to as the 5' direction. Similarly, the right-hand end or direction of a nucleic acid sequence (single-stranded or double-stranded) is the 3' end or direction. Generally, a region or sequence that is 5' to a reference point in a nucleic acid is referred to as "upstream" and a region or sequence that is 3' to a reference point in a nucleic acid is referred to as "downstream". Generally, the 5' direction or end of an mRNA is where the initiation or start codon is located, and the 3' end or direction is where the stop codon is located. In some aspects, nucleotides that are upstream of the reference point in a nucleic acid may be designated by a negative number, and nucleotides that are downstream of the reference point may be designated by a positive number. For example, a reference point (e.g., an exon-exon junction in an mRNA) may be designated as a "zero" site, with the nucleotide immediately adjacent to the reference point upstream being designated as a "minus one," e.g., "-1," and the nucleotide immediately adjacent to the reference point downstream being designated as a "plus one," e.g., "+1."

[0161]

[0224] In some embodiments, the ASO is complementary to (and binds to) a targeted portion of an OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA, that is downstream (in the 3' direction) (e.g., in the direction designated by a positive number relative to the 5' splice site) of the included exon in the OPA1 pre-mRNA. In some embodiments, the ASO is complementary to a targeted portion of an OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA, that is within a region of about +1 to about +500 relative to the 5' splice site (or 3' end) of the included exon. In some embodiments, the ASO can be complementary to a targeted portion of an OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA, that is within a region of +6 to +40,000 nucleotides relative to the 5' splice site (or 3' end) of the included exon.In some embodiments, the ASO is about +1 to about +40,000, about +1 to about +30,000, about +1 to about +20,000, about +1 to about +15,000, about +1 to about +10,000, about +1 to about +5,000, about +1 to about +4,000, about +1 to about +3,000, about +1 to about +2,000, about +1 to about +1,000, about + 1 to about +500, about +1 to about +490, about +1 to about +480, about +1 to about +470, about +1 to about +460, about +1 to about +450, about +1 to about +440, about +1 to about +430, about +1 to about +420, about +1 to about +410, about +1 to about +400, about +1 to about +390, about +1 to about +380, about +1 to about +370, about +1 to about +360, about +1 to about +350, about +1 to about +340, About +1 to about +330, about +1 to about +320, about +1 to about +310, about +1 to about +300, about +1 to about +290, about +1 to about +280, about +1 to about +270, about +1 to about +260, about +1 to about +250, about +1 to about +240, about +1 to about +230, about +1 to about +220, about +1 to about +210, about +1 to about +200, about +1 to about +190, about +1 to about +180, about +1 to about +17 is complementary to a targeting portion within a region of 0, about +1 to about +160, about +1 to about +150, about +1 to about +140, about +1 to about +130, about +1 to about +120, about +1 to about +110, about +1 to about +100, about +1 to about +90, about +1 to about +80, about +1 to about +70, about +1 to about +60, about +1 to about +50, about +1 to about +40, about +1 to about +30, or about +1 to about +20. In some embodiments, the ASO is complementary to a targeting portion that is within a region of about +1 to about +100, about +100 to about +200, about +200 to about +300, about +300 to about +400, or about +400 to about +500 relative to the 5' splice site (or 3' end) of the encompassing exon.

[0162]

[0225] In some embodiments, the ASO is complementary to (or binds to) a targeted portion of an OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA, that is upstream (in the 5' direction) (e.g., in the direction designated by a negative number relative to the 5' splice site) of the 5' splice site (or 3' end) of the included exon in the OPA1 pre-mRNA, e.g., the OPA1 NMD exon-containing pre-mRNA. In some embodiments, the ASO is complementary to a targeted portion of an OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA, that is within a region of about -4 to about -270 relative to the 5' splice site (or 3' end) of the included exon. In some embodiments, the ASO can be complementary to a targeted portion of an OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA, that is within a region of -1 to -40,000 nucleotides relative to the 5' splice site (or 3' end) of the included exon.In some embodiments, the ASO is about -1 to about -40,000, about -1 to about -30,000, about -1 to about -20,000, about -1 to about -15,000, about -1 to about -10,000, about -1 to about -5,000, about -1 to about -4,000, about -1 to about -3,000, about -1 to about -2,000, about -1 to about -1,000, about - 1 to about -500, about -1 to about -490, about -1 to about -480, about -1 to about -470, about -1 to about -460, about -1 to about -450, about -1 to about -440, about -1 to about -430, about -1 to about -420, about -1 to about -410, about -1 to about -400, about -1 to about -390, about -1 to about -380, about -1 to about -370, about -1 to about -360, about -1 to about -350, about -1 to about -340, About -1 to about -330, about -1 to about -320, about -1 to about -310, about -1 to about -300, about -1 to about -290, about -1 to about -280, about -1 to about -270, about -1 to about -260, about -1 to about -250, about -1 to about -240, about -1 to about -230, about -1 to about -220, about -1 to about -210, about -1 to about -200, about -1 to about -190, about -1 to about -180, about -1 to about -17 0, about -1 to about -160, about -1 to about -150, about -1 to about -140, about -1 to about -130, about -1 to about -120, about -1 to about -110, about -1 to about -100, about -1 to about -90, about -1 to about -80, about -1 to about -70, about -1 to about -60, about -1 to about -50, about -1 to about -40, about -1 to about -30, or about -1 to about -20.

[0163]

[0226] In some embodiments, the ASO is complementary to a targeted portion of an OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA, that is upstream (in the 5' direction) (e.g., in the direction designated by a negative number) of the 3' splice site (or 5' end) of the included exon in the OPA1 pre-mRNA. In some embodiments, the ASO is complementary to a targeted portion of an OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA, that is within a region of about -1 to about -500 relative to the 3' splice site (or 5' end) of the included exon. In some embodiments, the ASO is complementary to a targeted portion of an OPA1 pre-mRNA that is within a region of -1 to -40,000 relative to the 3' splice site of the included exon.In some embodiments, the ASO is about -1 to about -40,000, about -1 to about -30,000, about -1 to about -20,000, about -1 to about -15,000, about -1 to about -10,000, about -1 to about -5,000, about -1 to about -4,000, about -1 to about -3,000, about -1 to about -2,000, about -1 to about -1,000, about -1 to about -50 0, about -1 to about -490, about -1 to about -480, about -1 to about -470, about -1 to about -460, about -1 to about -450, about -1 to about -440, about -1 to about -430, about -1 to about -420, about -1 to about -410, about -1 to about -400, about -1 to about -390, about -1 to about -380, about -1 to about -370, about -1 to about -360, about -1 to about -350, about -1 to about -340, about -1 to about about -330, about -1 to about -320, about -1 to about -310, about -1 to about -300, about -1 to about -290, about -1 to about -280, about -1 to about -270, about -1 to about -260, about -1 to about -250, about -1 to about -240, about -1 to about -230, about -1 to about -220, about -1 to about -210, about -1 to about -200, about -1 to about -190, about -1 to about -180, about -1 to about -170, The ASO is complementary to a targeting moiety within a region of about -1 to about -160, about -1 to about -150, about -1 to about -140, about -1 to about -130, about -1 to about -120, about -1 to about -110, about -1 to about -100, about -1 to about -90, about -1 to about -80, about -1 to about -70, about -1 to about -60, about -1 to about -50, about -1 to about -40, about -1 to about -30, or about -1 to about -20. In some embodiments, the ASO is complementary to a targeting moiety within a region of about -1 to about -100, about -100 to about -200, about -200 to about -300, about -300 to about -400, or about -400 to about -500 relative to the 3' splice site of the encompassing exon.

[0164]

[0227] In some embodiments, the ASO is complementary to a targeted portion of an OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA, that is downstream (in the 3' direction) (e.g., in the direction designated by a positive number) of the 3' splice site (5' end) of the included exon in the OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA. In some embodiments, the ASO is complementary to a targeted portion of an OPA1 pre-mRNA that is within a region of about +1 to about +40,000 relative to the 3' splice site of the included exon. In some embodiments, the ASO is at least about +1 to about +40,000, about +1 to about +30,000, about +1 to about +20,000, about +1 to about +15,000, about +1 to about +10,000, about +1 to about +5,000, about +1 to about +4,000, about +1 to about +3,000, about +1 to about +2,000, about +1 to about +1,000, about +1 to about +500, About +1 to about +490, about +1 to about +480, about +1 to about +470, about +1 to about +460, about +1 to about +450, about +1 to about +440, about +1 to about +430, about +1 to about +420, about +1 to about +410, about +1 to about +400, about +1 to about +390, about +1 to about +380, about +1 to about +370, about +1 to about +360, about +1 to about +350, about +1 to about +340, about +1 to about +330 , about +1 to about +320, about +1 to about +310, about +1 to about +300, about +1 to about +290, about +1 to about +280, about +1 to about +270, about +1 to about +260, about +1 to about +250, about +1 to about +240, about +1 to about +230, about +1 to about +220, about +1 to about +210, about +1 to about +200, about +1 to about +190, about +1 to about +180, about +1 to about +170, about +1 to about +16 or about +1 to about +10.

[0165]

[0228] In some embodiments, the targeting portion of an OPA1 pre-mRNA, e.g., an OPA1 NMD exon-containing pre-mRNA, is within a region of +100 relative to the 5' splice site (3' end) of the included exon to -100 relative to the 3' splice site (5' end) of the included exon. In some embodiments, the targeting portion of an OPA1 NMD exon-containing pre-mRNA is within an NMD exon. In some embodiments, the targeting portion of an OPA1 NMD exon-containing pre-mRNA includes the boundary between the pseudoexon and the intron.

[0166]

[0229] ASOs can be any length suitable for effective enhancement of specific binding and splicing. In some embodiments, ASOs are 8-50 nucleobases. For example, ASOs can be 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 nucleobases long. In some embodiments, ASOs are more than 50 nucleobases long. In some embodiments, the ASO is 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 nucleobases, 8-15 nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 nucleobases, 10-50 nucleobases, 10-40 nucleobases, 10-35 nucleobases, 10-30 nucleobases, 10-25 nucleobases, 10-20 nucleobases, 10-15 nucleobases, 11-50 nucleobases, 11-40 nucleobases, 11-35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11-15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12 ~30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, 12-15 nucleobases, 13-50 nucleobases, 13-40 nucleobases, 13-35 nucleobases, 13-30 nucleobases, 13-25 nucleobases, 13-20 nucleobases, 14-50 nucleobases, 14-40 nucleobases, 14-35 nucleobases, 14-30 nucleobases, 14-25 nucleobases, 14-20 nucleobases , 15-50 nucleobases, 15-40 nucleobases, 15-35 nucleobases, 15-30 nucleobases, 15-25 nucleobases, 15-20 nucleobases, 20-50 nucleobases, 20-40 nucleobases, 20-35 nucleobases, 20-30 nucleobases, 20-25 nucleobases, 25-50 nucleobases, 25-40 nucleobases, 25-35 nucleobases, or 25-30 nucleobases in length. In some embodiments, the ASO is 18 nucleotides in length. In some embodiments, the ASO is 15 nucleotides in length. In some embodiments, the ASO is 25 nucleotides in length.

[0167]

[0230] In some embodiments, two or more ASOs are used that have different chemistries but are complementary to the same targeting portion of the processed mRNA or pre-mRNA, hi some embodiments, two or more ASOs are used that are complementary to different targeting portions of the processed mRNA or pre-mRNA.

[0168]

[0231] In some embodiments, the antisense oligonucleotides of the present disclosure are chemically linked to one or more moieties or conjugates, such as targeting moieties or other conjugates that enhance the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholesteryl moieties, aliphatic chains, such as dodecanediol or undecyl residues, polyamines or polyethylene glycol chains, or adamantane acetic acid. Oligonucleotides containing lipophilic moieties and preparation methods are described in the published literature. In embodiments, the antisense oligonucleotides are conjugated to moieties, such as, but not limited to, abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), lipids, or polyhydrocarbon compounds. The conjugate can be linked to one or more of any nucleotides, including antisense oligonucleotides, at any of several positions of the sugar, base, or phosphate group, for example, using a linker, as understood in the art and described in the literature. The linker can include a bivalent or trivalent branched linker. In an embodiment, the conjugate is attached to the 3' end of the antisense oligonucleotide. Methods for preparing oligonucleotide conjugates are described, for example, in U.S. Patent No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides," incorporated herein by reference.

[0169]

[0232] In some embodiments, the agents disclosed herein include a cell-penetrating peptide conjugated to an antisense oligomer, e.g., an ASO disclosed herein. The term "cell-penetrating peptide" or "CPP" may be used interchangeably and refer to a cationic cell-penetrating peptide, also called a delivery peptide, carrier peptide, or peptide transduction domain. The peptide has the ability to induce cell permeability to at least 70%, 80%, 90%, or 95%, or 100% of the cells of a given cell culture population, and can allow the movement of macromolecules into multiple tissues in vivo upon systemic administration. Non-limiting examples of cell-penetrating peptides that can be used in the agents disclosed herein are listed in Table 9. The synthesis, structure, and delivery properties of morpholino oligomers are detailed in U.S. Patent Application Publication No. 2010 / 0016215, and in Jearawiriyapaisarn et al. (2008), Mol Ther., September 2008;16(9):1624-1629, both of which are incorporated herein in their entireties.

[0170]

[0233] In some embodiments, the agents disclosed herein comprise a cell-penetrating peptide conjugated to a phosphoramidate morpholino oligomer, the phosphoramidate morpholino oligomer having the sequence of any of the ASOs disclosed herein. The term "morpholino oligomer" or "PMO" (phosphoramidate- or phosphorodiamidate morpholino oligomer) refers to an oligonucleotide composed of morpholino subunit structures, (i) linked together by phosphorus-containing linkages that are 1-3 atoms long, preferably 2 atoms long and preferably uncharged or cationic, connecting the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, and (ii) each morpholino ring has a purine or pyrimidine base-pairing moiety available for base-specific hydrogen bonding to a base in a polynucleotide. Changes can be made to the phosphorodiamidate linkages, provided that the changes do not interfere with binding or activity. For example, the oxygen attached to the phosphorus can be replaced by sulfur (thiophosphorodiamidate). The 5' oxygen may be substituted with amino or lower alkyl substituted amino. The pendant nitrogen attached to the phosphorus may be unsubstituted, mono- or di-substituted with (optionally substituted) lower alkyl. See also the discussion of cationic linkages below. The purine or pyrimidine base pairing moiety is typically adenine, cytosine, guanine, uracil, thymidine or inosine. The synthesis, structure and binding properties of morpholino oligomers are detailed in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063 and 5,506,337, and PCT Publication No. WO 2008036127, all of which are incorporated herein by reference.

[0171]

[0234] In some embodiments, the nucleic acid targeted by the ASO is the processed mRNA of OPA1 in a cell. In some embodiments, the nucleic acid targeted by the ASO is an OPA1 pre-mRNA, e.g., an NMD exon-containing pre-mRNA, expressed in a cell, e.g., a eukaryotic cell. In some embodiments, the term "cell" may refer to a population of cells. In some embodiments, the cell is in a subject. In some embodiments, the cell is isolated from a subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is a pathology or disease-related cell or cell line. In some embodiments, the cell is in vitro (e.g., in cell culture). Pharmaceutical Compositions

[0235] The pharmaceutical compositions or preparations comprising the described compositions and agents for use in any of the described methods, such as antisense oligonucleotides, can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the open literature.In embodiments, the pharmaceutical compositions or preparations for treating subjects comprise an effective amount of any antisense oligomer as described herein, or its pharmaceutically acceptable salt, solvate, hydrate, or ester.The pharmaceutical preparations comprising antisense oligomers can further comprise pharmaceutically acceptable excipients, diluents, or carriers.

[0172]

[0236] Pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and represent a reasonable benefit / risk ratio. (See, e.g., S. M. Berge et al., J. Pharmaceutical Sciences, 66:1-19 (1977), incorporated herein by reference for this purpose. Salts may be added in the form of a salt during the final isolation and purification of the compounds. They can be prepared in situ or separately by reacting the free base form with a suitable organic acid. Examples of pharma-ceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other documented methodologies such as ion exchange. Other pharma-ceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, etc. salts, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc.Further pharma-ceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed where appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates.

[0173]

[0237] In some embodiments, the composition is formulated into any of many possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. In embodiments, the composition is formulated as a suspension in an aqueous, non-aqueous, or mixed medium. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. Suspensions may also contain stabilizers. In embodiments, pharmaceutical formulations or compositions of the present disclosure include, but are not limited to, solutions, emulsions, microemulsions, foams, or liposome-containing formulations (e.g., cationic or non-cationic liposomes).

[0174]

[0238] The pharmaceutical compositions or formulations described herein may optionally include one or more penetration enhancers, carriers, excipients, or other active or inactive ingredients known to those skilled in the art or described in the published literature. In embodiments, liposomes also include sterically stabilized liposomes, e.g., liposomes that include one or more specialized lipids. These specialized lipids result in liposomes with enhanced circulation life. In embodiments, the sterically stabilized liposomes include one or more glycolipids or are derivatized with one or more hydrophilic polymers, e.g., polyethylene glycol (PEG) moieties. In some embodiments, a surfactant is included in the pharmaceutical formulation or composition. The use of surfactants in drug products, formulations, and emulsions is well known in the art. In embodiments, the present disclosure uses a penetration enhancer to provide efficient delivery of antisense oligonucleotides, e.g., 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.

[0175]

[0239] In some embodiments, the pharmaceutical formulation comprises more than one antisense oligonucleotide. In embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent. Combination therapy

[0240] In some aspects, methods, compositions and kits related to combination therapy are provided herein. In some embodiments, the combination therapy disclosed herein involves utilizing an agent that modulates the translational control element of the processed mRNA that encodes the target protein, for example, the OPA1 protein, and an agent that modulates the splicing of the pre-mRNA transcribed from the gene that encodes the target protein, for example, the OPA1 gene. In some aspects, compositions, methods and kits related to combination therapy are provided herein that utilize an agent that targets at least a portion of the 5'UTR of the processed mRNA that encodes the target protein, for example, the OPA1 protein, and an agent that modulates the splicing of the pre-mRNA transcribed from the gene that encodes the target protein, for example, the OPA1 gene.

[0176]

[0241] Provided herein is a method for increasing expression of a target protein in a cell having a processed mRNA that optionally includes a translational control element that encodes the target protein and inhibits translation of the processed mRNA, the method comprising delivering to the cell (1) a first agent or a first nucleic acid sequence encoding the first agent, and (2) a second agent or a second nucleic acid sequence encoding the second agent, wherein the first agent modulates splicing of a pre-mRNA transcribed from a target gene that encodes the target protein, and the second agent modulates the structure of the translational control element of the processed mRNA that encodes the target protein, thereby increasing expression of the target protein in the cell. In some embodiments, provided herein are methods of treatment comprising administering to a subject in need of treatment (1) a first agent or a first nucleic acid sequence encoding the first agent, and (2) a second agent or a second nucleic acid sequence encoding the second agent, wherein the first agent modulates splicing of a pre-mRNA transcribed from a target gene that encodes a target protein, and the second agent modulates the structure of a translational control element of a processed mRNA that encodes the target protein, thereby increasing expression of the target protein in cells of the subject.

[0177]

[0242] In some cases, provided herein is a method of increasing expression of a target protein in a cell having a processed mRNA that includes a translational control element that encodes the target protein and inhibits translation of the processed mRNA, the method comprising delivering to the cell (1) a first agent or a first nucleic acid sequence encoding the first agent, and (2) a second agent or a second nucleic acid sequence encoding the second agent, wherein the first agent modulates splicing of a pre-mRNA transcribed from a target gene that encodes the target protein, and the second agent (a) binds to a targeted portion of the processed mRNA, (b) modulates the interaction of the translational control element with a factor involved in translation of the processed mRNA, or (c) a combination of (a) and (b), thereby increasing expression of the target protein in the cell. In some embodiments, provided herein are methods of treatment comprising administering to a subject in need of treatment (1) a first agent or a first nucleic acid sequence encoding the first agent, and (2) a second agent or a second nucleic acid sequence encoding the second agent, wherein the first agent modulates splicing of a pre-mRNA transcribed from a target gene encoding a target protein, and the second agent (a) binds to a targeted portion of the processed mRNA, (b) modulates the interaction of a translational control element with a factor involved in the translation of the processed mRNA, or (c) a combination of (a) and (b), thereby increasing expression of the target protein in cells of the subject.

[0178]

[0243] In some embodiments, provided herein is a method of modulating expression of a target protein in a cell, comprising contacting a cell with (1) a first agent or a first nucleic acid sequence encoding the first agent, and (2) a second agent or a second nucleic acid sequence encoding the second agent, wherein the first agent comprises a first antisense oligomer having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6-275 and 280-299, and the second agent comprises a second antisense oligomer that binds to the 5'UTR of a processed mRNA encoding the target protein, and the target protein is OPA1 protein. In some cases, provided herein is a method of treatment comprising administering to a subject in need of treatment (1) a first agent or a first nucleic acid sequence encoding the first agent, and (2) a second agent or a second nucleic acid sequence encoding the second agent, wherein the first agent comprises a first antisense oligomer having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6-275 and 280-299, and the second agent comprises a second antisense oligomer that binds to the 5'UTR of a processed mRNA encoding a target protein, and the target protein is OPA1 protein.

[0179]

[0244] In some aspects, provided herein is a pharmaceutical composition comprising: (1) a first therapeutic agent or a first nucleic acid sequence encoding the first therapeutic agent; and (2) a second therapeutic agent or a second nucleic acid sequence encoding the second therapeutic agent, wherein the first therapeutic agent comprises a first antisense oligomer having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6-275 and 280-299, and the second therapeutic agent comprises a second antisense oligomer that binds to the 5'UTR of a processed mRNA encoding the OPA1 protein.

[0180]

[0245] In some aspects, provided herein is a pharmaceutical composition comprising: (1) a first therapeutic agent or a first nucleic acid sequence encoding the first therapeutic agent; (2) a second therapeutic agent or a second nucleic acid sequence encoding the second therapeutic agent; and (3) a pharma- ceutically acceptable carrier or excipient, wherein the first therapeutic agent modulates splicing of a pre-mRNA transcribed from a target gene encoding a target protein; and the second therapeutic agent (a) binds to a targeted portion of the processed mRNA that encodes the OPA1 protein and comprises a translational control element; (b) modulates the interaction of the translational control element with a factor involved in translation of the processed mRNA; or (c) a combination of (a) and (b), wherein the translational control element inhibits translation of the processed mRNA.

[0181]

[0246] In some aspects, provided herein is a pharmaceutical composition comprising: (1) a first therapeutic agent or a first nucleic acid sequence encoding the first therapeutic agent; (2) a second therapeutic agent or a second nucleic acid sequence encoding the second therapeutic agent; and (3) a pharma- ceutically acceptable carrier or excipient; wherein the first therapeutic agent modulates splicing of a pre-mRNA transcribed from a target gene encoding a target protein; and the second therapeutic agent modulates the structure of a translational control element of a processed mRNA encoding the target protein, and the translational control element inhibits translation of the processed mRNA.

[0182]

[0247] In some of these cases, the first antisense oligomer has at least 80%, 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 36, 236, 242, 250, 280-283, 288, and 290-292. In some of these cases, the first antisense oligomer has at least 80%, 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 227-242, 250, 280-283, 288, and 290-292. In some of these, the first antisense oligomer has at least 80%, 90%, or 100% sequence identity to SEQ ID NO: 267. In some of these, the first antisense oligomer has at least 80%, 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 236, 242, 250, 280-283, 288, and 290-292. In some of these, the second antisense oligomer has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 608-1253. In some of these, the second antisense oligomer has at least 80%, 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 827-847, 932-937, 953, 968, and 988-1023.

[0183]

[0248] In some embodiments, the agent disclosed in the present disclosure can be used in combination with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents can include small molecules. For example, the one or more additional therapeutic agents can include small molecules described in WO2016128343A1, WO2017053982A1, WO2016196386A1, WO201428459A1, WO201524876A2, WO2013119916A2, and WO2014209841A2, which are incorporated herein in their entirety. In some embodiments, the one or more additional therapeutic agents include ASOs that can be used to correct intron retention. Treatment of the subject

[0249] Any of the compositions provided herein can be administered to an individual. An "individual" can be used interchangeably with a "subject" or a "patient." An individual can be a mammal, e.g., 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 embodiments, an individual is a human. In embodiments, an individual is a fetus, embryo, or child. In other embodiments, an individual can be another eukaryotic organism, e.g., a plant. In some embodiments, a composition provided herein is administered to a cell ex vivo.

[0184]

[0250] In some embodiments, the compositions provided herein are administered to an individual as a method of treating a disease or disorder. In some embodiments, the individual has a genetic disease, such as any of the diseases described herein. In some embodiments, the individual is at risk of having a disease, such as any of the diseases described herein. In some embodiments, the individual is at increased risk of having a disease or disorder caused by an insufficient amount of protein or insufficient activity of a protein. When an individual is at "increased risk" of having a disease or disorder caused by an insufficient amount of protein or insufficient activity of a protein, the method involves a preventative or prophylactic treatment. For example, an individual may be at increased risk of having such a disease or disorder due to a family history of the disease. Typically, an individual at increased risk of having such a disease or disorder will benefit from a prophylactic treatment (e.g., by preventing or delaying the onset or progression of the disease or disorder). In an embodiment, a fetus is treated in utero, for example, by administering an ASO composition to the fetus directly or indirectly (e.g., via the mother).

[0185]

[0251] In some cases, the subject pharmaceutical compositions and methods are applicable to the treatment of a condition or disease associated with an OPA1 deficiency. In some cases, the subject pharmaceutical compositions and methods are applicable to the treatment of an ocular disease or condition. In some cases, the subject pharmaceutical compositions and methods are useful for treating optic atrophy type 1, autosomal dominant optic atrophy (ADOA), 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 external ophthalmoplegia; hearing loss; ataxia; motor neuropathy; sensory neuropathy; myopathy; Beer's syndrome; brain dysfunction; encephalopathy; peripheral neuropathy; fatal pediatric mitochondrial encephalopathy; hypertrophic cardiomyopathy; spastic ataxia syndrome; sensory motor peripheral neuropathy; hypotonia; gastrointestinal motility and swallowing disorders; optic atrophy; optic atrophy plus syndrome; mitochondrial DNA depletion syndrome 14; late onset cardiomyopathy; diabetic cardiomyopathy; Alzheimer's disease; focal segmental glomerulosclerosis; kidney disease; Huntington's disease; cognitive function in healthy aging. The present invention is applicable to the treatment of: cognitive impairment; prion diseases; late onset dementia and Parkinsonism; mitochondrial myopathy; Leigh syndrome; Friedreich's ataxia; Parkinson's disease; MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes): pyruvate dehydrogenase complex deficiency; chronic kidney disease; Leber's hereditary optic neuropathy; obesity; age-related generalized neurodegeneration; skeletal muscle atrophy; cardiac and cerebral ischemic disorders; widespread hepatic apoptosis; NARP (neuropathy, ataxia, and retinitis pigmentosa); MERRF (myoclonic epilepsy with ragged-red fibers); Pearson / Kerns-Sayre syndrome; MIDD (maternally inherited diabetes and deafness); mitochondrial trifunctional protein deficiency; Fuchs endothelial dystrophy; macular telangiectasia; retinitis pigmentosa; Leber's congenital amaurosis; hereditary maculopathy; Stargardt's disease; or Sohrsby's fundus degeneration.

[0186]

[0252] In some embodiments, the compositions and methods provided herein are applicable to the treatment of mitochondrial disorders, e.g., alleviating one or more ocular symptoms of primary mitochondrial disorders. In some cases, the compositions and methods provided herein are applicable to the treatment of optic neuropathy (e.g., DOA or dominant optic atrophy, LHON or Leber's hereditary optic neuropathy), CPEO (chronic progressive external ophthalmoplegia), or pigmentary retinopathy (e.g., NARP (neuropathy, ataxia, retinitis pigmentosa), MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes), MERRF (myoclonus epilepsy with ragged-red fibers), Leigh syndrome, Pearson / Kerns-Sayre syndrome, MIDD (maternally inherited diabetes mellitus and deafness), or mitochondrial triprotein deficiency).

[0187]

[0253] In some embodiments, the compositions and methods provided herein are applicable to the treatment of age-related ophthalmic diseases associated with mitochondrial dysfunction, such as glaucoma, age-related macular degeneration, diabetic retinopathy, Fuchs endothelial corneal dystrophy, or macular telangiectasia.

[0188]

[0254] In some embodiments, the compositions and methods provided herein are applicable to the treatment of inherited ophthalmic diseases associated with mitochondrial dysfunction, such as retinitis pigmentosa (e.g., CERKL retinitis pigmentosa), Leber congenital amaurosis, or hereditary maculopathy (e.g., Stargardt disease or Sohrsby fundus degeneration).

[0189]

[0255] Autosomal dominant optic atrophy (ADOA) is the most common inherited optic neuropathy and is characterized by retinal ganglion cell loss. 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 may play a pivotal role in maintaining mitochondrial structure and function. Most OPA1 mutations can lead to haploinsufficiency resulting in approximately 50% reduction in normal OPA1 protein levels. Approximately 1 in 30,000 people are affected worldwide, with a higher incidence of approximately 1 in 10,000 in Denmark due to a founder effect. ADOA presents symptoms within the first decade of life. 80% of ADOA patients show symptoms before the age of 10. The disease causes progressive, irreversible vision loss, with up to 46% of patients registered as legally blind.

[0190]

[0256] In some cases, the therapeutic agent comprises an oligonucleotide. In some cases, the therapeutic agent comprises a vector, e.g., a viral vector, that expresses an oligonucleotide that binds to a target region of a pre-mRNA that encodes a target peptide sequence. The methods provided herein can be adapted to contact a cell with an agent, e.g., a vector that encodes an oligonucleotide, such that the agent binds to the pre-mRNA in the cell and modulates pre-mRNA processing. In some cases, the viral vector comprises an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, a herpes simplex viral (HSV) viral vector, a retroviral vector, or any applicable viral vector. In some cases, the therapeutic agent comprises a gene editing tool that is configured to modify a gene that encodes a target peptide sequence such that a region of the gene that encodes an inefficient translation region is deleted. In some cases, the gene editing tool comprises a vector, e.g., a viral vector, for gene editing based on CRISPR-Cas9, TALEN, zinc finger, or other applicable technology.

[0191]

[0257] The suitable administration route of the ASO of the present disclosure may vary according to the cell type that ASO is desired to be delivered to.Multiple tissues and organs are affected by ADOA, and the most significant affected tissue is the eye.The ASO of the present disclosure can be administered parenterally to patients, for example, by intravitreal injection, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

[0192]

[0258] In embodiments, antisense oligonucleotides are administered by any method known in the art together with one or more agents that can facilitate the penetration of the antisense oligonucleotides of interest across the blood-brain barrier.For example, delivery of agents by administration of adenoviral vectors to motor neurons of 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.Direct delivery of vectors to the brain, for example, the striatum, thalamus, hippocampus, or substantia nigra, is described in U.S. Patent No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain," which is incorporated herein by reference.

[0193]

[0259] In some embodiments, the antisense oligonucleotide is linked or conjugated to an agent that provides desirable pharmaceutical or pharmacodynamic properties. In embodiments, the antisense oligonucleotide is coupled to an agent known in the art that enhances penetration or transport across the blood-brain barrier, such as an antibody against the transferrin receptor. In embodiments, the antisense oligonucleotide is linked to a viral vector, for example, to make the antisense compound more effective or to increase transport across the blood-brain barrier. In some embodiments, osmotic blood-brain barrier disruption is achieved by the addition of sugars, such as mesoerythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myoinositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, This is supported by infusions of adonitol, 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 blood-brain barrier penetration are described, for example, in U.S. Pat. No. 9,193,969, entitled "Compositions and methods for selective delivery of oligonucleotide molecules to specific neuron types," U.S. Pat. No. 4,866,042, entitled "Method for the delivery of genetic material across the blood brain barrier," U.S. Pat. No. 6,294,520, entitled "Material for passage through the blood-brain barrier," and U.S. Pat. No. 6,936,589, entitled "Parental delivery systems," each of which is incorporated herein by reference.

[0194]

[0260] In some embodiments, subjects treated using the methods and compositions are evaluated for improvement in their condition using any method known and described in the art. Methods for identifying additional ASOs that induce exon skipping

[0261] Methods for identifying or determining ASOs that induce exon skipping of OPA1 NMD exon-containing pre-mRNA are also within the scope of the present disclosure. For example, the method can include identifying or determining ASOs that induce pseudoexon skipping of OPA1 NMD exon-containing pre-mRNA. ASOs that specifically hybridize to different nucleotides in the target region of the pre-mRNA can be screened to identify or determine ASOs that improve the rate and / or extent of splicing of the target intron. In some embodiments, the ASOs can block or interfere with the binding site of a splicing repressor / silencer. Any method known in the art can be used to identify (determine) ASOs that result in a desired effect (e.g., pseudoexon skipping, protein or functional RNA production) when hybridized to the target region of an exon. These methods can also be used to identify ASOs that induce exon skipping of an included exon by binding to a target region in an intron adjacent to an included exon or a non-included exon. One example of a method that can be used is provided below.

[0195]

[0262] A round of screening, referred to as ASO "walking", may be performed using ASOs designed to hybridize to a target region of the pre-mRNA. For example, the ASOs used for ASO walking may be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 3' splice site of the included exon (e.g., a portion of the sequence of the exon located upstream of the target / included exon) to approximately 100 nucleotides downstream of the 3' splice site of the target / included exon, and / or from approximately 100 nucleotides upstream of the 5' splice site of the included exon to approximately 100 nucleotides downstream of the 5' splice site of the target / included exon (e.g., a portion of the sequence of the exon located downstream of the target / included exon). For example, a first ASO of 15 nucleotides in length may be designed to specifically hybridize to nucleotides +6 to +20 relative to the 3' splice site of the target / included exon. The second ASO may be designed to specifically hybridize to nucleotides +11 to +25 relative to the 3' splice site of the target / inclusive exon. The ASO is designed to span the target region of the pre-mRNA. In embodiments, the ASO can be tiled more closely, for example, every 1, 2, 3, or 4 nucleotides. Additionally, the ASO can be tiled from 100 nucleotides downstream of the 5' splice site to 100 nucleotides upstream of the 3' splice site. In some embodiments, the ASO can be tiled from about 1,160 nucleotides upstream of the 3' splice site to about 500 nucleotides downstream of the 5' splice site. In some embodiments, the ASO can be tiled from about 500 nucleotides upstream of the 3' splice site to about 1,920 nucleotides downstream of the 3' splice site.

[0196]

[0263] One or more ASOs, or a control ASO (an ASO with a scrambled sequence, which is a sequence not expected to hybridize to the target region), are delivered, for example, by transfection, to a disease-relevant cell line expressing a target pre-mRNA (e.g., an NMD exon-containing pre-mRNA described herein). The exon skipping effect of each ASO may be assessed by any method known in the art, for example, by reverse transcription (RT)-PCR using primers spanning the splice junction, as described in Example 2.4. A reduction or absence of longer RT-PCR products in ASO-treated cells compared to control ASO-treated cells, produced using primers spanning a region containing an inclusion exon (e.g., including an exon adjacent to an NMD exon), indicates enhanced splicing of the target NMD exon. In some embodiments, exon skipping efficiency (or splicing efficiency of splicing introns containing NMD exons), the ratio of spliced ​​pre-mRNA to unspliced ​​pre-mRNA, the rate of splicing, or the extent of splicing can be improved using the ASOs described herein. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine whether each ASO has achieved the desired effect (e.g., enhanced functional protein production). Any method known in the art for evaluating and / or quantifying protein production can be used, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0197]

[0264] A second round of screening, termed ASO "microwalking", may be performed using ASOs designed to hybridize to target regions of the pre-mRNA. The ASOs used for ASO microwalking are tiled nucleotide by nucleotide to further refine the nucleotide acid sequence of the pre-mRNA that results in exon skipping (or enhanced splicing of NMD exons) when hybridized with the ASO.

[0198]

[0265] The region defined by the ASO promoting splicing of the target intron is explored in more detail by ASO "microwalking" with ASOs spaced at one nucleotide steps apart, and by longer ASOs, typically 18-25 nucleotides.

[0199]

[0266] As described above for ASO walking, ASO microwalking is performed by delivering one or more ASOs, or a control ASO (an ASO with a scrambled sequence that is not expected to hybridize to the target region), for example by transfection, to a disease-related cell line expressing a target pre-mRNA. The splicing-inducing effect of each ASO may be evaluated by any method known in the art, for example by reverse transcription (RT)-PCR using primers spanning the NMD exon, as described herein (see, for example, Example 2.4). Reduction or absence of longer RT-PCR products produced using primers spanning the NMD exon in ASO-treated cells compared to those in control ASO-treated cells indicates enhanced exon skipping (or splicing of the target intron containing the NMD exon). In some embodiments, exon skipping efficiency (or splicing efficiency of splicing introns containing NMD exons), the ratio of spliced ​​pre-mRNA to unspliced ​​pre-mRNA, the rate of splicing, or the extent of splicing can be improved using the ASOs described herein. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine whether each ASO has achieved the desired effect (e.g., enhanced functional protein production). Any method known in the art for evaluating and / or quantifying protein production can be used, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0200]

[0267] ASOs that result in exon skipping (or enhanced splicing of introns containing NMD exons) and increased protein production when hybridized to a region of a pre-mRNA 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 disease. The suitable route of administration of the ASO may vary depending on the disease and / or cell type to which delivery of the ASO is desired. The ASO may be administered, for example, by intravitreal, intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous, or intravenous injection. After administration, cells, tissues, and / or organs of the model animal can be evaluated to determine the efficacy of ASO treatment, for example, by assessing splicing (e.g., efficiency, speed, extent) and protein production by methods known in the art and described herein. The animal model may also be any phenotypic or behavioral indicator of disease or disease severity.

[0201]

[0268] Methods for identifying or validating NMD-induced exons in the presence of an NMD inhibitor, such as cycloheximide, are also within the scope of this disclosure. An exemplary method is provided in Example 2.2. Methods for identifying additional ASOs that modulate mRNA translation

[0269] Methods for identifying or determining ASOs that regulate the translation of OPA1 processed mRNA transcripts are also within the scope of the present disclosure. For example, the method can include identifying or determining ASOs that regulate the translation of OPA1 processed mRNA transcripts. ASOs that specifically hybridize to different nucleotides in the targeting portion of the processed mRNA can be screened to identify or determine ASOs that improve the rate and / or efficiency of translation of the processed mRNA. In some embodiments, the ASOs can interfere with the interaction of one or more translation factors with the processed mRNA. Any method known in the art can be used to identify (determine) ASOs that produce a desired effect (e.g., increase the rate and / or efficiency of translation of the processed mRNA) when hybridized to the targeting portion of the processed mRNA. One example of a method that can be used is provided below.

[0202]

[0270] A round of screening, referred to as ASO "walking", may be performed using ASOs designed to hybridize to a target region of an mRNA transcript, e.g., a processed mRNA transcript. For example, the ASOs used for ASO walking may be tiled every 5 nucleotides from approximately 100, 200, 300, 400 or 500 nucleotides upstream of the region of interest (e.g., the 5'-UTR of the processed mRNA) to approximately 100 nucleotides downstream of the region of interest. For example, a first ASO of 15 nucleotides in length may be designed to specifically hybridize to the first 15 nucleotides at the 5' end of the processed mRNA, e.g., nucleotides +1 to +15 relative to the 5' end of exon 1. A second ASO may be designed to specifically hybridize to nucleotides +6 to +20 relative to the 5' end of exon 1. The ASOs are designed to span the targeted portion of the mRNA transcript. In embodiments, the ASOs can be tiled more closely, for example, every 1, 2, 3, or 4 nucleotides.

[0203]

[0271] One or more ASOs, or a control ASO (an ASO with a scrambled sequence, which is a sequence not expected to hybridize to the target region), are delivered, for example, by transfection, to a disease-related cell line expressing a target processed mRNA (e.g., a processed mRNA of OPA, or a processed mRNA comprising the 5'-UTR of OPA1 mRNA and a sequence encoding a reporter protein). The translational regulatory effect of each ASO may be evaluated by any method known in the art, for example, by evaluating the expression level of the protein encoded by the processed mRNA. In some cases, when the targeting moiety involves the 5'-UTR of the target processed mRNA, the translational regulatory effect of the ASO may be evaluated by an assay in which the 5'-UTR of the target processed mRNA is linked to a coding sequence of a reporter protein (e.g., luciferase) and the expression of the reporter protein is examined in cells treated with the ASO or the control ASO. An increase in reporter protein levels (e.g., luciferase expression levels in a luciferase assay as described in Example 1.3) in ASO-treated cells compared to those in control ASO-treated cells indicates enhanced translation of processed mRNA. In some embodiments, for evaluation of the effect of ASO on translational regulation of processed mRNA, the level of processed mRNA in cells is monitored, and the protein level is normalized by the level of processed mRNA so that any effect of ASO treatment on the level of processed mRNA can be excluded. In some cases, in the above reporter protein assay, it is the level of reporter protein in the cells that is investigated. Any method known in the art for evaluating and / or quantifying protein production can be used, such as Western blotting, flow cytometry, immunofluorescence microscopy, or ELISA.

[0204]

[0272] A second round of screening, termed ASO "microwalking", may be performed using an ASO designed to hybridize to a target region of the processed mRNA (e.g., the OPA1 processed mRNA, or a processed mRNA comprising the 5'-UTR of OPA1 mRNA and a sequence encoding a reporter protein). The ASOs used for ASO microwalking are tiled nucleotide by nucleotide to further refine the nucleotide acid sequence of the processed mRNA that results in an increased rate and / or efficacy of translation of the processed mRNA when hybridized with the ASO.

[0205]

[0273] The regions defined by ASOs that promote mRNA translation can be explored in more detail by ASO “microwalking” with ASOs spaced at one-nucleotide steps apart, and by longer ASOs, typically 18–25 nucleotides.

[0206]

[0274] As described above with respect to ASO walking, ASO microwalking is performed by delivering, e.g., by transfection, one or more ASOs, or a control ASO (an ASO with a scrambled sequence that is a sequence not expected to hybridize to the target region), to a disease-relevant cell line expressing the target processed mRNA (e.g., OPA1 processed mRNA, or a processed mRNA comprising the 5'-UTR of OPA1 mRNA and a sequence encoding a reporter protein). The translational regulatory effect of each ASO may be assessed by any method known in the art, for example, by Western blotting, Jess blotting, or bioluminescence quantification when luciferase is used as the reporter protein, as described herein (see, e.g., Example 1.1).

[0207]

[0275] ASOs that result in increased mRNA translation and increased protein production when hybridized to a region of processed mRNA may be tested in vivo using animal models, for example, transgenic mouse models with full-length human genes knocked in, or in humanized mouse models of disease. The suitable route of administration of ASOs may vary depending on the disease and / or cell type to which the ASO is desired to be delivered. ASOs may be administered, for example, by intravitreal, intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous, or intravenous injection. After administration, cells, tissues, and / or organs of the model animals may be evaluated to determine the efficacy of ASO treatment, for example, by assessing splicing (e.g., efficiency, speed, extent) and protein production by methods known in the art and described herein. The animal model may also be any phenotypic or behavioral indicator of disease or disease severity. Specific embodiment (A)

[0276] Embodiment A1. A method of treating optic atrophy type 1 in a subject by increasing expression of a target protein or functional RNA by cells of the subject in need of such treatment, wherein the cells have an mRNA containing a nonsense-mediated RNA decay-guided exon (NMD exon mRNA), the NMD exon mRNA encoding the target protein or functional RNA, the method comprising contacting the cells of the subject with a therapeutic agent that binds to a targeting portion of the NMD exon mRNA encoding the target protein or functional RNA, thereby excluding the nonsense-mediated RNA decay-guided 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 expression of the target protein or functional RNA in the cells of the subject.

[0208]

[0277] Embodiment A2. The method of embodiment A1, wherein the target protein is OPA1.

[0209]

[0278] Embodiment A3. A method of increasing expression of OPA1 protein by a cell having an mRNA that contains a nonsense-mediated RNA decay-guided exon (NMD exon mRNA), said mRNA encoding an OPA1 protein, comprising contacting the cell with an agent that binds to a targeting portion of the NMD exon mRNA encoding the OPA1 protein, thereby excluding the nonsense-mediated RNA decay-guided exon from the NMD exon mRNA encoding the OPA1 protein, thereby increasing the level of the mRNA encoding the OPA1 protein and increasing expression of the OPA1 protein in the cell.

[0210]

[0279] Embodiment A4. The method of any one of embodiments A1 to A3, wherein the nonsense-mediated RNA decay-guided exon is excised from the NMD exon mRNA encoding a target protein or functional RNA.

[0211]

[0280] Embodiment A5. The method of any one of embodiments A1 to A4, wherein the target protein does not comprise an amino acid sequence encoded by a nonsense-mediated RNA decay-directed exon.

[0212]

[0281] Embodiment A6. The method of any one of embodiments A1 to A5, wherein the target protein is a full-length target protein.

[0213]

[0282] Embodiment A7. The method of any one of embodiments A1 to A6, wherein the agent is an antisense oligomer (ASO) complementary to a targeted portion of the NMD exon mRNA.

[0214]

[0283] Embodiment A8. The method of any one of embodiments A1 to A7, wherein the mRNA is a precursor mRNA.

[0215]

[0284] Embodiment A9. The method of any one of embodiments A1-A8, wherein the contacting step comprises contacting the therapeutic agent with mRNA, wherein the mRNA is nuclear in a cell.

[0216]

[0285] Embodiment A10. The method of any one of embodiments A1 to A9, wherein the target protein or functional RNA corrects a deficiency of the target protein or functional RNA in the subject.

[0217]

[0286] Embodiment A11. The method of any one of embodiments A1 to A10, wherein the cell is in or derived from a subject having a pathology caused by a defective amount or activity of OPA1 protein.

[0218]

[0287] Embodiment A12. The method of any one of embodiments A1 to A11, wherein the deficient amount of the target protein is caused by haploinsufficiency of the target protein, the subject has a first allele that encodes a functional target protein and a second allele in which the target protein is not produced or a second allele that encodes a non-functional target protein, and the antisense oligomer binds to a targeted portion of an NMD exon mRNA transcribed from the first allele.

[0219]

[0288] Embodiment A13. The subject has a pathology caused by a disorder resulting from a deficiency in the amount or function of a target protein, and the subject: (a) (i) the target protein is produced at a reduced level compared to production from a wild-type allele; (ii) the target protein is produced in a form that has reduced functionality compared to the equivalent wild-type protein, or (iii) the target protein is not produced; a first mutant allele, and (b) (i) the target protein is produced at a reduced level compared to production from a wild-type allele; (ii) the target protein is produced in a form that has reduced functionality compared to the equivalent wild-type protein, or (iii) the target protein is not produced; A second mutant allele, having when the subject has a first variant allele (a)(iii), the second variant allele is (b)(i) or (b)(ii); and when the subject has a second variant allele (b)(iii), the first variant allele is (a)(i) or (a)(ii), and the NMD exon mRNA is transcribed from either the first variant allele that is (a)(i) or (a)(ii) and / or the second variant allele that is (b)(i) or (b)(ii). The method according to any one of embodiments A1 to A11.

[0220]

[0289] Embodiment A14. The method of embodiment A13, wherein the target protein is produced in a form that has reduced functionality compared to the equivalent wild-type protein.

[0221]

[0290] Embodiment A15. The method of embodiment A13, wherein the target protein is produced in a form that is fully functional compared to the comparable wild-type protein.

[0222]

[0291] Embodiment A16. The method of any one of embodiments A1 to A15, wherein the targeted portion of the NMD exon mRNA is within a nonsense-mediated RNA decay-inducible exon.

[0223]

[0292] Embodiment A17. The method of any one of embodiments A1 to A15, wherein the targeted portion of the NMD exon mRNA is either upstream or downstream of the nonsense-mediated RNA decay-directed exon.

[0224]

[0293] Embodiment A18. The method of 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 to SEQ ID NO: 2 or 3.

[0225]

[0294] Embodiment A19. The method of 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 to SEQ ID NO:1.

[0226]

[0295] Embodiment A20. The method of any one of embodiments A1 to A17, wherein the targeted portion of the NMD exon mRNA comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 consecutive nucleic acids of SEQ ID NO: 2 or 3.

[0227]

[0296] Embodiment A21. The method of any one of embodiments A1 to A20, wherein the agent 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 8 contiguous nucleic acids of SEQ ID NO: 2 or 3.

[0228]

[0297] Embodiment A22. The method of any one of embodiments A1 to A15, wherein the targeted portion of the NMD exon mRNA is within nonsense-mediated RNA decay-directed exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1.

[0229]

[0298] Embodiment A23. The method of any one of embodiments A1 to A15, wherein the targeted portion of the NMD exon mRNA is upstream or downstream of nonsense-mediated RNA decay-directed exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1.

[0230]

[0299] Embodiment A24. The method of any one of embodiments A1 to A15, wherein the targeted portion of the NMD exon mRNA comprises exon-intron junction exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1.

[0231]

[0300] Embodiment A25. The method of any one of embodiments A1 to A24, wherein the target protein produced is a full-length protein or a wild-type protein.

[0232]

[0301] Embodiment A26. The total amount of mRNA encoding a target protein or functional RNA produced in a cell contacted with an antisense oligomer is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 1.1 to about 10 ... fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold.

[0233]

[0302] Embodiment A27. The total amount of mRNA encoding a target protein or functional RNA produced in a cell contacted with an antisense oligomer is 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%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 5 ... The method of any one of embodiments A1-A25, wherein the amount of the saturation is increased by 0% to about 150%, by about 50% to about 200%, by about 50% to about 250%, by about 100% to about 150%, by about 100% to about 200%, by about 100% to about 250%, by about 150% to about 200%, by about 150% to about 250%, by about 200% to about 250%, by at least about 10%, by at least about 20%, by at least about 50%, by at least about 100%, by at least about 150%, by at least about 200%, by at least about 250%, or by at least about 300%.

[0234]

[0303] Embodiment A28. The total amount of target protein produced by cells contacted with antisense oligomers is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 ... The method of any one of embodiments A1 to A25, wherein the increase is about 8-fold, about 2-fold, about 3-fold, about 6-fold, about 3-fold, about 7-fold, about 3-fold, about 8-fold, about 3-fold, about 9-fold, about 4-fold, about 4-fold, about 4-fold, about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold.

[0235]

[0304] Embodiment A29. The total amount of target protein produced by cells contacted with an antisense oligomer is 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%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 20 ... %, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%.

[0236]

[0305] Embodiment A30. The method of any one of embodiments A1-29, wherein the agent is an antisense oligomer (ASO), and the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

[0237]

[0306] Embodiment A31. The method of any one of embodiments A1 to A30, wherein the agent is an antisense oligomer (ASO), and the antisense oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

[0238]

[0307] Embodiment A32. The method of any one of embodiments A1 to A31, wherein the agent is an antisense oligomer (ASO), and the antisense oligomer comprises at least one modified sugar moiety.

[0239]

[0308] Embodiment A33. The method of embodiment A32, wherein each sugar moiety is a modified sugar moiety.

[0240]

[0309] Embodiment A34. The agent is an antisense oligomer (ASO), and the antisense oligomer is selected from the group consisting of 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 nucleobases, 8-15 nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 nucleobases, 10-50 nucleobases, 10-40 nucleobases, 10-35 nucleobases, 10-30 nucleobases. The method of any one of embodiments A1 to A33, wherein the nucleotide sequence comprises from 1 to 25 nucleobases, from 10 to 20 nucleobases, from 10 to 15 nucleobases, from 11 to 50 nucleobases, from 11 to 40 nucleobases, from 11 to 35 nucleobases, from 11 to 30 nucleobases, from 11 to 25 nucleobases, from 11 to 20 nucleobases, from 11 to 15 nucleobases, from 12 to 50 nucleobases, from 12 to 40 nucleobases, from 12 to 35 nucleobases, from 12 to 30 nucleobases, from 12 to 25 nucleobases, from 12 to 20 nucleobases, or from 12 to 15 nucleobases.

[0241]

[0310] Embodiment A35. The method of any one of embodiments A1 to A34, wherein the agent 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 targeted portion of the NMD exon mRNA encoding the protein.

[0242]

[0311] Embodiment A36. The method of any one of embodiments A1 to A35, further comprising assessing OPA1 mRNA or protein expression.

[0243]

[0312] Embodiment A37. The method of any one of embodiments A1 to A36, wherein optic atrophy type 1 is treated and the antisense oligomer binds to a targeting portion of the OPA1 NMD exon mRNA, wherein the targeting portion is within SEQ ID NO: 2 or 3.

[0244]

[0313] Embodiment A38. The method of any one of embodiments A1 to A37, wherein the subject is a human.

[0245]

[0314] Embodiment A39. The method of any one of embodiments A1 to A38, wherein the subject is a non-human animal.

[0246]

[0315] Embodiment A40. The method of any one of embodiments A1-A39, wherein the subject is a fetus, embryo, or child.

[0247]

[0316] Embodiment A41. The method of any one of embodiments A1 to A40, wherein the cells are ex vivo.

[0248]

[0317] Embodiment A42. The method of any one of embodiments A1-A41, wherein the therapeutic agent is administered by intrathecal, intraventricular, intraperitoneal, intramuscular, subcutaneous, or intravenous injection into the subject.

[0249]

[0318] Embodiment A43. The method of any of embodiments A1-A42, further comprising administering a second therapeutic agent to the subject.

[0250]

[0319] Embodiment A44. The method of embodiment A43, wherein the second therapeutic agent is a small molecule.

[0251]

[0320] Embodiment A45. The method of embodiment A43, wherein the second therapeutic agent is an ASO.

[0252]

[0321] Embodiment A46. The method of any one of embodiments A43-A45, wherein the second therapeutic agent corrects intron retention.

[0253]

[0322] Embodiment A47. An antisense oligomer when used in a method according to any of embodiments A1 to A46.

[0254]

[0323] Embodiment A48. An antisense oligomer comprising a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO:2 or 3.

[0255]

[0324] Embodiment A49. A pharmaceutical composition comprising an antisense oligomer according to embodiment A47 or A48 and an excipient.

[0256]

[0325] Embodiment A50. A method of treating a subject in need thereof, comprising administering to the subject the pharmaceutical composition of embodiment A49, wherein the administering is by intravitreal, intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous, or intravenous injection.

[0257]

[0326] Embodiment A51. A composition comprising a Therapeutic Agent for use in a method of increasing expression of a target protein or functional RNA by a cell to treat optic atrophy type 1 in a subject in need thereof associated with a defective protein or defective functional RNA, wherein the defective protein or defective functional RNA is defective in amount or activity in the subject, and the target protein is (a) a defective protein, or (b) a compensatory protein that functionally enhances or replaces a defective protein in a subject. and the functional RNA is (c) a defective RNA, or (d) a compensatory functional RNA that functionally enhances or replaces a defective functional RNA in a subject; and The composition, wherein the therapeutic agent enhances the exclusion of nonsense-mediated RNA decay-induced 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 a subject.

[0258]

[0327] Embodiment A52. A composition comprising a therapeutic agent for use in a method of treating a condition associated with OPA1 protein in a subject in need thereof, the method comprising increasing expression of OPA1 protein by a cell of the subject, the cell having an mRNA that contains a nonsense-mediated RNA decay-guided exon (NMD exon mRNA), the mRNA encoding the OPA1 protein, the method comprising contacting the cell with a therapeutic agent, thereby excluding the nonsense-mediated RNA decay-guided exon from the NMD exon mRNA encoding the OPA1 protein, thereby increasing the level of the mRNA encoding the OPA1 protein and increasing expression of the OPA1 protein in the cell of the subject.

[0259]

[0328] Embodiment A53. The composition of embodiment A52, wherein the condition is a disease or disorder.

[0260]

[0329] Embodiment A54. The composition of embodiment A53, wherein the disease or disorder is optic atrophy type 1.

[0261]

[0330] Embodiment A55. The composition of any one of embodiments A52-54, wherein the OPA1 protein and NMD exon mRNA are encoded by the OPA1 gene.

[0262]

[0331] Embodiment A56. The composition of any one of embodiments A51 to A55, wherein the nonsense-mediated RNA decay-guided exon is excised from the NMD exon mRNA encoding the OPA1 protein.

[0263]

[0332] Embodiment A57. The composition of any one of embodiments A51 to A56, wherein the OPA1 protein does not contain an amino acid sequence encoded by a nonsense-mediated RNA decay-directed exon.

[0264]

[0333] Embodiment A58. The composition of any one of embodiments A51 to A57, wherein the OPA1 protein is a full-length OPA1 protein.

[0265]

[0334] Embodiment A59. The composition of any one of embodiments A51 to A58, wherein the therapeutic agent is an antisense oligomer (ASO) complementary to a targeted portion of an NMD exon mRNA.

[0266]

[0335] Embodiment A60. The composition of any of embodiments A51-A59, wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer targets a portion of the NMD exon mRNA that is within the nonsense-mediated RNA decay-guided exon.

[0267]

[0336] Embodiment A61. The composition of any one of embodiments A51-A59, wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer targets a portion of the NMD exon mRNA that is upstream or downstream of the nonsense-mediated RNA decay-guided exon.

[0268]

[0337] Embodiment A62. The composition of any one of embodiments A51-A61, wherein the target protein is OPA1.

[0269]

[0338] Embodiment A63. The composition of embodiment A62, wherein the NMD exon mRNA comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 2 or 3.

[0270]

[0339] Embodiment A64. The composition of embodiment A62, wherein the NMD exon mRNA is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO:1.

[0271]

[0340] Embodiment A65. The composition of embodiment A62, wherein the targeted portion of the NMD exon mRNA comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 consecutive nucleic acids of SEQ ID NO: 2 or 3.

[0272]

[0341] Embodiment A66. The composition of any one of embodiments A62 to A65, wherein the targeted portion of the NMD exon mRNA is within nonsense-mediated RNA decay-directed exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1.

[0273]

[0342] Embodiment A67. The composition of any one of embodiments A62 to A65, wherein the targeted portion of the NMD exon mRNA is upstream or downstream of nonsense-mediated RNA decay-directed exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1.

[0274]

[0343] Embodiment A68. The composition of any one of embodiments A62 to A65, wherein the targeted portion of the NMD exon mRNA comprises an exon-intron junction of exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1.

[0275]

[0344] Embodiment A69. The composition of any one of embodiments A62-A68, wherein the therapeutic agent 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 a region comprising at least 8 contiguous nucleic acids of SEQ ID NO:2 or 3.

[0276]

[0345] Embodiment A70. The composition of any one of embodiments A51 to A69, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.

[0277]

[0346] Embodiment A71. The composition of any one of embodiments A51 to A70, wherein the target protein produced is a full-length protein or a wild-type protein.

[0278]

[0347] Embodiment A72. The composition of any one of embodiments A51 to A71, wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises a backbone modification comprising a phosphorothioate or phosphorodiamidate linkage.

[0279]

[0348] Embodiment A73. The composition of any of embodiments A51-A72, wherein the therapeutic agent is an antisense oligomer (ASO), and said antisense oligomer is an antisense oligonucleotide.

[0280]

[0349] Embodiment A74. The composition of any of embodiments A51-A73, wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

[0281]

[0350] Embodiment A75. The composition of any of embodiments A51-A74, wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises at least one modified sugar moiety.

[0282]

[0351] Embodiment A76. The composition of embodiment A75, wherein each sugar moiety is a modified sugar moiety.

[0283]

[0352] Embodiment A77. The therapeutic agent is an antisense oligomer (ASO), wherein the antisense oligomer is selected from the group consisting of 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 nucleobases, 8-15 nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 nucleobases, 10-50 nucleobases, 10-40 nucleobases, 10-35 nucleobases, 10-30 nucleobases. The composition according to any of embodiments A51 to A76, comprising from 1 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

[0284]

[0353] Embodiment A78. A pharmaceutical composition comprising a therapeutic agent of any of the compositions of embodiments A51-A77 and an excipient.

[0285]

[0354] Embodiment A79. A method of treating a subject in need thereof, comprising administering to the subject the pharmaceutical composition of embodiment A78, wherein the administering is by intravitreal, intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous, or intravenous injection.

[0286]

[0355] Embodiment A80. The method of any of embodiments A51-A79, further comprising administering a second therapeutic agent to the subject.

[0287]

[0356] Embodiment A81. The method of embodiment A80, wherein the second therapeutic agent is a small molecule.

[0288]

[0357] Embodiment A82. The method of embodiment A80, wherein the second therapeutic agent is an ASO.

[0289]

[0358] Embodiment A83. The method of any one of embodiments A80-A82, wherein the second therapeutic agent corrects intron retention.

[0290]

[0359] Embodiment A84. A pharmaceutical composition comprising an antisense oligomer that hybridizes to a target sequence of an OPA1 mRNA transcript, the OPA1 mRNA transcript containing a nonsense-mediated RNA decay-inducible exon, and induces exclusion of the nonsense-mediated RNA decay-inducible exon from the OPA1 mRNA transcript; and a pharmaceutically acceptable excipient.

[0291]

[0360] Embodiment A85. The pharmaceutical composition of embodiment A84, wherein the OPA1 mRNA transcript is an OPA1 NMD exon mRNA transcript.

[0292]

[0361] Embodiment A86. The pharmaceutical composition of embodiment A84 or A85, wherein the targeted portion of the NMD exon mRNA is within the nonsense-mediated RNA decay-directed exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1.

[0293]

[0362] Embodiment A87. The pharmaceutical composition of embodiment A84 or A85, wherein the targeted portion of the NMD exon mRNA is upstream or downstream of nonsense-mediated RNA decay-directed exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1.

[0294]

[0363] Embodiment A88. The pharmaceutical composition of embodiment A84 or A85, wherein the targeted portion of the NMD exon mRNA comprises the exon-intron junction exon 6x of OPA1, exon 7x of OPA1, or exon 28x of OPA1.

[0295]

[0364] Embodiment A89. The pharmaceutical composition of any one of embodiments A84 to A88, wherein the OPA1 NMD exon mRNA transcript is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1.

[0296]

[0365] Embodiment A90. The pharmaceutical composition of embodiment A84 or A88, wherein the OPA1 NMD exon mRNA transcript comprises a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2 or 3.

[0297]

[0366] Embodiment A91. The pharmaceutical composition of embodiment A84, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate or phosphorodiamidate linkage.

[0298]

[0367] Embodiment A92. The pharmaceutical composition of embodiment A84, wherein the antisense oligomer is an antisense oligonucleotide.

[0299]

[0368] Embodiment A93. The pharmaceutical composition of embodiment A84, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

[0300]

[0369] Embodiment A94. The pharmaceutical composition of embodiment A84, wherein the antisense oligomer comprises at least one modified sugar moiety.

[0301]

[0370] Embodiment A95. The antisense oligomer comprises 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases. The pharmaceutical composition of embodiment A84, comprising an acid base, 10-20 nucleobases, 10-15 nucleobases, 11-50 nucleobases, 11-40 nucleobases, 11-35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11-15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12-15 nucleobases.

[0302]

[0371] Embodiment A96. The pharmaceutical composition of embodiment A84 or A85, wherein 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 targeted portion of the OPA1 NMD exon mRNA transcript.

[0303]

[0372] Embodiment A97. The pharmaceutical composition of embodiment A84 or A85, wherein the targeted portion of the OPA1 NMD exon mRNA transcript is within SEQ ID NO: 2 or 3.

[0304]

[0373] Embodiment A98. The pharmaceutical composition of embodiment A84, wherein the antisense oligomer comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO: 2 or 3.

[0305]

[0374] Embodiment A99. The pharmaceutical composition of embodiment A84, wherein the antisense oligomer comprises a nucleotide sequence identical to a region comprising at least 8 contiguous nucleic acids, SEQ ID NO:2 or 3.

[0306]

[0375] Embodiment A100. The pharmaceutical composition of any one of embodiments A84 to A99, formulated for intravitreal, intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous or intravenous injection.

[0307]

[0376] Embodiment A101. The method of any of embodiments A84-A100, further comprising administering a second therapeutic agent to the subject.

[0308]

[0377] Embodiment A102. The method of embodiment A101, wherein the second therapeutic agent is a small molecule.

[0309]

[0378] Embodiment A103. The method of embodiment A101, wherein the second therapeutic agent is an ASO.

[0310]

[0379] Embodiment A104. The method of any one of embodiments A101-A103, wherein the second therapeutic agent corrects intron retention.

[0311]

[0380] Embodiment A105. A method of inducing processing of a defective OPA1 mRNA transcript to facilitate removal of a nonsense-mediated RNA decay-induced exon to produce a fully processed OPA1 mRNA transcript encoding a functional form of an OPA1 protein, comprising: (a) contacting an antisense oligomer with a target cell of a subject; (b) hybridizing an antisense oligomer to a defective OPA1 mRNA transcript, the defective OPA1 mRNA transcript being capable of encoding a functional form of OPA1 protein and containing at least one nonsense-mediated RNA decay-inducible exon; (c) removing at least one nonsense-mediated RNA decay-inducible exon from the defective OPA1 mRNA transcript to produce a fully processed OPA1 mRNA transcript encoding a functional form of the OPA1 protein; and (d) translating a functional form of OPA1 protein from the fully processed OPA1 mRNA transcript. The method comprising:

[0312]

[0381] Embodiment A106. A method of treating a subject having a condition caused by a defective amount or activity of OPA1 protein, comprising administering to the subject an antisense oligomer comprising a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a region comprising at least 8 consecutive nucleic acids of SEQ ID NO:2 or 3.

[0313]

[0382] Embodiment A107. A method of treating optic atrophy type 1 in a subject by increasing expression of a target protein or functional RNA by cells of the subject in need thereof, wherein the cells have an mRNA containing a nonsense-mediated RNA decay-guided exon (NMD exon mRNA), the NMD exon mRNA encoding the target protein or functional RNA, the method comprising contacting the cells of the subject with a therapeutic agent that modulates splicing of the NMD exon mRNA encoding the target protein or functional RNA, thereby excluding the nonsense-mediated RNA decay-guided 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 expression of the target protein or functional RNA in the cells of the subject.

[0314]

[0383] Embodiment A108. A method of increasing expression of an OPA1 protein by a cell having an mRNA that contains a nonsense-mediated RNA decay-guided exon (NMD exon mRNA), said mRNA encoding an OPA1 protein, comprising contacting the cell with an agent that modulates splicing of the NMD exon mRNA encoding the OPA1 protein, thereby excluding the nonsense-mediated RNA decay-guided exon from the NMD exon mRNA encoding the OPA1 protein, thereby increasing the level of the mRNA encoding the OPA1 protein and increasing expression of the OPA1 protein in the cell.

[0315]

[0384] Embodiment A109. The agent is (a) binds to a targeted portion of an NMD exon mRNA that encodes a target protein or functional RNA; (b) binds to one or more components of the spliceosome; or (c) a combination of (a) and (b); The method according to embodiment A107 or A108.

[0316]

[0385] Embodiment B1. A method of modulating expression of a target protein by a cell having an mRNA that contains a nonsense-mediated RNA decay-directed exon (NMD exon) and encodes the target protein, comprising contacting the cell with a therapeutic agent, whereby the therapeutic agent modulates splicing of the NMD exon from the mRNA, thereby modulating the level of processed mRNA encoding the target protein and modulating expression of the target protein in the cell, wherein the target gene is selected from the group consisting of OPA1 protein.

[0317]

[0386] Embodiment B2. A method of treating a disease or condition in a subject by modulating expression of a target protein in a cell of the subject in need of such treatment, comprising contacting a cell of the subject with a therapeutic agent that modulates splicing of a nonsense-mediated mRNA decay-directed exon (NMD exon) from an mRNA in the cell, wherein the mRNA comprises an NMD exon and encodes a target protein, thereby modulating the level of processed mRNA encoding the target protein and modulating expression of the target protein in the cell of the subject, wherein the target protein is selected from the group consisting of OPA1 protein.

[0318]

[0387] Embodiment B3. The therapeutic agent comprises: (a) binds to a targeting portion of an mRNA encoding a target protein; (b) modulating the binding of factors involved in splicing of the NMD exon; or (c) a combination of (a) and (b); The method according to embodiment B1 or B2.

[0319]

[0388] Embodiment B4. The method of embodiment B3, wherein the therapeutic agent interferes with the binding of a factor involved in splicing of the NMD exon to the region of the targeting moiety.

[0320]

[0389] Embodiment B5. The method of embodiment B3 or B4, wherein the targeting moiety is proximal to an NMD exon.

[0321]

[0390] Embodiment B6. The method of any one of embodiments B3-B5, wherein the targeting moiety is up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream of the 5' end of the NMD exon.

[0322]

[0391] Embodiment B7. The method of any one of embodiments B3-B6, wherein the targeting moiety is at least about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, 50, 40, 300, 200, 100, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 2, 1 nucleotide upstream of the 5' end of the NMD exon.

[0323]

[0392] Embodiment B8. The method of any one of embodiments B3-B5, wherein the targeting moiety is up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream of the 3' end of the NMD exon.

[0324]

[0393] Embodiment B9. The method of any one of embodiments B3-B5 or B8, wherein the targeting moiety is at least about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, 50, 40, 300, 200, 100, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 2, 1 nucleotide downstream of the 3' end of the NMD exon.

[0325]

[0394] Embodiment B10. The method of any one of embodiments B3-B5, wherein the targeting moiety is at most about 1500, about 1000, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 80, about 70, about 60, about 50 nucleotides upstream of a genomic site selected from the group consisting of GRCh38 / hg38:chr3 193628509; and GRCh38 / hg38:chr3 193603500.

[0326]

[0395] Embodiment B11. The method of any one of embodiments B3-B5 or B10, wherein the targeting moiety is about 1500, about 1000, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 80, about 70, about 60, about 50 nucleotides upstream of a genomic site selected from the group consisting of GRCh38 / hg38:chr3 193628509; and GRCh38 / hg38:chr3 193603500.

[0327]

[0396] Embodiment B12. The method of any one of embodiments B3-B5, wherein the targeting moiety is at most about 1500, about 1000, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 80, about 70, about 60, about 50 nucleotides downstream of a genomic site selected from the group consisting of GRCh38 / hg38:chr3 193628616; and GRCh38 / hg38:chr3 193603557.

[0328]

[0397] Embodiment B13. The method of any one of embodiments B3-B5 or B12, wherein the targeting moiety is about 1500, about 1000, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 80, about 70, about 60, about 50 nucleotides downstream of a genomic site selected from the group consisting of GRCh38 / hg38:chr3 193628616; and GRCh38 / hg38:chr3 193603557.

[0329]

[0398] Embodiment B14. The method of any one of embodiments B3 to B13, wherein the targeting moiety is located in an intron region between two canonical exon regions of an mRNA encoding a target protein, and the intron region contains an NMD exon.

[0330]

[0399] Embodiment B15. The method of any one of embodiments B3-B14, wherein the targeting moiety at least partially overlaps with an NMD exon.

[0331]

[0400] Embodiment B16. The method of any one of embodiments B3-B15, wherein the targeting moiety at least partially overlaps with an intron upstream or downstream of the NMD exon.

[0332]

[0401] Embodiment B17. The method of any one of embodiments B3-B16, wherein the targeting moiety comprises a 5' NMD exon-intron junction or a 3' NMD exon-intron junction.

[0333]

[0402] Embodiment B18. The method of any one of embodiments B3-B16, wherein the targeting moiety is within an NMD exon.

[0334]

[0403] Embodiment B19. The method of any one of embodiments B1-B18, wherein the targeting moiety 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 contiguous nucleotides of an NMD exon.

[0335]

[0404] Embodiment B20. The method of any one of embodiments B1 to B19, wherein the mRNA encoding the target protein comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 4 or 5.

[0336]

[0405] Embodiment B21. The method of any one of embodiments B1 to B20, wherein the mRNA encoding the target protein is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO:1.

[0337]

[0406] Embodiment B22. The method of any one of embodiments B3 to B21, wherein the targeting portion of the mRNA comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 consecutive nucleic acids of SEQ ID NO: 4 or 5.

[0338]

[0407] Embodiment B23. The method of any one of embodiments B1 to B22, wherein the agent 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 8 contiguous nucleic acids of SEQ ID NO: 4 or 5.

[0339]

[0408] Embodiment B24. The method of any one of embodiments B3 to B23, wherein the targeted portion of the mRNA is within a nonsense-mediated RNA decay-inducible exon selected from the group consisting of GRCh38 / hg38:chr3 193628509 193628616; and GRCh38 / hg38:chr3 193603500 193603557.

[0340]

[0409] Embodiment B25. The method of any one of embodiments B3 to B23, wherein the targeted portion of the mRNA is upstream or downstream of a nonsense-mediated RNA decay-inducible exon selected from the group consisting of GRCh38 / hg38:chr3 193628509 193628616; and GRCh38 / hg38:chr3 193603500 193603557.

[0341]

[0410] Embodiment B26. The method of any one of embodiments B3 to B23, wherein the targeting portion of the mRNA comprises an exon-intron junction of an exon selected from the group consisting of GRCh38 / hg38:chr3 193628509 193628616; and GRCh38 / hg38:chr3 193603500 193603557.

[0342]

[0411] Embodiment B27. The method of any one of embodiments B1 to B26, wherein the target protein produced is a full-length protein or a wild-type protein.

[0343]

[0412] Embodiment B28. The method of any one of embodiments B1-B27, wherein the therapeutic agent promotes exclusion of an NMD exon from a pre-mRNA encoding a target protein.

[0344]

[0413] Embodiment B29. The exclusion of NMD exons from pre-mRNA encoding a target protein in cells contacted with a therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about The method of embodiment B28, wherein the increase is 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold.

[0345]

[0414] Embodiment B30. The method of embodiment B28 or B29, wherein the therapeutic agent increases the level of processed mRNA encoding the target protein in the cell.

[0346]

[0415] Embodiment B31. The level of processed mRNA encoding a target protein produced in a cell contacted with a therapeutic agent is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 10 times, about 1.1 ... The method of any one of embodiments B28 to B30, wherein the increase is about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold.

[0347]

[0416] Embodiment B32. The method of any one of embodiments B28-B31, wherein the therapeutic agent increases expression of a target protein in the cell.

[0348]

[0417] Embodiment B33. The level of the target protein produced in cells contacted with the therapeutic agent is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 ... fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold increase.

[0349]

[0418] Embodiment B34. The method of any one of embodiments B2 to B33, wherein the disease or condition is induced by a loss-of-function mutation in the target protein.

[0350]

[0419] Embodiment B35. The method of embodiment B34, wherein the disease or condition is associated with haploinsufficiency of a gene encoding a target protein and the subject has a first allele that encodes a functional target protein and a second allele in which the target protein is not produced or is produced at a reduced level, or a second allele that encodes a non-functional or partially functional target protein.

[0351]

[0420] Embodiment B36. The method of any one of embodiments B2 to B35, wherein the disease or condition is selected from the group consisting of optic atrophy type 1.

[0352]

[0421] Embodiment B37. The method of any one of embodiments B34-B36, wherein the therapeutic agent promotes exclusion of an NMD exon from a pre-mRNA encoding a target protein, increasing expression of the target protein in the cell.

[0353]

[0422] Embodiment B38. The method of any one of embodiments B1-B27, wherein the therapeutic agent inhibits exclusion of an NMD exon from a pre-mRNA encoding a target protein.

[0354]

[0423] Embodiment B39. The exclusion of NMD exons from pre-mRNA encoding a target protein in cells contacted with a therapeutic agent is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 times The method according to embodiment B38, wherein the amount of the ionized blood glucose level is reduced by about one-fifth, about one-half to about one-sixth, about one-half to about one-seventh, about one-half to about one-eighth, about one-half to about one-ninth, about one-third to about one-sixth, about one-third to about one-seventh, about one-third to about one-eighth, about one-third to about one-ninth, about one-quarter to about one-seventh, about one-quarter to about one-eighth, about one-quarter to about one-ninth, about 1.1 times or less, about 1.5 times or less, about half or less, about 2.5 times or less, about one-third or less, about 3.5 times or less, about one-quarter or less, about one-fifth or less, or about one-tenth or less.

[0355]

[0424] Embodiment B40 The method of embodiment B38 or B39, wherein the therapeutic agent reduces the level of processed mRNA encoding the target protein in the cell.

[0356]

[0425] Embodiment B41. The level of processed mRNA encoding a target protein in cells contacted with a therapeutic agent is about 1.1 to about 10 times lower, about 1.5 to about 10 times lower, about 2 to about 10 times lower, about 3 to about 10 times lower, about 4 to about 10 times lower, about 1.1 to about 5 times lower, about 1.1 to about 6 times lower, about 1.1 to about 7 times lower, about 1.1 to about 8 times lower, about 1.1 to about 9 times lower, about 1 / 2 to about 5 times lower, about The method of any one of embodiments B38 to B40, wherein the amount of the saturation is reduced by a factor of 2 to about 6, about a factor of 2 to about 7, about a factor of 2 to about 8, about a factor of 2 to about 9, about a factor of 3 to about 6, about a factor of 3 to about 7, about a factor of 3 to about 8, about a factor of 3 to about 9, about a factor of 4 to about 7, about a factor of 4 to about 8, about a factor of 4 to about 9, about 1.1 or less, about 1.5 or less, about 1 / 2 or less, about 2.5 or less, about 1 / 3 or less, about 3.5 or less, about 1 / 4 or less, about 1 / 5 or less, or about 1 / 10 or less.

[0357]

[0426] Embodiment B42. The method of any one of embodiments B38-B41, wherein the therapeutic agent reduces expression of a target protein in the cell.

[0358]

[0427] Embodiment B43. The level of the target protein produced in cells contacted with the therapeutic agent is about 1.1 to about 10 times lower, about 1.5 to about 10 times lower, about 2 to about 10 times lower, about 3 to about 10 times lower, about 4 to about 10 times lower, about 1.1 to about 5 times lower, about 1.1 to about 6 times lower, about 1.1 to about 7 times lower, about 1.1 to about 8 times lower, about 1.1 to about 9 times lower, about 2 to about 5 times lower, about 2 to about 6 times lower, about 2 to about 10 ... The method of any one of embodiments B38 to B42, wherein the amount of the saturation is reduced by about 1 / 2 to about 1 / 8, about 1 / 2 to about 1 / 9, about 1 / 3 to about 1 / 6, about 1 / 3 to about 1 / 7, about 1 / 3 to about 1 / 8, about 1 / 3 to about 1 / 9, about 1 / 4 to about 1 / 7, about 1 / 4 to about 1 / 8, about 1 / 3 to about 1 / 9, about 1 / 4 to about 1 / 7, about 1 / 4 to about 1 / 8, about 1 / 4 to about 1 / 9, about 1.1 or less, about 1.5 or less, about 1 / 2 or less, about 2.5 or less, about 1 / 3 or less, about 3.5 or less, about 1 / 4 or less, about 1 / 5 or less, or about 1 / 10 or less.

[0359]

[0428] Embodiment B44. The method of any one of embodiments B2 to B27 or B38 to B43, wherein the disease or condition is induced by a gain-of-function mutation in the target protein.

[0360]

[0429] Embodiment B45. The method of embodiment B44, wherein the subject has an allele that causes the target protein to be produced at increased levels or that encodes a mutant target protein that exhibits increased activity in the cell.

[0361]

[0430] Embodiment B46 The method of embodiment B44 or B45, wherein the therapeutic agent inhibits exclusion of an NMD exon from a pre-mRNA encoding a target protein, thereby reducing expression of the target protein in the cell.

[0362]

[0431] Embodiment B47. The method of any one of embodiments B1-B46, wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises a backbone modification comprising a phosphorothioate or phosphorodiamidate linkage.

[0363]

[0432] Embodiment B48. The method of any one of embodiments B1-B47, wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

[0364]

[0433] Embodiment B49. The method of any one of embodiments B1-B48, wherein the therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer comprises at least one modified sugar moiety.

[0365]

[0434] Embodiment B50. The method of embodiment B49, wherein each sugar moiety is a modified sugar moiety.

[0366]

[0435] Embodiment B51. The therapeutic agent is an antisense oligomer (ASO), and the antisense oligomer is selected from the group consisting of 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 nucleobases, 8-15 nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 nucleobases, 10-50 nucleobases, 10-40 nucleobases, 10-35 nucleobases, 10-30 nucleobases. The method of any one of embodiments B1 to B50, wherein the nucleotide sequence comprises from 10 to 25 nucleobases, from 10 to 20 nucleobases, from 10 to 15 nucleobases, from 11 to 50 nucleobases, from 11 to 40 nucleobases, from 11 to 35 nucleobases, from 11 to 30 nucleobases, from 11 to 25 nucleobases, from 11 to 20 nucleobases, from 11 to 15 nucleobases, from 12 to 50 nucleobases, from 12 to 40 nucleobases, from 12 to 35 nucleobases, from 12 to 30 nucleobases, from 12 to 25 nucleobases, from 12 to 20 nucleobases, or from 12 to 15 nucleobases.

[0367]

[0436] Embodiment B52. The method of any one of embodiments B3-B51, wherein the therapeutic agent 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 targeted portion of the mRNA.

[0368]

[0437] Embodiment B53. The method of any one of embodiments B1 to B52, further comprising assessing the mRNA or expression level of the target protein.

[0369]

[0438] Embodiment B54. The method of any one of embodiments B1-B53, wherein the subject is a human.

[0370]

[0439] Embodiment B55. The method of any one of embodiments B1-B53, wherein the subject is a non-human animal.

[0371]

[0440] Embodiment B56. The method of any one of embodiments B2-B54, wherein the subject is a fetus, embryo, or child.

[0372]

[0441] Embodiment B57. The method of any one of embodiments B1-B56, wherein the cells are ex vivo.

[0373]

[0442] Embodiment B58. The method of any one of embodiments B2-B56, wherein the therapeutic agent is administered by intravitreal, intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous, intravitreal or intravenous injection into the subject.

[0374]

[0443] Embodiment B59. The method of any one of embodiments B2-B56 or B58, further comprising administering a second therapeutic agent to the subject.

[0375]

[0444] Embodiment B60. The method of any one of embodiments B1-B59, wherein the second therapeutic agent is a small molecule.

[0376]

[0445] Embodiment B61. The method of any one of embodiments B1-B59, wherein the second therapeutic agent is an antisense oligomer.

[0377]

[0446] Embodiment B62. The method of any one of embodiments B1-B61, wherein the second therapeutic agent corrects intron retention.

[0378]

[0447] Embodiment B63. The method of any one of embodiments B2 to B62, wherein the disease or condition is optic atrophy type 1. Further specific embodiments

[0448] Embodiment 1. A method of modulating expression of an OPA1 protein in a cell having a pre-mRNA transcribed from the OPA1 gene and including a nonsense-mediated RNA decay-induced exon (NMD exon), comprising contacting the cell with an agent or a vector encoding the agent, whereby the agent modulates splicing of the NMD exon from the pre-mRNA, thereby modulating the level of processed mRNA processed from the pre-mRNA and modulating expression of an OPA1 protein in the cell, wherein the agent comprises an antisense oligomer having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6-275 and 280-299.

[0379]

[0449] Embodiment 2. The agent is (a) binds to a targeting portion of a pre-mRNA; (b) modulating the binding of factors involved in splicing of the NMD exon; or (c) a combination of (a) and (b); 2. The method of embodiment 1.

[0380]

[0450] Embodiment 3. The method of embodiment 2, wherein the agent interferes with the binding of a factor involved in splicing of the NMD exon to the region of the targeting moiety.

[0381]

[0451] Embodiment 4. The method of embodiment 2, wherein the targeting portion of the pre-mRNA is proximal to an NMD exon.

[0382]

[0452] Embodiment 5. The method of embodiment 2, wherein the targeting portion of the pre-mRNA is up to about 1500, about 1000, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 80, about 70, about 60, or about 50 nucleotides upstream of the 5' end of the NMD exon.

[0383]

[0453] Embodiment 6. The method of embodiment 2, wherein the targeting portion of the pre-mRNA is at least about 1500, about 1000, about 800, about 700, ab...

Claims

1. 1. A pharmaceutical composition for use in a method for increasing expression of an OPA1 protein in a cell having a processed mature mRNA that encodes the OPA1 protein and that includes a translational control element that inhibits translation of the processed mature mRNA, the composition comprising an agent or a vector encoding the agent, wherein the agent: (a) binds to a targeting portion of the processed mature mRNA, and / or (b) regulating the formation of secondary mRNA structures of translational control elements; the translational control element comprises a stem, a stem-loop, a G-quadruplex, or any combination thereof; The pharmaceutical composition thereby increases the expression of OPA1 protein in cells.

2. The translational control element is (i) in the 5' untranslated region (5'UTR) of the processed mature mRNA; and / or (ii) the pharmaceutical composition of claim 1, comprising at least a portion of the 5'UTR of the processed mature mRNA.

3. 2. The pharmaceutical composition of claim 1, wherein the translational control element comprises a secondary mRNA structure that is base-paired with at least one nucleotide of the major start codon of the processed mature mRNA, and the agent inhibits base-pairing with at least one nucleotide of the major start codon of the processed mature mRNA.

4. 2. The pharmaceutical composition of claim 1, wherein the processed mature mRNA is a full-length mature mRNA and the targeting portion is within exon 1 of the full-length mature mRNA.

5. 2. The pharmaceutical composition of claim 1, wherein the targeting site does not comprise a canonical or non-canonical start site.

6. the agent binds to a targeting portion of the processed mature mRNA; (i) the targeted portion of the processed mature mRNA is at least 42 nucleotides upstream of the major start codon of the processed mature mRNA; and / or (ii) the targeted portion of the processed mature mRNA is at most 234 nucleotides upstream of the major start codon of the processed mature mRNA; The pharmaceutical composition of claim 1.

7. 2. The pharmaceutical composition of claim 1, wherein the primary start codon is defined by genomic coordinates GRCh38 chr3:193,593,378 to 193,593,380.

8. The pharmaceutical composition of claim 1 , wherein the translational control element comprises an upstream start codon.

9. 10. The pharmaceutical composition of claim 1, wherein the agent promotes the formation of a secondary mRNA structure involving base pairing with at least one nucleotide of the upstream start codon.

10. 2. The pharmaceutical composition of claim 1, wherein the translational control element comprises a G-quadruplex formed by a G-rich sequence in the processed mature mRNA.

11. 11. The pharmaceutical composition of claim 10, wherein the G-rich sequence comprises at least a portion of the 5' untranslated region (5'UTR) of the processed mature mRNA.

12. 11. The pharmaceutical composition of claim 10, wherein the G-rich sequence comprises a sequence represented by the formula Gx-Ny-Gx-Ny-Gx-Ny-Gx, where x is an integer of 3 or greater, y is an integer of 1 to 7, and N is A, C, G, or U.

13. 13. The pharmaceutical composition of claim 12, wherein the G-rich sequence comprises the sequence GGGAGCCGGGCUGGGGCUCACACGGGGGG.

14. 2. The pharmaceutical composition of claim 1, wherein the targeting portion of the processed mature mRNA has a sequence comprising at least 8 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1263-1271.

15. The pharmaceutical composition of claim 1 , wherein the agent comprises an antisense oligomer.

16. 16. The pharmaceutical composition of claim 15, wherein the antisense oligomer has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 608-1253.

17. The pharmaceutical composition of claim 1, wherein the agent increases the expression of OPA1 protein in a cell by increasing the translation efficiency and / or rate of the processed mature mRNA.

18. 16. The pharmaceutical composition of claim 15, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate or phosphorodiamidate linkage.

19. 16. The pharmaceutical composition of claim 15, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, or a 2'-O-methoxyethyl moiety.

20. 16. The pharmaceutical composition of claim 15, wherein the antisense oligomer comprises at least one modified sugar moiety.

21. 16. The pharmaceutical composition of claim 15, wherein the antisense oligomer consists of 8 to 50 nucleobases.

22. 16. The pharmaceutical composition of claim 15, wherein the antisense oligomer consists of 8 to 25 nucleobases.

23. The pharmaceutical composition of claim 15 , wherein the vector comprises a viral vector encoding the agent.

24. 24. The pharmaceutical composition of claim 23, wherein the viral vector comprises an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, a herpes simplex viral (HSV) viral vector, or a retroviral vector.

25. the translation efficiency and / or rate of translation of processed mature mRNA encoding OPA1 protein in cells contacted with the agent or agent-encoding vector is increased by about 1.1 compared to the translation efficiency and / or rate of translation of said processed mature mRNA in control cells not contacted with the agent or agent-encoding vector; and / or the level of OPA1 protein expressed in cells contacted with the agent or the vector encoding the agent is increased compared to the level of OPA1 protein expressed in control cells not contacted with the agent or the vector encoding the agent, wherein the level of OPA1 protein expressed in cells contacted with the agent or the vector encoding the agent may be increased by about 1.1 compared to the level of OPA1 protein expressed in control cells not contacted with the agent or the vector encoding the agent; The pharmaceutical composition according to any one of claims 1 to 24.

26. The pharmaceutical composition according to any one of claims 1 to 24, wherein the OPA1 protein translated from the processed mature mRNA is a functional OPA1 protein.

27. 27. The pharmaceutical composition of claim 26, wherein the OPA1 protein translated from the processed mature mRNA has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1272-1280.

28. 25. The pharmaceutical composition of any one of claims 1 to 24, for use in treating a disease or condition associated with a deficient amount of OPA1 protein, deficient activity of OPA1 protein, or a loss-of-function mutation in the OPA1 gene.

29. The disease or condition may be 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 external ophthalmoplegia; hearing loss; ataxia; motor neuropathy; sensory neuropathy; myopathy; Beer's syndrome; cerebral dysfunction; encephalopathy; peripheral neuropathy; fatal pediatric mitochondrial encephalopathy; hypertrophic cardiomyopathy; spastic ataxia syndrome; sensorimotor peripheral neuropathy; hypotonia; gastrointestinal motility disorders and dysphagia; optic atrophy; optic atrophy plus syndrome; mitochondrial DNA depletion syndrome 14; late-onset cardiomyopathy; diabetic cardiomyopathy; Alzheimer's disease; focal segmental Glomerulosclerosis; kidney disease; Huntington's disease; cognitive decline in healthy aging; prion diseases; late-onset dementia and Parkinsonism; mitochondrial myopathy; Leigh syndrome; Friedreich's ataxia; Parkinson's disease; MELAS (mitochondrial encephalopathy, 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 disorders; widespread liver apoptosis; NARP (neuropathy, ataxia, retinitis pigmentosa); MERRF (myoclonic epilepsy with ragged-red fibers); Pearson / Kerns-Sayre syndrome; MIDD (maternally inherited diabetes and deafness); mitochondrial trifunctional transcription 25. The pharmaceutical composition of any one of claims 1 to 24, for use in treating a disease or condition selected from the group consisting of: corneal protein deficiency; Fuchs' endothelial corneal dystrophy; macular telangiectasia; retinitis pigmentosa; Leber's congenital amaurosis; hereditary maculopathy; Stargardt's disease; or Sohrsby's fundus degeneration.

30. A pharmaceutical composition comprising an agent or a vector encoding an agent, wherein the agent comprises an antisense oligomer having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 608-1253.

31. A pharmaceutical composition comprising a drug or a vector encoding the drug, wherein the drug comprises an antisense oligomer that binds to a targeting portion of the processed mature mRNA encoding the OPA1 protein, and the targeting portion of the processed mature mRNA comprises at least one nucleotide of the major start codon of the processed mature mRNA or is within the 5'UTR of the processed mature mRNA.

32. A pharmaceutical composition comprising a drug or a vector encoding the drug, wherein the drug regulates the structure of the translational control element of the processed mature mRNA encoding the OPA1 protein, thereby increasing the expression of the OPA1 protein, and the translational control element inhibits translation of the processed mature mRNA.

33. A pharmaceutical composition comprising an agent or a vector encoding the agent, wherein the agent increases translation of processed mature mRNA in a cell, the processed mature mRNA encodes an OPA1 protein and comprises a translational control element that inhibits translation of the processed mature mRNA, the agent modulates the structure of the translational control element, thereby increasing the translational efficiency and / or rate of translation of the processed mature mRNA, and the agent (a) binds to a targeting portion of the processed mature mRNA, (b) modulates the interaction of the translational control element with a factor involved in translation of the processed mature mRNA, or (c) is a combination of (a) and (b).

34. 1. Use of a first agent and a second agent, or nucleic acids encoding the first agent and the second agent, in the manufacture of a medicament for increasing expression of a target protein in a cell having processed mature mRNA that encodes the target protein and that includes a translational control element that inhibits translation of the processed mature mRNA, comprising: A use in which a first agent modulates the splicing of a pre-mRNA transcribed from a target gene that encodes a target protein, and a second agent modulates the structure of a translational control element of the processed mature mRNA that encodes the target protein, thereby increasing expression of the target protein in a cell.

35. 35. The use of claim 34, wherein the second agent (a) binds to a targeted portion of the processed mature mRNA, (b) modulates the interaction of a translational control element with a factor involved in the translation of the processed mature mRNA, or (c) a combination of (a) and (b).

36. 1. Use of a first agent and a second agent, or nucleic acids encoding the first agent and the second agent, in the manufacture of a medicament for modulating expression of a target protein in a cell, comprising: A use wherein the first agent comprises a first antisense oligomer having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6-275 and 280-299, and the second agent comprises a second antisense oligomer that binds to the 5'UTR of a processed mature mRNA encoding a target protein, wherein the target protein is OPA1 protein.

37. (1) a first therapeutic agent or a nucleic acid encoding a first therapeutic agent for use in combination with the administration of a second therapeutic agent or a nucleic acid encoding a second therapeutic agent; or (2) A first therapeutic agent or a nucleic acid encoding a first therapeutic agent for use in combination with the administration of a first therapeutic agent or a nucleic acid encoding a first therapeutic agent.

1. A pharmaceutical composition comprising: A pharmaceutical composition, wherein a first therapeutic agent comprises a first antisense oligomer having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6-275 and 280-299, and a second therapeutic agent comprises a second antisense oligomer that binds to the 5'UTR of a processed mature mRNA encoding a target protein, wherein the target protein is OPA1 protein.

38. An antisense oligomer having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 608-1253, wherein the antisense oligomer comprises a backbone modification, a sugar moiety modification, or a combination thereof.

39. A composition comprising a first antisense oligomer having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6-275 and 280-299, and a second antisense oligomer that binds to the 5'UTR of a processed mature mRNA encoding an OPA1 protein, wherein the first antisense oligomer and the second antisense oligomer comprise a backbone modification, a sugar moiety modification, or a combination thereof.

40. A pharmaceutical composition comprising a therapeutic agent or a vector encoding the therapeutic agent and a pharmaceutically acceptable carrier or excipient, wherein the therapeutic agent regulates the structure of the translational control element of the processed mature mRNA encoding the OPA1 protein, thereby increasing the expression of the OPA1 protein, and the translational control element inhibits translation of the processed mature mRNA.

41. A pharmaceutical composition comprising a therapeutic agent or a vector encoding a therapeutic agent and a pharmaceutically acceptable carrier or excipient, wherein the therapeutic agent (a) binds to a targeting portion of processed mature mRNA that encodes OPA1 protein and contains a translational control element, (b) modulates the interaction between the translational control element and factors involved in the translation of the processed mature mRNA, or (c) is a combination of (a) and (b), thereby increasing the expression of OPA1 protein in cells and the translational control element inhibits translation of the processed mature mRNA.

42. 1. A pharmaceutical composition comprising: (1) a first therapeutic agent or a first nucleic acid sequence encoding the first therapeutic agent; (2) a second therapeutic agent or a second nucleic acid sequence encoding the second therapeutic agent; and (3) a pharmaceutically acceptable carrier or excipient, wherein the first therapeutic agent modulates splicing of a pre-mRNA transcribed from a target gene encoding a target protein; and the second therapeutic agent (a) binds to a targeted portion of a processed mature mRNA that encodes an OPA1 protein and that contains a translational control element; (b) modulates the interaction of the translational control element with a factor involved in the translation of the processed mature mRNA; or (c) a combination of (a) and (b), wherein the translational control element inhibits translation of the processed mature mRNA.

43. A pharmaceutical composition comprising: (1) a first therapeutic agent or a first nucleic acid sequence encoding the first therapeutic agent; (2) a second therapeutic agent or a second nucleic acid sequence encoding the second therapeutic agent; and (3) a pharmaceutically acceptable carrier or excipient, wherein the first therapeutic agent modulates splicing of a pre-mRNA transcribed from a target gene encoding a target protein; and the second therapeutic agent modulates the structure of a translational control element of a processed mature mRNA encoding the target protein, and the translational control element inhibits translation of the processed mature mRNA.