Antisense oligonucleotides for exon skipping, compositions and pharmaceutical formulations thereof

Abstract

Provided are novel antisense oligonucleotides ("ASO") that generate or promote exon hopping during precursor mRNA splicing, pharmaceutical compositions containing such ASO, and uses thereof. An ASO (e.g., a binary ASO) comprises a targeting sequence and a 5 '-splice site decoy sequence operably linked to the 5'end and / or 3' end of the targeting sequence. The length of the bait sequence can be 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides, and a part of the bait sequence is complementary to the single-stranded 5'end of the U1snRNA. The target sequence hybridizes to a target region, such as an exon of interest, a flanking intron sequence upstream or downstream of the exon of interest, or an intron-exon junction upstream or downstream of the exon of interest.

Description

[0001] Priority Claim

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 377,946, filed Sep. 30, 2022, which is incorporated herein by reference in its entirety, including the drawings. Background of the Invention

[0003] The primary tool for exon skipping is antisense oligonucleotides (ASOs). ASOs can generate or enhance exon skipping by binding to splice sites or splicing regulatory elements. The splicing process can be regulated by different cis-acting elements and trans-acting factors that affect splice site selection. ASOs either block the binding of RNA-binding proteins (RBPs) or snRNAs to their specific sites or disrupt secondary RNA structures, and thus ASOs promote or inhibit pre-mRNA splicing depending on the sequence they target. Matlin A, et al. Understanding alternative splicing: towards a cellular code. May 2005. Nat. Rev. Mol. Cell Bio. 6:386–398. doi:10.1038 / nrm1645. Aartsma-Rus A, et al. Exonic sequences provide better targets for antisense oligonucleotides than splice site sequences in the modulation of Duchenne muscular dystrophy splicing. Oligonucleotides. Apr 2010;20(2):69-77. doi:10.1089 / oli.2009.0215.

[0004] Examples of human genetic diseases associated with mutations that disrupt the reading frame are Duchenne muscular dystrophy (DMD), a progressive form of muscular dystrophy that causes progressive weakness and loss (atrophy) of skeletal and cardiac muscle. Muscle weakness worsens with age and progresses to the arms, legs, and trunk. Most children with DMD are wheelchair-bound by the age of 13. Cardiac and respiratory muscle problems begin in adolescence and lead to severe, life-threatening complications.

[0005] DMD is caused by mutations in the DMD gene. The DMD gene encodes the protein dystrophin. Dystrophin is mainly produced in skeletal muscle and cardiomyocytes, but is also produced in small amounts in neurons in specific parts of the brain. DMD is inherited in an X-linked recessive pattern. Becker muscular dystrophy (BMD) is also caused by mutations in the DMD gene. The symptoms of BMD patients are milder than those of DMD patients. SUMMARY OF THE INVENTION

[0006] Certain aspects of the technology of the present invention generally relate to novel antisense oligonucleotides (ASOs), methods of using such ASOs to generate or promote exon skipping of a target exon during precursor mRNA (pre-mRNA) splicing, compositions (such as pharmaceutical compositions) comprising such ASOs, and methods of using such compositions to treat diseases and / or their complications. ASO-mediated exon skipping may be a method of manipulating the expression of a target gene. The expression of a target gene can be manipulated by ASO-induced exon skipping, resulting in (e.g., but not limited to): correcting the reading frame caused by a frameshift mutation, skipping the toxic part of a gene, silencing a gene, generating a dominant negative subtype, or altering the structure and function of a gene. Exon skipping of a target gene may be beneficial for treating diseases and / or their complications, although the target gene may or may not be the cause of the disease. The disease may be a genetic or non-genetic disease (such as some non-genetic cancers, metabolic diseases, or infectious diseases) disclosed herein. Genetic diseases may or may not be associated with splicing defects.

[0007] In some aspects, the technology of the present invention provides oligonucleotides, which may also be referred to as bipartite ASOs, comprising or consisting of: a targeting sequence (also referred to as an "ASO targeting sequence") and a 5'-splice site decoy sequence (also referred to as an "ASO decoy", "ASO decoy sequence", "decoy sequence", or "decoy") operably linked to the 5'-end and / or 3'-end of the targeting sequence. The decoy sequence may comprise a nucleotide sequence of 5, 6, 7, 8, 9, 10, or 11 nucleotides that is complementary to a part or all of the single-stranded 5'-end of U1 snRNA; and the targeting sequence hybridizes to a sequence selected from the group consisting of: a target exon, a flanking intron sequence upstream of the target exon, a flanking intron sequence downstream of the target exon, an intron-exon junction upstream of the target exon, and an intron-exon junction downstream of the target exon.

[0008] In certain embodiments of ASOs containing the same targeting sequence, the ASO containing the decoy sequence may have a higher exon skipping effect and / or efficiency than the ASO consisting of the targeting sequence. The exon skipping effect and / or efficiency can be quantified by the percentage of exclusion of the target exon in the total transcripts of each gene (exclusion %, also referred to as exon skipping percentage). In certain embodiments, the improvement quantified by the increase in exclusion % can exceed about 14-fold, about 15-fold, about 16-fold, or about 17-fold.

[0009] In some embodiments, the ASO decoy sequence comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID No. 1 to 23 and SEQ ID No. 347 to 353.

[0010] Examples of target exons include, but are not limited to, exon 7 of the endogenous SMN1 and SMN2 genes, exons 45, 51, and 53 of the endogenous DMD gene, exon 17 of the endogenous APP gene, exon 41 of the endogenous CEP290 gene, exon 19 of the endogenous HER2 gene, exon 10 of the SCA3 gene, exon 10 of the endogenous PKM gene, and exon 6 of the endogenous MDM4 gene.

[0011] In some embodiments, the ASO targeting sequence comprises or consists of a nucleotide sequence selected from the group consisting of: the nucleotide sequences of SEQ ID No. 25, 29, 33, 37, 41, 45, 49, 75, 92, 124, 156, 188, 220, 252, 284, and 316.

[0012] The targeting sequence can be directly or via a linker (e.g., but not limited to having a length of 1, 2, 3, 4, or 5 nucleotides) linked to the decoy sequence.

[0013] In some embodiments, the ASO (e.g., bipartite ASO) comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID No. 26 to 27, 30 to 31, 34 to 35, 38 to 39, 42 to 43, 46 to 47, 50 to 73, 76 to 90, 93 to 122, 125 to 154, 157 to 186, 189 to 218, 221 to 250, 253 to 282, 285 to 314, 317 to 346, and 354 to 356.

[0014] In some embodiments, an ASO (e.g., a bipartite ASO) comprises at least one nucleotide analogue. Examples of nucleotide analogues include, but are not limited to, 2'-O-methoxyethyl (MOE)-modified oligonucleotides having a phosphodiester or phosphorothioate backbone, and phosphorodamidate morpholino oligomers.

[0015] In some aspects, the technology of the present invention provides a composition comprising an ASO (e.g., a bipartite ASO) disclosed herein and a pharmaceutically acceptable carrier. In certain embodiments, the composition is a pharmaceutical formulation or composition.

[0016] In some aspects, the technology of the present invention provides a vector encoding an ASO (e.g., a bipartite ASO) disclosed herein.

[0017] In some aspects, the technology of the present invention provides a method for generating or promoting exon skipping of a target exon during pre-mRNA splicing, which comprises contacting the pre-mRNA in a cell or a subject with an ASO (e.g., a bipartite ASO) disclosed herein, a composition (e.g., a pharmaceutical composition) comprising an ASO (e.g., a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (e.g., a bipartite ASO) disclosed herein. In some embodiments, the method further comprises delivering an ASO (e.g., a bipartite ASO) disclosed herein to the cell or administering it to the subject. ASO-mediated exon skipping may be a method of manipulating the expression of a target gene. The expression of the target gene can be manipulated by the ASO inhibiting the splicing of an exon, an intron, or a specific splicing site of the target gene, resulting in (e.g., but not limited to): restoring the reading frame of the target defective gene, generating different subtypes of the target gene (e.g., a dominant negative subtype), skipping the toxic portion of the gene, silencing the gene, and / or altering the structure and function of the gene.

[0018] In some aspects, the technology of the present invention provides a method for improving the exon skipping efficacy and / or efficiency of a target sequence, which comprises obtaining one or more ASOs (e.g., bipartite ASOs), the one or more ASOs comprising a target sequence and a decoy sequence operably linked to the 5'-end and / or 3'-end of the target sequence, the decoy sequence comprising a nucleotide sequence of 5, 6, 7, 8, 9, 10 or 11 nucleotides that is complementary to a part or all of the single-stranded 5'-end of U1 snRNA. The decoy sequence may be similar to the optimal 5' splice site. In certain embodiments, the method further comprises screening the one or more ASOs (e.g., bipartite ASOs) according to the exon skipping efficacy and / or efficiency of the target exon of the one or more ASOs (e.g., bipartite ASOs). The exon skipping effect and / or efficiency can be quantified by the percentage of exclusion of the target exon in the total transcripts of each gene (exclusion %). The target sequence may be capable of hybridizing to a sequence selected from the group consisting of: the target exon, the flanking intron sequence upstream of the target exon, the flanking intron sequence downstream of the target exon, the intron-exon junction upstream of the target exon, and the intron-exon junction downstream of the target exon in a cell or a subject.

[0019] In some aspects, the technology of the present invention provides a method for treating a disease and / or its complication in a subject, which comprises administering to the subject the ASOs (e.g., bipartite ASOs) disclosed herein, a composition (e.g., a pharmaceutical composition) comprising the ASOs (e.g., bipartite ASOs) disclosed herein, and / or a vector encoding the ASOs (e.g., bipartite ASOs) disclosed herein. The ASOs (e.g., bipartite ASOs) may generate or promote the skipping of the target exon during pre-mRNA splicing. ASO-mediated exon skipping may be a method for manipulating the expression of a target gene. The expression of the target gene can be manipulated by the ASO inhibiting the splicing of the exon, intron or specific splice site of the target gene, resulting in (e.g., but not limited to): restoring the reading frame of the target defective gene, generating different subtypes of the target gene (e.g., dominant negative subtypes), skipping the toxic part of the gene, silencing the gene and / or altering the structure and function of the gene. Manipulating the expression of the target gene by the ASO may be beneficial for treating a disease and / or its complication, although the target gene may or may not be the cause of the disease. The disease may be a genetic disease or a non-genetic disease (e.g., some non-genetic cancers, metabolic diseases or infectious diseases) disclosed herein. The genetic disease may or may not be associated with a splicing defect-related mutation.

[0020] Examples of diseases and / or their complications include, but are not limited to, diseases and / or their complications that may benefit from exon skipping on one or more genes selected from the group consisting of: SMN1, SMN2, DMD, APP, CEP290, HER2, SCA3, PKM, and MDM4 genes. For example but not limited to, diseases and / or their complications can be treated by exon skipping of one or more exons selected from the group consisting of: exon 7 of the endogenous SMN1 and SMN2 genes, exons 45, 51, and 53 of the endogenous DMD gene, exon 17 of the endogenous APP gene, exon 41 of the endogenous CEP290 gene, exon 19 of the endogenous HER2 gene, exon 10 of the SCA3 gene, exon 10 of the endogenous PKM gene, and exon 6 of the endogenous MDM4 gene. Examples of diseases can include but are not limited to Duchenne muscular dystrophy (DMD), Alzheimer's disease, Joubert Syndrome, Spinocerebellar ataxia type 3 (SCA3), cancer, such as but not limited to breast cancer, HER2-positive cholangiocarcinoma, colorectal cancer, non-small cell lung cancer, bladder cancer, prostate cancer, lung cancer, cervical cancer, kidney cancer, papillary thyroid cancer, colon cancer, colorectal cancer, glioma, ovarian cancer, gastric cancer, hepatoblastoma, fibrolamellar hepatocellular carcinoma, soft tissue sarcoma, osteosarcoma, chronic lymphocytic leukemia, acute myeloid leukemia, mantle cell lymphoma, Pediatric Burkitt lymphoma, salivary gland cancer, liver cancer, and melanoma. In certain embodiments, ASOs (such as duplex ASOs), compositions comprising ASOs (such as duplex ASOs) (such as pharmaceutical compositions), and / or vectors encoding ASOs (such as duplex ASOs) are administered in a therapeutically effective amount.

[0021] In some aspects, the technology of the present invention provides the use of the ASOs (e.g., bisegmented ASOs) disclosed herein, compositions (e.g., pharmaceutical compositions) comprising the ASOs (e.g., bisegmented ASOs) disclosed herein, and / or vectors encoding the ASOs (e.g., bisegmented ASOs) disclosed herein for generating or promoting exon skipping of a target exon. ASO-mediated exon skipping may be a method of manipulating the expression of a target gene. The expression of the target gene can be manipulated by the ASO inhibiting the splicing of an exon, intron, or specific splicing site of the target gene, resulting in (e.g., but not limited to): restoring the reading frame of the target defective gene, generating different subtypes of the target gene (e.g., dominant negative subtypes), skipping the toxic portion of the gene, silencing the gene, and / or altering the structure and function of the gene. Examples of the target exon and the target gene include but are not limited to those disclosed herein.

[0022] In some aspects, the technology of the present invention provides the use of the ASOs (e.g., bisegmented ASOs) disclosed herein, compositions (e.g., pharmaceutical compositions) comprising the ASOs (e.g., bisegmented ASOs) disclosed herein, and / or vectors encoding the ASOs (e.g., bisegmented ASOs) disclosed herein for treating a disease and / or its complications. Examples of the disease include but are not limited to those disclosed herein.

[0023] In some aspects, the technology of the present invention provides the ASOs (e.g., bisegmented ASOs) disclosed herein, compositions (e.g., pharmaceutical compositions) comprising the ASOs (e.g., bisegmented ASOs) disclosed herein, and / or vectors encoding the ASOs (e.g., bisegmented ASOs) disclosed herein for generating or promoting exon skipping of a target exon. ASO-mediated exon skipping may be a method of manipulating the gene expression of a target gene. The expression of the target gene can be manipulated by the ASO inhibiting the splicing of an exon, intron, or specific splicing site of the target gene, resulting in (e.g., but not limited to): restoring the reading frame of the target defective gene, generating different subtypes of the target gene (e.g., dominant negative subtypes), skipping the toxic portion of the gene, silencing the gene, and / or altering the structure and function of the gene. Examples of the target exon and the target gene include but are not limited to those disclosed herein.

[0024] In some aspects, the technology of the present invention provides the ASOs (e.g., bisegmented ASOs) disclosed herein, compositions (e.g., pharmaceutical compositions) comprising the ASOs (e.g., bisegmented ASOs) disclosed herein, and / or vectors encoding the ASOs (e.g., bisegmented ASOs) disclosed herein for treating a disease and / or its complications. Examples of the disease and / or its complications include but are not limited to those disclosed herein.

[0025] In some aspects, the technology of the present invention provides a kit that includes an ASO (such as a bipartite ASO) disclosed herein, a composition (such as a pharmaceutical composition) that includes an ASO (such as a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (such as a bipartite ASO) disclosed herein, for generating or promoting exon skipping of a target exon. ASO-mediated exon skipping may be a method of manipulating the expression of a target gene. The expression of a target gene can be manipulated by an ASO inhibiting the splicing of an exon, intron, or specific splicing site of the target gene, resulting in (for example but not limited to): restoring the reading frame of a target defective gene, generating different subtypes of a target gene (such as a dominant negative subtype), skipping a toxic portion of a gene, silencing a gene, and / or altering the structure and function of a gene. Examples of target exons and target genes include but are not limited to those disclosed herein.

[0026] In some aspects, the technology of the present invention provides a kit that includes an ASO (such as a bipartite ASO) disclosed herein, a composition (such as a pharmaceutical composition) that includes an ASO (such as a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (such as a bipartite ASO) disclosed herein, for treating a disease and / or its complications. Examples of diseases and / or their complications include but are not limited to those disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A to Figure 1 B show some schematic diagrams of an ASO (such as a bipartite ASO) of the technology of the present invention, the ASO including: 1) a "targeting" sequence that is fully or substantially complementary to a target region in a target nucleic acid (such as a precursor mRNA), and 2) a "decoy" sequence adjacent to the targeting sequence (i.e., the decoy sequence is in the flanking region of the targeting sequence). Figure 1 A shows two schematic diagrams of a bipartite ASO, where the decoy sequence is located upstream (left side) of the 5' end of the targeting sequence and immediately downstream (right side) of the 3' end of the targeting sequence, respectively. Figure 1 B shows Figure 1 A possible mechanism of how the bipartite ASO of A generates or promotes the skipping of a target exon, where the decoy sequence is located immediately upstream of the 5' end of the targeting sequence, without being bound by any particular theory. The decoy sequence may resemble an optimal 5' splice site and be complementary to a partial or all of the free single-stranded sequence at the 5' end of U1 snRNA, so the decoy sequence can interfere with the recognition of the nearby authentic 5' splice site by U1 snRNA.

[0028] Figure 2 A to Figure 2Panel D shows the effect of the presence and / or location of decoy sequence 11 (SEQ ID No. 21) on exon 7 skipping in the SMN1 and SMN2 genes in HEK293 cells using an ASO containing one of three targeting sequences (2203, 1938, and 0120). Figure 2 Panel A shows a schematic diagram of the approximate binding positions of three tested targeting sequences (2203, 1938, and 0120) of the SMN1 and SMN2 genes. Figure 2 Panel B shows the exon 7 skipping effect of an ASO containing targeting sequence 2203 in the SMN1 and SMN2 genes. Figure 2 The left panel of B shows a representative semi - quantitative fluorescence RT - PCR imaging analysis of the SMN1 and SMN2 genes in HEK293 cells transfected with ASO (12.5 nM, 25 nM, or 50 nM) that have the targeting sequence 2203 alone (2203), the targeting sequence 2203 with decoy sequence 11 linked to the 5' end (2203 - L11), and the targeting sequence 2203 with decoy sequence 11 linked to the 3' end (2203 - R11), respectively. Figure 2 The middle panel of B shows the quantification (exclusion %) of exon 7 skipping in SMN1 using the same ASOs, presented as mean ± standard deviation (n = 3, **P < 0.01 compared to 2203). For the three columns at each concentration, the 2203 data are shown on the left, the 2203 - L11 data are shown in the middle, and the 2203 - R11 data are shown on the right. Figure 2 The right panel of B shows the quantification (exclusion %) of exon 7 skipping in SMN2 using the same ASOs, presented as mean ± standard deviation (n = 3, **P < 0.01 compared to 2203). For the three columns at each concentration, the 2203 data are shown on the left, the 2203 - L11 data are shown in the middle, and the 2203 - R11 data are shown on the right. Buffer was used as a negative control. Figure 2 Panel C shows the exon 7 skipping effect of an ASO containing targeting sequence 1938 in the SMN1 and SMN2 genes. Figure 2 The left panel of C shows a representative semi - quantitative fluorescence RT - PCR imaging analysis of the SMN1 and SMN2 genes in HEK293 cells transfected with ASO (12.5 nM, 25 nM, or 50 nM) that have the targeting sequence 1938 alone (1938), the targeting sequence 1938 with decoy sequence 11 linked to the 5' end (1938 - L11), and the targeting sequence 1938 with decoy sequence 11 linked to the 3' end (1938 - R11), respectively. Figure 2The middle panel of C shows the quantification (percent exclusion) of exon 7 skipping in SMN1 using the same ASO, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01 compared to 1938), where for the three columns at each concentration, the 1938 data is shown on the left, the 1938-L11 data is shown in the middle, and the 1938-R11 data is shown on the right. Figure 2 The right panel of C shows the quantification (percent exclusion) of exon 7 skipping in SMN2 using the same ASO, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01 compared to 1938), where for the three columns at each concentration, the 1938 data is shown on the left, the 1938-L11 data is shown in the middle, and the 1938-R11 data is shown on the right. Buffer was used as a negative control. Figure 2 D shows the exon 7 skipping effect of ASOs containing targeting sequence 0120 in SMN1 and SMN2. Figure 2 The left panel of D shows representative semi-quantitative fluorescent RT-PCR imaging analysis of SMN1 and SMN2 in HEK293 cells transfected with ASOs (12.5 nM, 25 nM, or 50 nM) having the targeting sequence 0120 alone (0120), the targeting sequence 0120 with the decoy sequence 11 linked to the 5'-end (0120-L11), and the targeting sequence 0120 with the decoy sequence 11 linked to the 3'-end (0120-R11), respectively. Figure 2 The middle panel of D shows the quantification (percent exclusion) of exon 7 skipping in SMN1 using the same ASO, presented as mean ± standard deviation (n = 3, **P < 0.01 compared to 0120), where for the three columns at each concentration, the 0120 data is shown on the left, 0120-L11 is shown in the middle, and 0120-R11 is shown on the right. Figure 2 The right panel of D shows the quantification (percent exclusion) of exon 7 skipping in SMN2 using the same ASO, presented as mean ± standard deviation (n = 3, **P < 0.01 compared to 0120), where for the three columns at each concentration, the 0120 data is shown on the left, the 0120-L11 data is shown in the middle, and the 0120-R11 data is shown on the right. Buffer was used as a negative control.

[0029] Figure 3 A to Figure 3 B depicts the effect of the presence and / or position of the decoy sequence 11 on exon 51 skipping in the DMD gene in rhabdomyosarcoma (RD) cells using ASOs containing one of four targeting sequences (148, 155, 165, and eteplirsen (Etep)). Figure 3Panel A shows the exon 51 skipping effect of an ASO containing one of three targeting sequences (148, 155, and 165) in the DMD gene. Figure 3 The left panel of Panel A shows a representative semi-quantitative fluorescence RT-PCR imaging analysis in which RD cells were transfected with an ASO (50 nM) having one of the following three targeting sequences: a targeting sequence without a decoy sequence (148, 155, and 165), a decoy sequence 11 linked to the 5'-end of the targeting sequence (148-L11, 155-L11, and 165-L11), or a decoy sequence 11 linked to the 3'-end of the targeting sequence (148-R11, 155-R11, and 165-R11). Figure 3 The right panel of Panel A shows the quantification (exclusion %) of exon 51 skipping in DMD in RD cells transfected with an ASO (50 nM), presented as mean ± standard deviation (n = 4, *P < 0.05 (148-L11 vs. 148), **P < 0.01 (155-L11 vs. 155, 165-L11 vs. 165)), having one of the following three targeting sequences: a targeting sequence without a decoy sequence (148, 155, and 165), a decoy sequence 11 linked to the 5'-end of the targeting sequence (148-L11, 155-L11, and 165-L11), or a decoy sequence 11 linked to the 3'-end of the targeting sequence (148-R11, 155-R11, and 165-R11). Buffer was used as a negative control. Figure 3 Panel B shows the exon 51 skipping effect of an ASO containing the targeting sequence Etep in the DMD gene. Figure 3 The left panel of Panel B shows a representative semi-quantitative fluorescence RT-PCR imaging analysis in which RD cells were transfected with an ASO (12.5 nM, 25 nM, or 50 nM) having a targeting sequence Etep without a decoy sequence (Etep), a decoy sequence 11 linked to the 5'-end of the targeting sequence (Etep-L11), or a decoy sequence 11 linked to the 3'-end of the targeting sequence (Etep-R11). Figure 3 The right panel of Panel B shows the quantification (exclusion %) of exon 51 skipping in DMD in RD cells transfected with an ASO (12.5 nM, 25 nM, or 50 nM), presented as mean ± standard deviation (**P < 0.01 (Etep-L11 vs. Etep)), having a targeting sequence without a decoy sequence (Etep, the left three columns for each concentration), a decoy sequence 11 linked to the 5'-end of the targeting sequence (Etep-L11, the middle three columns for each concentration), or a decoy sequence 11 linked to the 3'-end of the targeting sequence (Etep-R11, the right three columns for each concentration). Buffer was used as a negative control.

[0030] Figure 4 Show the effect of the presence, length, and / or sequence of various decoy sequences (6a to 6f, 7a to 7e, 8a to 8d, 9a to 9c, 10a to 10b, 11, 12, and 13) linked to the 5'-end of the targeting sequence Etep on exon 51 skipping in the DMD gene in RD cells. Figure 4 The upper panel above shows a representative semi-quantitative fluorescent RT-PCR analysis of the DMD gene in RD cells transfected with an ASO (25 nM) that has the targeting sequence Etep without a decoy sequence (Etep) or has different lengths of decoy sequences (L6a to L6f, L7a to L7e, L8a to L8d, L9a to L9c, L10a to L10b, L11, L12, and L13) linked to the 5'-end of the targeting sequence Etep. Buffer was used as a negative control. Figure 4 The lower panel below shows Figure 4 the quantification (exclusion %) of exon 51 skipping in DMD in RD cells transfected with the same ASOs (25 nM) (Etep, Etep-L6a to Etep-L6f, Etep-L7a to Etep-L7e, Etep-L8a to Etep-L8d, Etep-L9a to Etep-L9c, Etep-L10a to Etep-L10b, Etep-L11, Etep-L12, and Etep-L13) tested in the upper panel above, presented as mean ± standard deviation (n = 3, # P < 0.05 (all relative to Etep); *P < 0.05, **P < 0.01 (all relative to Etep-L11)). Buffer was used as a negative control.

[0031] Figure 5 Show the effect of the presence, length, and / or sequence of various decoy sequences (7a to 7e, 8a to 8d, 9a to 9c, 10a to 10b, and 11) linked to the 5'-end of the targeting sequence 000A on exon 51 skipping in the DMD gene in RD cells. Figure 5 The upper panel above shows a representative semi-quantitative fluorescent RT-PCR analysis of the DMD gene in RD cells transfected with an ASO (25 nM) that has the targeting sequence 000A without a decoy sequence (000A) or has different lengths of decoy sequences (L7a to L7e, L8a to L8d, L9a to L9c, L10a to L10b, and L11) linked to the 5'-end of the targeting sequence 000A. Buffer was used as a negative control. Figure 5 The lower panel below shows Figure 5Quantification (exclusion %) of exon 51 skipping in DMD in RD cells transfected with the same ASOs (25 nM) (000A, 000A-L7a to 000A-L7e, 000A-L8a to 000A-L8d, 000A-L9a to 000A-L9c, 000A-L10a to 000A-L10b, and 000A-L11) as tested in the upper figure above, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01, all relative to 000A). Buffer was used as negative control.

[0032] Figure 6 Illustration of the effect of the presence, length, and / or sequence of various bait sequences (7c, 8c, 9b, and 10a for targeting sequence Etep, and 8c and 9a to 9c for targeting sequence 000A) linked to the 5'-end of the targeting sequence Etep or 000A on exon 51 skipping in the DMD gene in the tibialis anterior and gastrocnemius of DMD humanized mice. Figure 6 The upper left figure above shows a representative semi-quantitative fluorescent RT-PCR analysis of DMD exon 51 skipping in the tibialis anterior of DMD humanized mice administered with an ASO having a targeting sequence Etep (Etep) without a bait sequence, or having bait sequences of different lengths linked to the 5'-end of the targeting sequence Etep or 000A (L7c, L8c, L9b, and L10a for targeting sequence Etep, and L8c and L9a to L9c for targeting sequence 000A). Saline was used as negative control. Figure 6 The upper right figure above shows a representative semi-quantitative fluorescent RT-PCR analysis of DMD exon 51 skipping in the gastrocnemius of DMD humanized mice administered with an ASO having a targeting sequence Etep (Etep) without a bait sequence, or having bait sequences of different lengths linked to the 5'-end of the targeting sequence Etep or 000A (L7c, L8c, L9b, and L10a for targeting sequence Etep, and L8c and L9a to L9c for targeting sequence 000A). Saline was used as negative control. Figure 6 The lower left figure shows administration of Figure 6 Quantification (exclusion %) of exon 51 skipping in the tibialis anterior of DMD humanized mice with the same ASOs (Etep, Etep-L7c, Etep-L8c, Etep-L9b, Etep-L10a, 000A-L8c, and 000A-L9a to 000A-L9c) as tested in the upper left figure above, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 (all relative to Etep)). Saline was used as negative control. Figure 6The lower right figure shows the administration of Figure 6 Quantification (exclusion %) of exon 51 skipping in the gastrocnemius muscle of DMD humanized mice tested in the upper right figure above for the same ASOs (Etep, Etep-L7c, Etep-L8c, Etep-L9b, Etep-L10a, 000A-L8c, and 000A-L9a to 000A-L9c), presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01 (all relative to Etep)). Saline was used as a negative control.

[0033] Figure 7 Illustrates the effect of the presence, position, length, and / or sequence of various bait sequences (7a to 7e, 8a to 8d, 9a to 9c, 10a to 10b, and 11) linked to the 5'-end or 3'-end of the targeting sequence Vilto on exon 53 skipping in the DMD gene in RD cells. Figure 7 The upper left figure shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 53 skipping in the DMD gene in RD cells transfected with ASO (25 nM) having either the targeting sequence Vilto alone (Vilto) or the targeting sequence Vilto with different lengths of bait sequences linked to the 5'-end (Vilto-L7a to Vilto-L7e, Vilto-L8a to Vilto-L8d, Vilto-L9a to Vilto-L9c, Vilto-L10a to Vilto-L10b, and Vilto-L11). Buffer was used as a negative control. Figure 7 The upper right figure shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 53 skipping in the DMD gene in RD cells transfected with ASO (25 nM) having either the targeting sequence Vilto alone (Vilto) or the targeting sequence Vilto with different lengths of bait sequences linked to the 3'-end (Vilto-R7a to Vilto-R7e, Vilto-R8a to Vilto-R8d, Vilto-R9a to Vilto-R9c, Vilto-R10a to Vilto-R10b, and Vilto-R11). Buffer was used as a negative control. Figure 7 The lower left figure shows in the use of Figure 7Quantification (exclusion %) of exon 53 skipping in the DMD gene in RD cells transfected with the same ASOs (25 nM) (Vilto, Vilto-L7a to Vilto-L7e, Vilto-L8a to Vilto-L8d, Vilto-L9a to Vilto-L9c, Vilto-L10a to Vilto-L10b, and Vilto-L11) tested in the upper left panel, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, all relative to Vilto). Buffer was used as a negative control. Figure 7 The lower right panel shows Figure 7 Quantification (exclusion %) of exon 53 skipping in the DMD gene in RD cells transfected with the same ASOs (25 nM) (Vilto, Vilto-R7a to Vilto-R7e, Vilto-R8a to Vilto-R8d, Vilto-R9a to Vilto-R9c, Vilto-R10a to Vilto-R10b, and Vilto-R11) tested in the upper right panel, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01, all relative to Vilto). Buffer was used as a negative control.

[0034] Figure 8 A to Figure 8 B illustrate the effect of the presence, location, length, and / or sequence of various bait sequences (7a to 7e, 8a to 8d, 9a to 9c, 10a to 10b, and 11) linked to the 5'-end or 3'-end of the targeting sequence 002A on the skipping of a portion of exon 45 in the DMD gene in RD cells. Figure 8 A shows a schematic diagram of the tested targeting sequence 002A activating the cryptic 5'-splice site in exon 45 of DMD, which causes a 32-nt loss at the end of exon 45. For some DMD patients, the 32-nt loss (here called partial skipping of exon 45) can restore the reading frame, the same as the skipping of the entire 176-nt exon 45, but with much less loss of the protein sequence encoded by exon 45. Figure 8 The upper left panel of B shows a representative semi-quantitative fluorescent RT-PCR analysis of partial exon 45 skipping in the DMD gene in RD cells transfected with ASO (25 nM) having either the targeting sequence 002A alone (002A) or the targeting sequence 002A with different lengths of bait sequences linked to the 5'-end (002A-L7a to 002A-L7e, 002A-L8a to 002A-L8d, 002A-L9a to 002A-L9c, 002A-L10a to 002A-L10b, and 002A-L11). Buffer was used as a negative control. Figure 8The upper right panel of B shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 45 skipping in the DMD gene in RD cells transfected with ASO (25 nM), which has a separate targeting sequence 002A (002A) or a targeting sequence 002A with different lengths of decoy sequences linked to the 3'-end (002A-R7a to 002A-R7e, 002A-R8a to 002A-R8d, 002A-R9a to 002A-R9c, 002A-R10a to 002A-R10b, and 002A-R11). Buffer was used as a negative control. Figure 8 The lower left panel of B shows Figure 8 Quantification (exclusion %) of exon 45 skipping in DMD in RD cells transfected with the same ASOs (25 nM) (002A, 002A-L7a to 002A-L7e, 002A-L8a to 002A-L8d, 002A-L9a to 002A-L9c, 002A-L10a to 002A-L10b, and 002A-L11) tested in the upper left panel of B, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01 (all relative to 002A)). Buffer was used as a negative control. Figure 8 The lower right panel of B shows Figure 8 Quantification (exclusion %) of exon 45 skipping in DMD in RD cells transfected with the same ASOs (25 nM) (002A, 002A-R7a to 002A-R7e, 002A-R8a to 002A-R8d, 002A-R9a to 002A-R9c, 002A-R10a to 002A-R10b, and 002A-R11) tested in the upper right panel of B, presented as mean ± standard deviation (n = 3, *P < 0.05, all relative to 002A). Buffer was used as a negative control.

[0035] Figure 9 A to Figure 9 B depicts the effect of the presence, position, length, and / or sequence of various decoy sequences (7a to 7e, 8a to 8d, 9a to 9c, 10a to 10b, and 11) linked to the 5'-end or 3'-end of the targeting sequence 014B on exon 17 skipping in the APP gene in HEK293 cells. Figure 9The left panel of A shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 17 skipping in the APP gene in HEK293 cells transfected with ASO (50 nM), which has a single targeting sequence 014B (014B) or a targeting sequence 014B with different lengths of decoy sequences linked to the 5'-end (014B-L7a to 014B-L7e, 014B-L8a to 014B-L8d, 014B-L9a to 014B-L9c, 014B-L10a to 014B-L10b, and 014B-L11). Buffer was used as a negative control. Figure 9 The right panel of A shows Figure 9 Quantification (exclusion %) of exon 17 skipping in the APP gene in HEK293 cells transfected with the same ASOs (50 nM) (014B, 014B-L7a to 014B-L7e, 014B-L8a to 014B-L8d, 014B-L9a to 014B-L9c, 014B-L10a to 014B-L10b, and 014B-L11) tested in the left panel of A, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 (all relative to 014B)). Buffer was used as a negative control. Figure 9 The left panel of B shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 17 skipping in the APP gene in HEK293 cells transfected with ASO (50 nM), which has a single targeting sequence 014B (014B) or a targeting sequence 014B with different lengths of decoy sequences linked to the 3'-end (014B-R7a to 014B-R7e, 014B-R8a to 014B-R8d, 014B-R9a to 014B-R9c, 014B-R10a to 014B-R10b, and 014B-R11). Buffer was used as a negative control. Figure 9 The right panel of B shows Figure 9 Quantification (exclusion %) of exon 17 skipping in the APP gene in HEK293 cells transfected with the same ASOs (50 nM) (014B, 014B-R7a to 014B-R7e, 014B-R8a to 014B-R8d, 014B-R9a to 014B-R9c, 014B-R10a to 014B-R10b, and 014B-R11) tested in the left panel of B, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 (all relative to 014B)). Buffer was used as a negative control.

[0036] Figure 10 A to Figure 10Panel B shows the effect of the presence, position, length, and / or sequence of various decoy sequences (7a to 7e, 8a to 8d, 9a to 9c, 10a to 10b, and 11) linked to the 5'- or 3'-end of targeting sequence 017B on exon 41 skipping in the CEP290 gene in HEK293 cells. Figure 10 The left panel of A shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 41 skipping in the CEP290 gene in HEK293 cells transfected with ASO (50 nM) that has the targeting sequence 017B alone (017B) or the targeting sequence 017B with decoy sequences of different lengths linked to the 5'-end (017B-L7a to 017B-L7e, 017B-L8a to 017B-L8d, 017B-L9a to 017B-L9c, 017B-L10a to 017B-L10b, and 017B-L11). Buffer was used as a negative control. Figure 10 The right panel of A shows Figure 10 Quantification (exclusion %) of exon 41 skipping in the CEP290 gene in HEK293 cells transfected with the same ASO (50 nM) tested in the left panel of A (017B, 017B-L7a to 017B-L7e, 017B-L8a to 017B-L8d, 017B-L9a to 017B-L9c, 017B-L10a to 017B-L10b, and 017B-L11), presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 (all relative to 017B)). Buffer was used as a negative control. Figure 10 The left panel of B shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 41 skipping in the CEP290 gene in HEK293 cells transfected with ASO (50 nM) that has the targeting sequence 017B alone (017B) or the targeting sequence 017B with decoy sequences of different lengths linked to the 3'-end (017B-R7a to 017B-R7e, 017B-R8a to 017B-R8d, 017B-R9a to 017B-R9c, 017B-R10a to 017B-R10b, and 017B-R11). Buffer was used as a negative control. Figure 10 The right panel of B shows Figure 10Quantification (exclusion %) of exon 41 skipping in the CEP290 gene in HEK293 cells transfected with the same ASOs (50 nM) (017B, 017B-R7a to 017B-R7e, 017B-R8a to 017B-R8d, 017B-R9a to 017B-R9c, 017B-R10a to 017B-R10b, and 017B-R11) as tested in the left panel of B, presented as mean ± standard deviation (n = 3, **P < 0.01, ***P < 0.001 (all relative to 017B)). Buffer was used as a negative control.

[0037] Figure 11 A to Figure 11 A to B show the effect of the presence, position, length, and / or sequence of various decoy sequences (7a to 7e, 8a to 8d, 9a to 9c, 10a to 10b, and 11) linked to the 5'-end or 3'-end of the targeting sequence 024B on exon 19 skipping in the HER2 gene (also known as ERBB2) in HeLa cells. Figure 11 The left panel of A shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 19 skipping in the HER2 gene in HeLa cells transfected with ASOs (50 nM) that have the individual targeting sequence 024B (024B) or the targeting sequence 024B with decoy sequences of different lengths linked to the 5'-end (024B-L7a to 024B-L7e, 024B-L8a to 024B-L8d, 024B-L9a to 024B-L9c, 024B-L10a to 024B-L10b, and 024B-L11). Buffer was used as a negative control. Figure 11 The right panel of A shows in Figure 11 Quantification (exclusion %) of exon 19 skipping in the HER2 gene in HeLa cells transfected with the same ASOs (50 nM) (024B, 024B-L7a to 024B-L7e, 024B-L8a to 024B-L8d, 024B-L9a to 024B-L9c, 024B-L10a to 024B-L10b, and 024B-L11) as tested in the left panel of A, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 (all relative to 024B)). Buffer was used as a negative control. Figure 11The left panel of B shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 19 skipping in the HER2 gene in HeLa cells transfected with ASO (50 nM), which has a single targeting sequence 024B (024B) or a targeting sequence 024B with different lengths of decoy sequences linked to the 3'-end (024B-R7a to 024B-R7e, 024B-R8a to 024B-R8d, 024B-R9a to 024B-R9c, 024B-R10a to 024B-R10b, and 024B-R11). Buffer was used as a negative control. Figure 11 The right panel of B shows Figure 11 the quantification (exclusion %) of exon 19 skipping in the HER2 gene in HeLa cells transfected with the same ASOs (50 nM) (024B, 024B-R7a to 024B-R7e, 024B-R8a to 024B-R8d, 024B-R9a to 024B-R9c, 024B-R10a to 024B-R10b, and 024B-R11) tested in the left panel of B, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01 (all relative to 024B)). Buffer was used as a negative control.

[0038] Figure 12 A to Figure 12 B illustrate the effect of the presence, position, length, and / or sequence of various decoy sequences (7a to 7e, 8a to 8d, 9a to 9c, 10a to 10b, and 11) linked to the 5'-end or 3'-end of the targeting sequence 015C on exon 10 skipping in the ATXN3 gene (also known as SCA3) in A549 cells. Figure 12 The left panel of A shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 10 skipping in the ATXN3 gene in A549 cells transfected with ASO (40 nM), which has a single targeting sequence 015C (015C) or a targeting sequence 015C with different lengths of decoy sequences linked to the 5'-end (015C-L7a to 015C-L7e, 015C-L8a to 015C-L8d, 015C-L9a to 015C-L9c, 015C-L10a to 015C-L10b, and 015C-L11). Buffer was used as a negative control. Figure 12 The right panel of A shows Figure 12Quantification (exclusion %) of exon 10 skipping in the ATXN3 gene in A549 cells transfected with the same ASOs (40 nM) (015C, 015C-L7a to 015C-L7e, 015C-L8a to 015C-L8d, 015C-L9a to 015C-L9c, 015C-L10a to 015C-L10b, and 015C-L11) as tested in the left panel of A, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 (all relative to 015C)). Buffer was used as a negative control. Figure 12 The left panel of B shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 10 skipping in the ATXN3 gene in A549 cells transfected with ASOs (40 nM) having the individual targeting sequence 015C (015C) or the targeting sequence 015C with different lengths of decoy sequences attached to the 3′ end (015C-R7a to 015C-R7e, 015C-R8a to 015C-R8d, 015C-R9a to 015C-R9c, 015C-R10a to 015C-R10b, and 015C-R11). Buffer was used as a negative control. Figure 12 The right panel of B shows Figure 12 Quantification (exclusion %) of exon 10 skipping in the ATXN3 gene in A549 cells transfected with the same ASOs (40 nM) (015C, 015C-R7a to 015C-R7e, 015C-R8a to 015C-R8d, 015C-R9a to 015C-R9c, 015C-R10a to 015C-R10b, and 015C-R11) as tested in the left panel of B, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 (all relative to 015C)). Buffer was used as a negative control.

[0039] Figure 13 A to Figure 13 B depicts the effect of the presence, location, length, and / or sequence of various decoy sequences (7a to 7e, 8a to 8d, 9a to 9c, 10a to 10b, and 11) attached to the 5′ or 3′ end of the targeting sequence 027B on exon 10 skipping in the PKM gene in RD cells. Figure 13The left panel of A shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 10 skipping in the PKM gene in RD cells transfected with ASO (50 nM), where the ASO has a single targeting sequence 027B (027B) or targeting sequence 027B with different lengths of decoy sequences linked to the 5'-end (027B-L7a to 027B-L7e, 027B-L8a to 027B-L8d, 027B-L9a to 027B-L9c, 027B-L10a to 027B-L10b, and 027B-L11). Buffer was used as a negative control. Figure 13 The right panel of A shows Figure 13 Quantification (exclusion %) of exon 10 skipping in the PKM gene in RD cells transfected with the same ASO (50 nM) (027B, 027B-L7a to 027B-L7e, 027B-L8a to 027B-L8d, 027B-L9a to 027B-L9c, 027B-L10a to 027B-L10b, and 027B-L11) as tested in the left panel of A, presented as mean ± standard deviation (n = 3, **P < 0.01, ***P < 0.001 (all relative to 027B)). Buffer was used as a negative control. Figure 13 The left panel of B shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 10 skipping in the PKM gene in RD cells transfected with ASO (50 nM), where the ASO has a single targeting sequence 027B (027B) or targeting sequence 027B with different lengths of decoy sequences linked to the 3'-end (027B-R7a to 027B-R7e, 027B-R8a to 027B-R8d, 027B-R9a to 027B-R9c, 027B-R10a to 027B-R10b, and 027B-R11). Buffer was used as a negative control. Figure 13 The right panel of B shows Figure 13 Quantification (exclusion %) of exon 10 skipping in the PKM gene in RD cells transfected with the same ASO (50 nM) (027B, 027B-R7a to 027B-R7e, 027B-R8a to 027B-R8d, 027B-R9a to 027B-R9c, 027B-R10a to 027B-R10b, and 027B-R11) as tested in the left panel of B, presented as mean ± standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001 (all relative to 027B)). Buffer was used as a negative control.

[0040] Figure 14 A to Figure 14Panel B shows the effect of the presence, position, length, and / or sequence of various decoy sequences (7a to 7e, 8a to 8d, 9a to 9c, 10a to 10b, and 11) linked to the 5'-end or 3'-end of the targeting sequence 029B on exon 6 skipping in the MDM4 gene in HEK293 cells. Figure 14 The left panel of A shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 6 skipping in the MDM4 gene in HEK293 cells transfected with ASO (50 nM) that has the targeting sequence 029B alone (029B) or the targeting sequence 029B with different lengths of decoy sequences linked to the 5'-end (029B-L7a to 029B-L7e, 029B-L8a to 029B-L8d, 029B-L9a to 029B-L9c, 029B-L10a to 029B-L10b, and 029B-L11). Buffer was used as a negative control. Figure 14 The right panel of A shows Figure 14 The quantification (exclusion %) of exon 6 skipping in the MDM4 gene in HEK293 cells transfected with the same ASO (50 nM) (029B, 029B-L7a to 029B-L7e, 029B-L8a to 029B-L8d, 029B-L9a to 029B-L9c, 029B-L10a to 029B-L10b, and 029B-L11) tested in the left panel of A, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 (all relative to 029B)). Buffer was used as a negative control. Figure 14 The left panel of B shows a representative semi-quantitative fluorescent RT-PCR analysis of exon 6 skipping in the MDM4 gene in HEK293 cells transfected with ASO (50 nM) that has the targeting sequence 029B alone (029B) or the targeting sequence 029B with different lengths of decoy sequences linked to the 3'-end (029B-R7a to 029B-R7e, 029B-R8a to 029B-R8d, 029B-R9a to 029B-R9c, 029B-R10a to 029B-R10b, and 029B-R11). Buffer was used as a negative control. Figure 14 The right panel of B shows Figure 14Quantification (exclusion %) of exon 6 skipping in the MDM4 gene in HEK293 cells transfected with the same ASOs (50 nM) (029B, 029B-R7a to 029B-R7e, 029B-R8a to 029B-R8d, 029B-R9a to 029B-R9c, 029B-R10a to 029B-R10b, and 029B-R11) as tested in the left panel for B, presented as mean ± standard deviation (n = 3, *P < 0.05, **P < 0.01 (all relative to 029B)). Buffer was used as a negative control. Detailed Description

[0041] Exon skipping is a potential treatment for a subject having a disease and / or its complication, who may benefit from manipulating the expression of a target gene by ASO-induced exon skipping. ASOs can be used to generate or promote exon skipping, but screening for ASOs that provide the desired efficiency and efficacy for exon skipping can be challenging. The expression of a target gene can be manipulated by ASO inhibition of splicing of an exon, intron, or specific splicing site of the target gene, resulting in (e.g., but not limited to): restoring the reading frame of a target defective gene, generating different subtypes of the target gene (e.g., dominant negative subtypes), skipping a toxic portion of a gene, silencing a gene, and / or altering the structure and function of a gene. Manipulating the expression of a target gene may be beneficial for treating a disease and / or its complication, although the target gene may or may not be the cause of the disease. The disease can be a genetic or non-genetic disease disclosed herein (e.g., some non-genetic cancers, metabolic diseases, or infectious diseases). A genetic disease may or may not be associated with a splicing defect.

[0042] Most human genes contain introns, which must be spliced out in the nucleus from nascent transcripts to produce mature mRNA, and then exported to the cytoplasm for protein translation by ribosomes. However, genes are often alternatively spliced, which not only contributes to the expansion of transcript diversity but also serves as a mechanism for regulating gene function. A common alternative splicing pattern is exon skipping. Although exon skipping is a natural phenomenon frequently observed during gene expression, it can be used as a method to manipulate gene expression for therapeutic purposes. Depending on the skipped exon, the results can vary significantly. When the skipped exon is symmetric, i.e., the length of the exon is divisible by 3, the internally truncated protein isoform only loses the amino acids encoded by the exon. If the skipped exon contains important parts, such as protein localization signals, motifs related to protein stability, or enzyme activity domains, the truncated protein isoform may change its localization or stability, lose its function, or become a protein with a dominant negative effect or a different function. If the skipped exon is a toxic exon, either natural or generated by mutation, exon skipping serves to restore the function of the gene. When the skipped exon is asymmetric, i.e., the length of the exon is not divisible by 3, the reading frame of the downstream exon will be disrupted, which is likely to generate a premature termination codon (PTC) in the next exon, usually triggering nonsense-mediated mRNA decay (NMD). For mRNA species whose reading frame is disrupted but escape NMD, C-terminal truncated protein isoforms will be produced, which usually results in the loss of their function. In summary, exon skipping can generate mRNA isoforms or proteins with similar, opposite, or different functions, or loss of function. Aartsma-Rus A, van Ommen GJ. Antisense-mediated exon skipping: a versatile tool with therapeutic and research applications. RNA 2007 Oct;13(10):1609-24. Doi: 10.1261 / rna.653607.

[0043] Exon skipping methods have a wide range of applications. First, it can be used to skip the toxic parts of abnormal genes. An example of a toxic exon is exon 10 of the ATXN3 gene in patients with SCA3, who carry an abnormal amplification of the CAG repeat sequence in the exon. Second, exon skipping can be used to restore the reading frame disrupted by frameshift mutations observed in a variety of genetic diseases. One such disease is called Duchenne muscular dystrophy. In addition, exon skipping can be used to disrupt the expression of harmful gene products, such as oncoproteins, viral proteins, or neurodegeneration-related peptides. Finally, exon skipping can be used to generate beneficial protein subtypes. For example, skipping of PDCD1 exon 3 produces a PD1 subtype that has the potential to treat cancer by acting as a PDL1 / 2 antibody. Toonen LJA, et al. Antisense Oligonucleotide-Mediated Removal of the Polyglutamine Repeat in Spinocerebellar Ataxia Type 3 Mice. Mol Ther Nucleic Acids. September 2017;8:232-242. doi:10.1016 / j.omtn.2017.06.019. Lucía Echevarría L, et al. Exon-skipping advances for Duchenne muscular dystrophy. Human Molecular Genetics. August 2018;27(R2):R163–R172. doi.org / 10.1093 / hmg / ddy171. Chang JL, et al. Targeting Amyloid-β Precursor Protein, APP, Splicing with Antisense Oligonucleotides Reduces Toxic Amyloid-β Production. Mol Ther. June 2018;26(6):1539-1551. doi:10.1016 / j.ymthe.2018.02.029. Sun J, et al. Modulation of PDCD1 exon 3 splicing. RNA Biol. December 2019;16(12):1794-1805. Doi:10.1080 / 15476286.2019.1659080。

[0044] As disclosed herein, without being bound by any particular theory, it has been found that compared to an ASO that contains a targeting sequence but does not contain any of the bait sequences disclosed herein, an ASO that contains or consists of a bait sequence (such as, but not limited to, the nucleotide sequences of SEQ ID No. 1 to 23 and SEQ ID No. 347 to 353) linked to a targeting sequence may be more effective and / or efficient in generating or promoting exon skipping. Without being bound by any particular theory, an ASO that contains the bait sequences described herein surprisingly and unexpectedly improves the exon skipping efficiency of the targeting sequence. In some embodiments, when the bait sequence is combined with a targeting sequence that is relatively inefficient in promoting exon skipping, the resulting ASO becomes efficient in modulating exon skipping. Alternatively, compared to an ASO that contains a targeting sequence but does not contain any of the bait sequences disclosed herein, an ASO that contains or consists of a bait sequence (such as the nucleotide sequences of SEQ ID No. 1 to 23 and SEQ ID No. 347 to 353) linked to a targeting sequence can achieve comparable efficacy and / or efficiency in generating or promoting exon skipping at a lower dose.

[0045] In certain embodiments, the exon skipping effect and / or efficiency can be quantified by the percentage of exclusion of the target exon in the total transcript of each gene (exclusion %). In certain embodiments, the exclusion % of an ASO that contains a bait sequence linked to a targeting sequence can be about 2.18-fold, about 2.3-fold, about 4-fold, more than about 10-fold, more than about 14-fold, more than about 15-fold, more than about 16-fold, or more than about 17-fold that of an ASO that consists of the targeting sequence. In certain embodiments, an ASO that contains a bait sequence linked to a targeting sequence can achieve an exclusion % comparable (such as at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%) to that of an ASO that consists of the targeting sequence when the dose does not exceed about 25% or does not exceed about 50% of the dose of the ASO that consists of the targeting sequence.

[0046] In certain embodiments, optimal ASOs comprise a decoy sequence (e.g., but not limited to, the nucleotide sequences of SEQ ID Nos. 1 to 23 and SEQ ID Nos. 347 to 353) linked to the 5'-end of the targeting sequence. In certain embodiments, optimal ASOs comprise a decoy sequence (e.g., but not limited to, the nucleotide sequences of SEQ ID Nos. 1 to 23 and SEQ ID Nos. 347 to 353) linked to the 3'-end of the targeting sequence. In certain embodiments, ASOs comprise a decoy sequence (e.g., but not limited to, the nucleotide sequences of SEQ ID Nos. 1 to 23 and SEQ ID Nos. 347 to 353) linked to both the 3'-end and 5'-end of the targeting sequence. In certain embodiments, the decoy sequence and the targeting sequence are linked without a linker.

[0047] Accordingly, in certain embodiments, provided herein are methods for generating or promoting exon skipping of a target exon during pre-mRNA splicing, which comprise contacting a pre-mRNA in a cell or a subject with an ASO (e.g., a bipartite ASO) disclosed herein, a composition (e.g., a pharmaceutical composition) comprising an ASO (e.g., a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (e.g., a bipartite ASO) disclosed herein. ASO-mediated exon skipping may be a method of manipulating the expression of a target gene. The expression of a target gene can be manipulated by inhibiting the splicing of an exon, an intron, or a specific splicing site of the target gene by an ASO, resulting in (e.g., but not limited to): restoring the reading frame of a target defective gene, generating different isoforms (e.g., dominant negative isoforms) of the target gene, skipping a toxic portion of a gene, silencing a gene, and / or altering the structure and function of a gene. In some embodiments, the method further comprises delivering an ASO (e.g., a bipartite ASO) disclosed herein to a cell or administering an ASO (e.g., a bipartite ASO) disclosed herein to a subject.

[0048] In certain embodiments, provided herein is also a method of improving exon skipping efficacy and / or efficiency of a target sequence, which comprises obtaining one or more ASOs (e.g., bipartite ASOs), the one or more ASOs comprising a target sequence and a decoy sequence (e.g., but not limited to the nucleotide sequences of SEQ ID No. 1 to 23 and SEQ ID No. 347 to 353) operably linked to the 5'-end and / or 3'-end of the target sequence. In certain embodiments, the method further comprises screening and / or optimizing the one or more ASOs (e.g., bipartite ASOs) according to the exon skipping efficacy and / or efficiency of their target exons. The exon skipping effect and / or efficiency can be quantified by the percentage of exclusion of the target exon in the total transcripts of each gene (exclusion %). The target sequence may be capable of hybridizing to a sequence selected from the group consisting of: the target exon, the flanking intron sequence upstream of the target exon, the flanking intron sequence downstream of the target exon, the intron-exon junction upstream of the target exon, and the intron-exon junction downstream of the target exon in a cell or a subject.

[0049] In certain embodiments, provided herein is also a method of treating a disease and / or its complication in a subject, which comprises administering to the subject the ASOs (e.g., bipartite ASOs) disclosed herein, a pharmaceutical composition comprising the ASOs (e.g., bipartite ASOs) disclosed herein, and / or a vector encoding the ASOs (e.g., bipartite ASOs) disclosed herein.

[0050] The present disclosure also provides novel ASOs, each of which comprises a decoy sequence linked to a targeting sequence. In certain embodiments, the decoy sequence is selected from the group consisting of the nucleotide sequences of SEQ ID No. 1 to 23 and SEQ ID No. 347 to 353. In certain embodiments, the ASO is capable of mediating exon skipping of one or more exons of a gene such as the DMD gene (NCBI Gene ID: 1756), the SMN1 gene (NCBI Gene ID: 6606), the SMN2 gene (NCBI Gene ID: 6607), the APP gene (NCBI Gene ID: 351), the CEP290 gene (NCBI Gene ID: 80184), the HER2 gene (NCBI Gene ID: 2064), the ATXN3 gene (NCBI Gene ID: 4287), the PKM gene (NCBI Gene ID: 5315), and the MDM4 gene (NCBI Gene ID: 4194). This process is referred to as exon skipping. In certain embodiments, the targeting sequence can target an exon, an intron, or a junction. In certain embodiments, the target exon is selected from the group consisting of exon 7 of the endogenous SMN1 and SMN2 genes, exon 51 of the endogenous DMD gene, exon 53 of the endogenous DMD gene, exon 45 of the endogenous DMD gene, exon 17 of the endogenous APP gene, exon 41 of the endogenous CEP290 gene, exon 19 of the endogenous HER2 gene, exon 10 of the ATXN3 gene, exon 10 of the endogenous PKM gene, and exon 6 of the endogenous MDM4 gene. In certain embodiments, the targeting sequence comprises or consists of a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 25, 29, 33, 37, 41, 45, 49, 75, 92, 124, 156, 188, 220, 252, 284, and 316. In certain embodiments, the ASO comprises or consists of a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 26 to 27, 30 to 31, 34 to 35, 38 to 39, 42 to 43, 46 to 47, 50 to 73, 76 to 90, 93 to 122, 125 to 154, 157 to 186, 189 to 218, 221 to 250, 253 to 282, 285 to 314, 317 to 346, and 354 to 356. In certain embodiments, the targeting sequence targets a targeted sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 24, 28, 32, 36, 40, 44, 48, 74, 91, 123, 155, 187, 219, 251, 283, and 315.

[0051] In some embodiments, an ASO or a composition comprising an ASO (e.g., a pharmaceutical composition) is suitable for treating diseases and / or their complications that can be treated by exon skipping of exon 45, 51, or 53 of the DMD gene. The ASO comprises or consists of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID No. 38 to 39, 42 to 43, 46 to 47, 50 to 73, 76 to 90, 93 to 122, and 125 to 154. In certain embodiments, the disease is Duchenne muscular dystrophy (DMD). DMD is caused by a frameshift mutation of the DMD gene, and the exon skipping strategy using the ASO or the composition comprising an ASO disclosed herein can be used to restore the reading frame and thus treat DMD.

[0052] In some embodiments, an ASO or a composition comprising an ASO (e.g., a pharmaceutical composition) is suitable for treating diseases and / or their complications that can be treated by exon skipping of exon 7 of the SMN1 and / or SMN2 gene. The ASO comprises or consists of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID No. 26 to 27, 30 to 31, 34 to 35, and 354 to 356.

[0053] In some embodiments, an ASO or a composition comprising an ASO (e.g., a pharmaceutical composition) is suitable for treating diseases and / or their complications that can be treated by exon skipping of exon 17 of the APP gene. The ASO comprises or consists of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID No. 157 to 186.

[0054] In some embodiments, an ASO or a composition comprising an ASO (e.g., a pharmaceutical composition) is suitable for treating diseases and / or their complications that can be treated by exon skipping of exon 41 of the CEP290 gene. The ASO comprises or consists of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID No. 189 to 218.

[0055] In some embodiments, an ASO or a composition comprising an ASO (e.g., a pharmaceutical composition) is suitable for treating diseases and / or their complications that can be treated by exon skipping of exon 19 of the HER2 gene. The ASO comprises or consists of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID No. 221 to 250.

[0056] In some embodiments, an ASO or a composition comprising an ASO (e.g., a pharmaceutical composition) is suitable for treating diseases and / or their complications that can be treated by exon skipping of exon 10 of the ATXN3 gene. The ASO comprises or consists of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID No. 253 to 282.

[0057] In some embodiments, an ASO or a composition comprising an ASO (e.g., a pharmaceutical composition) is suitable for treating a disease and / or its complications that can be treated by exon 10 skipping of the PKM gene. The ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 285 to 314 or consists of the same.

[0058] In some embodiments, an ASO or a composition comprising an ASO (e.g., a pharmaceutical composition) is suitable for treating a disease and / or its complications that can be treated by exon 6 skipping of the MDM4 gene. The ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 317 to 346 or consists of the same.

[0059] The following description is illustrative only in nature and is not intended to limit the technology of the present invention, its application, or its use. It should be understood that throughout the drawings, corresponding reference numerals indicate the same or corresponding parts and features. The description of specific examples indicated in various embodiments of the technology of the present invention is for illustrative purposes only and is not intended to limit the scope of the technology of the present invention disclosed herein. In addition, the reference to multiple embodiments having the stated features is not intended to exclude other embodiments having additional features or other embodiments with different combinations of the stated features.

[0060] Furthermore, the detailed description of the various embodiments herein refers to the accompanying drawings / figures, which illustrate the various embodiments in a pictorial manner. Although the embodiments are described in sufficient detail to enable those skilled in the art to practice the technology of the present invention, it should be understood that other embodiments may be achieved and logical and mechanical changes may be made without departing from the spirit and scope of the technology of the present invention. Therefore, the detailed description herein is presented for illustrative purposes only and not restrictively. For example, the steps or functions listed in the description, any method, system, or process may be performed in any order and are not limited to the order presented. In addition, any of its steps or functions may be outsourced to or performed by one or more third parties.

[0061] Furthermore, any reference to the singular includes multiple embodiments, and any reference to more than one component may include a singular embodiment.

[0062] Definitions

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the technology of the present invention, the following terms are defined as follows.

[0064] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, "an element" means one element or more than one element.

[0065] The term "about" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies at an acceptable level in this technology. In some embodiments, such variation can be up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. When the term "about" is used in conjunction with a numerical range, it adjusts the range by extending the boundaries of the recited numerical values upward or downward.

[0066] The term "administering" or "administer" includes delivering a therapeutic agent comprising an ASO of the technology of the present invention to a subject by local or systemic administration. Administration can be local (including ophthalmic and mucosal, including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer), intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration.

[0067] The term "isolated" refers to a material that is substantially or essentially free of components that are normally associated with it in its natural state. For example, an "isolated oligonucleotide" or "isolated oligomer" as used herein can refer to an oligomer that has been purified or removed from the sequences that flank it in its natural state, such as a DNA fragment removed from the sequences adjacent to the fragment in the genome.

[0068] When referring to cells, the term "isolated" can refer to purifying cells (e.g., fibroblasts, lymphocytes) from a source subject (e.g., a subject with an oligonucleotide repeat disease). In the case of mRNA or protein, "isolated" can refer to recovering mRNA or protein from a source (e.g., a cell).

[0069] The term "functional" with respect to a protein includes the corresponding wild-type protein, as well as truncated forms of the wild-type protein, such forms being derived from an mRNA transcript containing a sequence corresponding to a truncated form of the transcript that results from deleting one or more exons while having sufficient biological activity to reduce the adverse effects of a defective protein in a subject having a disease and / or its complications. As measured by conventional techniques in the art, a functional protein can have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers therebetween) of the in vitro or in vivo biological activity of the corresponding wild-type protein. A truncated protein having less than the full biological activity of the wild-type protein is sometimes referred to as a "semi-functional" protein. Animal models are also valuable resources for studying disease pathogenesis and provide a means for testing disease-related activities, such that in some instances, less than 100% biological activity may be sufficient to treat a disease.

[0070] The term "functional" with respect to dystrophin includes proteins derived from mRNA transcripts containing sequences corresponding to all exons 1 through 79 of the dystrophin gene, also known as wild-type proteins. It also includes truncated forms of dystrophin derived from mRNA transcripts containing sequences corresponding to truncated forms of the transcript (e.g., dystrophin mRNA transcripts having fewer than all of the exons 1 through 79 of the dystrophin gene), such as forms generated by some of the ASOs of the technology of the present invention. In other words, truncated forms of dystrophin mRNA may not include one or more exons of the corresponding dystrophin gene. Truncated forms of dystrophin mRNA may express truncated or shortened forms of dystrophin, also known as microdystrophin protein. Functional dystrophin generally refers to dystrophin that typically has sufficient biological activity to reduce the progressive degradation of muscle tissue (otherwise characteristic of Duchenne muscular dystrophy) compared to the altered or "defective" forms of dystrophin present in some subjects with DMD or related conditions. As measured by conventional techniques in the art, functional dystrophin may have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers therebetween) of the in vitro or in vivo biological activity of wild-type dystrophin. Dystrophin having less than the full biological activity of wild-type dystrophin is sometimes referred to as "semi-functional" dystrophin. As an example, dystrophin-related activity in in vitro muscle cultures can be measured according to myotube size, myofibrillar organization (or degree of disorganization), contractile activity, and spontaneous aggregation of acetylcholine receptors (see, e.g., Susan C. Brown et al. Dystrophic phenotype induced in vitro by antibody blockade of muscle α-dystroglycan-laminin interaction. January 1999. Journal of Cell Science. 112:209-216. doi:10.1242 / jcs.112.2.209). Animal models are also valuable resources for studying disease pathogenesis and provide means for testing dystrophin-related activity.Two animal models widely used in DMD research are the mdx mouse and the golden retriever muscular dystrophy (GRMD) dog, both of which are dystrophin-negative (see, e.g., C.A. Collins and J.E. Morgan. Duchenne's muscular dystrophy: animal models used to investigate pathogenesis and develop therapeutic strategies. August 2003. Int J Exp Pathol. 84:165-172. doi:10.1046 / j.1365-2613.2003.00354.x). These and other animal models can be used to measure the functional activity of various dystrophins.

[0071] The terms "DMD gene" and "dystrophin gene" are used interchangeably herein and refer to the gene encoding dystrophin. Similarly, the terms "DMD protein" and "dystrophin protein" and "dystrophin" are used interchangeably herein and refer to the translated protein product of the DMD gene.

[0072] "Exon skipping" generally refers to the process by which an entire exon or a portion thereof is removed from a given precursor RNA and is thus excluded from the mature RNA (such as the mature mRNA that is translated into a protein). Thus, the portion of the protein that was originally encoded by the skipped exon is absent from the expressed form of the protein, often resulting in an altered but still functional form of the protein. In some embodiments, the skipped exon is an abnormal exon from the human dystrophin gene, which may contain mutations or other alterations in its sequence that otherwise result in abnormal splicing. The terms "precursor mRNA (pre-mRNA)" and "pre-mRNA (precursor mRNA)" are used interchangeably and refer to the unprocessed or partially processed pre-mRNA synthesized by transcription from a DNA template in the nucleus.

[0073] In the context of the technology of the present invention, inducing and / or promoting exon skipping (or inducing or promoting exon skipping) as indicated herein means that at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the mRNA in one or more cells of a patient will be devoid of that exon. The level of exon skipping can be evaluated by PCR as described in the examples.

[0074] The terms "complementary" and "complementarity" generally refer to the interaction of two nucleotide sequences that hybridize to form a double-stranded molecule. In some embodiments, the terms "complementary" and "complementarity" refer to polynucleotides (i.e., nucleotide sequences) related by base-pairing rules. For example, the sequence "T-G-A-C (5'-3')" is complementary to the sequence "G-T-C-A (5'-3')". Complementarity can be "partial", where only some of the nucleic acid bases match the reference or target sequence (such as a target region) according to the base-pairing rules. Alternatively, there can be "complete" or "exact" or "total" or "full" complementarity between nucleic acids, which has the same meaning as "fully complementary" or "completely complementary", where all specified nucleic acid bases match according to the base-pairing rules. The degree of complementarity between nucleic acid strands has a significant effect on the hybridization efficiency and strength between the nucleic acid strands. Although complete complementarity is generally required, some embodiments can include one or more mismatches relative to the target region. Variations at any position included within the oligomer.

[0075] The term "substantially complementary" refers to a polynucleotide having at least 70% partial complementarity; i.e., at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the nucleic acid bases match according to the base-pairing rules. The mismatch can be one or more base substitutions (mutations), or one or more base deletions, or one or more base insertions.

[0076] The terms "antisense oligonucleotide", "antisense oligomer", "antisense compound", and "ASO" are used interchangeably and refer to an oligonucleotide comprising a targeting oligonucleotide sequence capable of hybridizing in a complementary manner to a target sequence (such as a target region) in a nucleic acid (RNA or DNA, typically RNA). In some embodiments, the ASO comprises a decoy sequence and a targeting sequence. In some embodiments, the targeting sequence of the ASO is fully or substantially complementary to the target region. In some embodiments, the target region is a precursor RNA sequence that is fully or partially complementary to the targeting sequence. The target region can be located in the target exon, the flanking intron sequence upstream of the target exon, the flanking intron sequence downstream of the target exon, the intron-exon junction upstream of the target exon, and the intron-exon junction downstream of the target exon. In some embodiments, the decoy sequence mimics the optimal 5' splice site, and thus it acts as a 5' splice site decoy to interfere with the recognition of the adjacent authentic 5' splice site by U1snRNA. In some embodiments, the ASO comprises one or more DNA nucleotides, one or more RNA nucleotides, and mixtures thereof. Thus, although the sequences disclosed herein are presented in DNA form, the sequences also include the corresponding RNA forms. In certain embodiments, one or more of the one or more DNA nucleotides and / or RNA nucleotides can be modified nucleotides. In some embodiments, the ASO further comprises one or more additional chemical moieties. In some embodiments, the additional chemical moiety is covalently linked to the 5' end and / or the 3' end of the decoy-targeting sequence moiety. In some embodiments, the additional chemical moiety is a cell-penetrating peptide.

[0077] The term "modified nucleotide" includes any chemical moiety that is structurally different from a natural nucleotide but capable of performing at least one function of a natural nucleotide. In some embodiments, the modified nucleotide comprises a modification at the sugar, base, and / or internucleotide linkage. In some embodiments, the modified nucleotide comprises a modified sugar, a modified nucleobase, and / or a modified internucleotide linkage. In some embodiments, the modified nucleotide has at least one function of a nucleotide, such as forming a subunit in a polymer capable of base pairing with a nucleic acid comprising at least a complementary base sequence.

[0078] The term "moiety" refers to a particular segment or functional group of a molecule. A chemical moiety is a generally recognized chemical entity that is incorporated into or attached to a molecule. In some embodiments, a moiety of a compound is a monovalent, divalent, or polyvalent group formed from the compound by removing one or more -H and / or its equivalents from the compound. In some embodiments, depending on the context, "moiety" can also refer to the compound or entity from which the moiety is derived.

[0079] "Exon" refers to a defined segment of nucleic acid that encodes a protein, or a nucleic acid sequence that remains in the mature form of an RNA molecule after any portion of the preprocessed (or precursor) RNA has been removed by splicing. The mature RNA molecule can be messenger RNA (mRNA) or a functional form of non-coding RNA, such as rRNA or tRNA. The human dystrophin gene has approximately 79 exons.

[0080] "Intron" refers to a region of nucleic acid (within a gene) that is not translated into protein. Introns are non-coding segments that are transcribed into precursor RNA (pre-RNA) and are then removed by splicing during the formation of mature RNA.

[0081] The term "in vitro" refers to an event that occurs in an artificial environment (e.g., in a test tube or reaction vessel, in cell culture, etc.) rather than within a living organism (e.g., an animal, a plant, and / or a microorganism).

[0082] The term "in vivo" refers to an event that occurs within a living organism (e.g., an animal, a plant, or a microorganism).

[0083] The term "gene" is intended to refer to genomic genes and also includes cDNA, mRNA precursors (i.e., pre-mRNA), and mRNA.

[0084] The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases (including inorganic acids and inorganic bases as well as organic acids and organic bases). For example, for a compound containing a basic nitrogen, the salt can be prepared from a pharmaceutically acceptable non-toxic acid (including inorganic acids and organic acids). Pharmaceutically acceptable acid addition salts of the compounds applicable to the technology of the present invention include salts of the following: acetic acid, benzenesulfonic acid (benzenesulfonate), benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, and the like. When the compound contains an acidic side chain, pharmaceutically acceptable base addition salts of the compounds applicable to the technology of the present invention include metal salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts made from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine.

[0085] The term "pharmaceutical composition" means a composition comprising a compound described herein and at least one component selected from pharmaceutically acceptable carriers, diluents, adjuvants, excipients or vehicles, such as preservatives, fillers, disintegrants, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants and dispensing agents, depending on the mode of administration and the nature of the dosage form.

[0086] The term "pharmaceutically acceptable carrier" is used to mean any carrier, diluent, adjuvant, excipient or vehicle that is non-toxic and safe for human use as described herein. Examples of suspending agents include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and tragacanth, or mixtures of these substances. The action of microorganisms can be ensured against by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and the like. Isotonic agents, such as sugars, sodium chloride and the like, may also be required. Prolonged absorption of injectable pharmaceutical forms can be achieved by using delaying agents, such as aluminum monostearate and gelatin. Examples of suitable carriers, diluents, solvents or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Examples of excipients include lactose, milk sugar, sodium citrate, calcium carbonate and dibasic calcium phosphate. Examples of disintegrants include starch, alginic acid and some complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc and high molecular weight polyethylene glycols.

[0087] The term "pharmaceutically acceptable" means being applicable, within the scope of reasonable medical judgment, to contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response and the like and being commensurate with a reasonable benefit / risk ratio.

[0088] When used in reference to upstream or downstream sequences, the term "immediately" means a direct covalent bond connection between two groups with no intervening nucleotides.

[0089] The term "flanking" is used to refer to nucleotide sequences that are directly connected to each other with no intervening nucleotides. For example, the pentanucleotide 5'-AAAAA-3' flanks the trinucleotide 5'-TTT-3' when they are linked as follows: 5'-AAAAATTT-3' or 5'-TTTAAAAA-3', but they do not flank when linked as follows: 5'-AAAAACTTT-3'. In the latter case, the C nucleotide is said to be "inserted" between the pentanucleotide and the trinucleotide.

[0090] As used herein, the terms "therapeutically-effective amount", "therapeutically effective amount", and "effective amount" are interchangeable and refer to the amount of a therapy (e.g., an ASO or a pharmaceutical composition thereof provided herein) sufficient to reduce and / or ameliorate the severity and / or duration of a given disease and / or symptoms associated therewith. This term also encompasses the amount necessary to reduce or ameliorate the advancement or progression of a given disease, reduce or ameliorate the recurrence, development, or onset of a given disease, and / or modify or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than an ASO provided herein).

[0091] The terms "decoy" and "decoy sequence", "ASO decoy", "ASO decoy sequence", and "5'-splice site decoy" are used interchangeably herein to refer to a nucleotide sequence that is fully or partially complementary to the single-stranded 5'-end of U1 snRNA. In some embodiments, the decoy sequence mimics the optimal 5'-splice site and thus serves as a 5'-splice site decoy to interfere with the recognition of an adjacent authentic 5'-splice site by U1 snRNA.

[0092] Overview

[0093] In various embodiments, the present disclosure provides ASOs for treating diseases and / or their complications that can be treated by exon skipping in a target gene (such as, but not limited to, SMN1, SMN2, DMD, APP, CEP290, HER2, SCA3, PKM, and MDM4). In certain embodiments, the ASO (such as a bipartite ASO) comprises two components: 1) a "targeting" sequence that is fully or substantially complementary to a target region in the target gene, and 2) a "decoy" sequence that is located immediately upstream of the 5'-end and / or immediately downstream of the 3'-end of the targeting sequence. In some embodiments, the decoy sequence is located immediately upstream of the 5'-end of the targeting sequence. In certain embodiments, the decoy sequence is located immediately downstream of the 3'-end of the targeting sequence. In some embodiments, the decoy sequence has low sequence complementarity with the corresponding flanking sequence at the 5'-end or 3'-end of the target region in the adjacent target nucleic acid (such as mRNA), such as less than about 80%.

[0094] In some embodiments, the ASO (such as a bipartite ASO) further comprises a linker between the decoy sequence and the targeting sequence.

[0095] In some embodiments, the ASO of the technology of the present invention (e.g., bipartite ASO) can bind to the transcript (e.g., precursor mRNA) of a target gene (e.g., SMN1, SMN2, DMD, APP, CEP290, HER2, SCA3, PKM, and MDM4), and change the splicing pattern of the transcript by inducing the skipping of one or more exons. In some embodiments, the ASO of the technology of the present invention (e.g., bipartite ASO) causes the skipping of one or more exons in the mRNA, wherein the exon skipping percentage (excluding %) increases by about 10% or higher, about 20% or higher, about 30% or higher, about 40% or higher, about 50% or higher, about 60% or higher, about 70% or higher, about 80% or higher, and about 90% or higher. The exon skipping percentage can be measured using techniques known in the prior art and as described throughout this application, for example, as described in the following "Analysis" section.

[0096] The presence of the bait sequence in the bipartite ASO can increase the exon skipping percentage by the targeting sequence. In some embodiments, compared with an ASO having the same targeting sequence but without the bait sequence, the presence of the bait sequence in the bipartite ASO can increase the exon skipping efficiency of the targeting sequence by at least about 2-fold. In some embodiments, compared with an ASO having the same targeting sequence but without the bait sequence, the presence of the bait sequence in the bipartite ASO increases the exon skipping efficiency of the targeting sequence by about 2-fold or higher, about 2.18-fold or higher, about 2.3-fold or higher, about 3-fold or higher, about 4-fold or higher, about 5-fold or higher, about 6-fold or higher, about 7-fold or higher, about 8-fold or higher, about 9-fold or higher, about 10-fold or higher, about 11-fold or higher, about 12-fold or higher, about 13-fold or higher, about 14-fold or higher, about 15-fold or higher, about 16-fold or higher, about 17-fold or higher, about 18-fold or higher, about 19-fold or higher, about 20-fold or higher, about 25-fold or higher, about 30-fold or higher, about 40-fold or higher, about 50-fold or higher, about 60-fold or higher, about 70-fold or higher, about 80-fold or higher, about 90-fold or higher, about 100-fold or higher, or more, including all values and ranges between these values. In certain embodiments, compared with an ASO that contains the targeting sequence but does not contain any bait sequence disclosed herein, an ASO that contains or consists of a bait sequence (e.g., the nucleotide sequences of SEQ ID Nos. 1 to 23 and SEQ ID Nos. 347 to 353) linked to the targeting sequence can achieve comparable efficacy and / or efficiency in generating or promoting exon skipping at a lower dose.

[0097] In certain embodiments, provided herein are compositions comprising the ASO (e.g., bipartite ASO) disclosed herein and a pharmaceutically acceptable carrier. In certain embodiments, the composition is a pharmaceutical formulation or composition.

[0098] In certain embodiments, provided herein are vectors encoding ASOs (e.g., bipartite ASOs) disclosed herein.

[0099] In certain embodiments, provided herein are methods for generating or promoting exon skipping of a target exon during precursor mRNA splicing, comprising contacting a precursor mRNA in a cell or subject with an ASO (e.g., bipartite ASO) disclosed herein, a composition (e.g., a pharmaceutical composition) comprising an ASO (e.g., bipartite ASO) disclosed herein, and / or a vector encoding an ASO (e.g., bipartite ASO) disclosed herein. ASO-mediated exon skipping may be a method of manipulating the expression of a target gene. The expression of a target gene can be manipulated by the ASO inhibiting the splicing of an exon, intron, or specific splicing site of the target gene, resulting in (e.g., but not limited to): restoring the reading frame of a target defective gene, generating different isoforms of a target gene (e.g., dominant negative isoforms), skipping a toxic portion of a gene, silencing a gene, and / or altering the structure and function of a gene.

[0100] In certain embodiments, provided herein are methods for improving the efficacy and / or efficiency of exon skipping of a target sequence, comprising obtaining one or more ASOs (e.g., bipartite ASOs) that comprise a target sequence and a bait sequence disclosed herein operably linked to the 5'-end and / or 3'-end of the target sequence. In certain embodiments, multiple ASOs (e.g., bipartite ASOs) may be provided for screening and optimization to provide one or more ASOs (e.g., bipartite ASOs) having improved exon skipping efficiency, e.g., but not limited to, by further comprising screening and / or optimizing the one or more ASOs (e.g., bipartite ASOs) according to the exon skipping efficacy and / or efficiency of the target exon of the one or more ASOs (e.g., bipartite ASOs).

[0101] In certain embodiments, methods for treating a disease and / or its complications in a subject are also provided herein, which comprise administering to the subject an ASO (e.g., a bipartite ASO) disclosed herein, a composition (e.g., a pharmaceutical composition) comprising an ASO (e.g., a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (e.g., a bipartite ASO) disclosed herein. The ASO (e.g., a bipartite ASO) may generate or promote exon skipping of a target exon during pre-mRNA splicing. ASO-mediated exon skipping may be a method of manipulating the expression of a target gene. The expression of the target gene can be manipulated by the ASO inhibiting the splicing of an exon, intron, or specific splicing site of the target gene, resulting in (e.g., but not limited to): restoring the reading frame of a target defective gene, generating different subtypes of the target gene (e.g., dominant negative subtypes), skipping a toxic portion of the gene, silencing the gene, and / or altering the structure and function of the gene. Manipulating the expression of the target gene may be beneficial for treating a disease and / or its complications, although the target gene may or may not be the cause of the disease. The disease may be a genetic or non-genetic disease disclosed herein (e.g., some non-genetic cancers, metabolic diseases, or infectious diseases). The genetic disease may or may not be associated with a splicing defect-related mutation.

[0102] In certain embodiments, a kit is also provided herein, which comprises an ASO (e.g., a bipartite ASO) disclosed herein, a composition (e.g., a pharmaceutical composition) comprising an ASO (e.g., a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (e.g., a bipartite ASO) disclosed herein, for generating or promoting exon skipping of a target exon. ASO-mediated exon skipping may be a method of manipulating the expression of a target gene. The expression of the target gene can be manipulated by the ASO inhibiting the splicing of an exon, intron, or specific splicing site of the target gene, resulting in (e.g., but not limited to): restoring the reading frame of a target defective gene, generating different subtypes of the target gene (e.g., dominant negative subtypes), skipping a toxic portion of the gene, silencing the gene, and / or altering the structure and function of the gene.

[0103] Examples of target genes and diseases treatable by exon skipping

[0104] Dystrophin and Duchenne muscular dystrophy

[0105] Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle wasting disease caused by a mutation in the DMD gene that eliminates the production of dystrophin. Dystrophin is a rod-shaped cytoplasmic protein and is an important part of a protein complex that connects the cytoskeleton of muscle fibers to the surrounding extracellular matrix via the cell membrane. Dystrophin contains multiple functional domains. For example, dystrophin contains an actin binding domain at about amino acids 14-240 and a central rod domain at about amino acids 253-3040. This large central domain is formed by 24 spectrin-like triple helical elements of about 109 amino acids, which have homology with α-actinin and spectrin. The repeats are usually separated by four proline-rich non-repeating segments (also called hinge regions). Repeats 15 and 16 are separated by a stretch of 18 amino acids, which appears to provide the main site for proteolytic cleavage of dystrophin. The sequence identity between most repeat sequences ranges from 10-25%. One repeat sequence contains three α-helices: 1, 2, and 3. α-Helices 1 and 3 are each formed by 7 helical turns, possibly interacting in a coiled-coil manner through a hydrophobic interface. α-Helix 2 has a more complex structure and is formed by segments of four and three helical turns separated by glycine or proline residues. Each repeat sequence is encoded by two exons, usually in the first part of α-helix 2, interrupted by an intron at a position located between amino acids 47 and 48. Another intron is found at different positions in the repeat sequence, usually scattered on helix-3. Anti-dystrophin also contains a cysteine-rich domain at approximately amino acids 3080-3360, including a cysteine-rich segment (i.e., 15 cysteines in 280 amino acids) that shows homology to the C-terminal domain of α-actinin from the slime mold (Dictyostelium discoideum). The carboxyl terminal domain is at about amino acids 3361-3685. The amino terminus of dystrophin binds to F-actin, and the carboxyl terminus binds to the dystrophin-associated protein complex (DAPC) at the sarcolemma. DAPC includes dystroglycan, sarcoglycan, integrin, and caveolin, and mutations in any of these components cause autosomal hereditary muscular dystrophy. When dystrophin is absent, DAPC is unstable, which causes member protein levels to decrease, and then causes progressive fiber damage and membrane leakage. In various forms of muscular dystrophy, such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), muscle cells produce altered and functionally defective forms of dystrophin, or do not produce dystrophin at all, which is mainly due to mutations in the gene sequence that cause incorrect splicing.As described above, a major expression defect of dystrophin or a complete lack of dystrophin or dystrophin-like proteins results in a rapid progression of muscle degeneration. In this regard, "defective" dystrophin may be characterized by the form of dystrophin produced in some subjects with DMD or BMD, as known in the prior art, or the absence of detectable dystrophin.

[0106] Over 7,000 different mutations have been reported in DMD patients. The majority of patients (about 65%) carry large deletions involving one or more exons, but large duplications (about 12%) and small mutations (20%) are also frequently reported. The commonality of these mutations is that they all result in non-functional dystrophin. For example, the deletion of exon 45 is one of the most common deletions in DMD patients, and the deletion of exons 44 and 45 is generally associated with BMD. Thus, if exon 44 could be skipped in the pre-messenger RNA (mRNA) transcripts of these DMD patients, this would restore the reading frame and enable the production of a partially functional BMD-like dystrophin. In fact, it appears that many patients carrying deletions in the region adjacent to exon 44 spontaneously skip exon 44, albeit at very low levels. This results in a slightly increased level of dystrophin compared to DMD patients carrying other deletions and is likely the reason for the less severe disease progression observed in these patients compared to DMD patients with other deletions.

[0107] Exon skipping for the treatment of DMD

[0108] Antisense-mediated exon skipping induces the skipping of one or more target exons and can be used to restore the defective reading frame. Mutations in the dystrophin gene are suitable for therapeutic exon skipping. For example, mutations in the following exons are suitable for exon 51 skipping, such as: 45 to 50, 47 to 50, 48 to 50, 49 to 50, 50, 52, 52 to 63 (Leiden Duchenne Muscular Dystrophy Mutation Database, Leiden University Medical Center, the Netherlands). It is possible to determine whether a patient has a mutation in the DMD gene that is suitable for exon skipping (see, for example, Aartsma-Rus et al. Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations. February 24, 2009. Hum Mut. 30:293-299. doi:10.1002 / humu.20918; Stephen Abbs et al. Best Practice Guidelines on molecular diagnostics in Duchenne / Becker muscular dystrophies. June 2010. Neuromusc Disorders. 20:422-427. doi:10.1016 / j.nmd.2010.04.005, the disclosures of which are incorporated herein by reference in their entireties). To develop exon skipping methods for patients with specific defects in the DMD gene, ASOs with targeting sequences for other exons of the targeted gene must be developed. For example, ASOs can be developed to skip exon 51, which is defective in 13%-14% of DMD patients.

[0109] The physiological or cellular responses associated with the treatment (in vivo or in vitro) encompassed by this technology will be apparent to those skilled in the art and may include alleviation of the following symptoms or pathologies: Duchenne muscular dystrophy (DMD) and related disorders such as Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy, congenital muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy; muscle wasting conditions or disorders such as AIDS, cancer or chemotherapy-related muscle wasting syndrome; and fibrosis or fibrosis-related disorders (e.g., skeletal muscle fibrosis). An "increase" in the response can be "statistically significant" compared to the response generated in a subject in need without administration of an ASO compound and / or treatment agent (e.g., compared to the "native" or "natural" expression rate of a particular subject or group) or compared to a control compound.

[0110] In some embodiments, in DMD patients, the DMD allele contains a mutation in an exon, and the disorder is treated at least in part by skipping of one or more exons of the DMD gene transcript. In some embodiments, in DMD patients, the DMD allele or DMD transcript has a mutation in an exon, which is a missense or nonsense mutation and / or a deletion, insertion, inversion, translocation or duplication. In some embodiments, in the treatment of muscular dystrophy, one or more exons within the DMD transcript are skipped, where the exon encodes a stretch of amino acids that is not important for DMD protein function, or whose skipping can provide a fully or at least partially functional DMD protein. Herein, the DMD protein refers to dystrophin. In some embodiments, in the treatment of muscular dystrophy, an ASO is capable of mediating skipping of DMD exon 51 or 53, thereby generating an mRNA from which an artificial internally truncated DMD protein isoform (e.g., a functional DMD protein variant) can be translated that provides at least partial improvement or full restoration of biological activity. In some embodiments, an internally truncated DMD protein variant generated by skipping one or more exons of the dystrophin DMD transcript is more functional than, for example, a C-terminally truncated DMD protein generated by a dystrophin DMD transcript with an out-of-frame deletion. In some embodiments, an internally truncated DMD protein variant generated by skipping one or more exons of the dystrophin DMD transcript is more resistant to nonsense-mediated decay, which can degrade, for example, a C-terminally truncated DMD protein generated by a dystrophin DMD transcript with an out-of-frame deletion. In some embodiments, restoring the reading frame can convert an out-of-frame mutation into an in-frame mutation; in some embodiments, in humans, this change can convert severe Duchenne muscular dystrophy into milder Becker muscular dystrophy.

[0111] The methods of the present invention can mitigate one or more characteristics of myogenic or muscle cells in a patient or mitigate one or more symptoms in a DMD patient having a deletion including but not limited to the following: exon 44, 44-46, 44-47, 44-48, 44-49, 44-51, 44-53 (correctable by exon 43 skipping) in the DMD gene, 19-45, 21-45, 43-45, 45, 47-54, 47-56 (correctable by exon 46 skipping), 51, 51-53, 51-55, 51-57 (correctable by exon 50 skipping), 13-50, 19-50, 29-50, 43-50, 45-50, 47-50, 48-50, 49-50, 50, 52 (correctable by exon 51 skipping), exon 8-51, 51, 53, 53-55, 53-57, 53-59, 53-60 (correctable by exon 52 skipping), and exon 10-52, 42-52, 43-52, 45-52, 47-52, 48-52, 49-52, 50-52, 52 (correctable by exon 53 skipping), occurring in a total of 68% of all DMD patients having a deletion (Aartsma-Rus et al. Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations. Feb 24, 2009. Hum Mut. 30:293-299. Doi:10.1002 / humu.20918). See U.S. Patent No. 9,499,818, which is incorporated herein by reference in its entirety.

[0112] The SMN Gene and Spinal Muscular Atrophy

[0113] The SMN1 and SMN2 genes are associated with a disease called spinal muscular atrophy (SMA). The two genes encode the same protein (survival of motor neuron, or SMN). One difference between the two genes is that SMN2 differs by a C to T transition in exon 7, which causes substantial skipping of this exon, such that SMN2 expresses only low levels of functional protein.

[0114] Other Target Genes and Related Diseases Treatable by Exon Skipping

[0115] Examples of other target genes include, but are not limited to, the APP gene, the CEP290 gene, the HER2 gene, the PKM gene, and the MDM4 gene. The APP gene is associated with Alzheimer's disease and the CEP290 gene is associated with Joubert syndrome. ASOs that promote exon 19 of the HER2 gene, exon 10 of the PKM gene, or exon 6 of the MDM4 gene have the potential to treat various types of cancer, including breast cancer, HER2-positive cholangiocarcinoma, colorectal cancer, non-small cell lung cancer, bladder cancer, prostate cancer, lung cancer, cervical cancer, renal cancer, papillary thyroid cancer, colon cancer, colorectal cancer, glioma, ovarian cancer, gastric cancer, hepatoblastoma, fibrolamellar hepatocellular carcinoma, soft tissue sarcoma, osteosarcoma, chronic lymphocytic leukemia, acute myeloid leukemia, mantle cell lymphoma, pediatric Burkitt lymphoma, salivary gland cancer, liver cancer, and melanoma. (See, e.g., Do-Youn Oh and Yung-Jue Bang .HER2-targeted therapies—a role beyond breast cancer. September 23, 2019. Nat. Rev. Clin. Oncol. 17:33-48. Doi:10.1038 / s41571-019-0268-3; K. Zahra et al. Pyruvate kinase M2 and cancer: the role of PKM2 in promoting tumorigenesis. March 2, 2020. Front. Oncol. 10:159. Doi:10.3389 / fonc.2020.00159; and D. Yu et al. Targeting MDMX for cancer therapy: rationale, strategies, and challenges. August 5, 2020. Front. Oncol. 10:1389. Doi:10.3389 / fonc.2020.01389.).

[0116] Dual ASOs for precursor mRNA splicing modulation via exon skipping

[0117] In some aspects, the technology of the present invention provides a method for generating or promoting exon skipping of a target exon during pre-mRNA splicing, which comprises contacting a pre-mRNA in a cell or a subject with an ASO (such as a bipartite ASO) disclosed herein, a composition (such as a pharmaceutical composition) comprising an ASO (such as a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (such as a bipartite ASO) disclosed herein, a vector encoding an ASO (such as a bipartite ASO) disclosed herein. In some embodiments, the method further comprises delivering an ASO (such as a bipartite ASO) disclosed herein to a cell or administering an ASO (such as a bipartite ASO) disclosed herein to a subject. ASO-mediated exon skipping may be a method for manipulating the expression of a target gene. The expression of a target gene can be manipulated by ASO inhibiting the splicing of an exon, an intron, or a specific splicing site of the target gene, resulting in (e.g., but not limited to): restoring the reading frame of a target defective gene, generating different subtypes of the target gene (such as a dominant negative subtype), skipping a toxic portion of the gene, silencing the gene, and / or altering the structure and function of the gene.

[0118] In some aspects, the technology of the present invention provides the use of an ASO (such as a bipartite ASO) disclosed herein, a composition (such as a pharmaceutical composition) comprising an ASO (such as a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (such as a bipartite ASO) disclosed herein for generating or promoting exon skipping of a target exon. ASO-mediated exon skipping may be a method for manipulating the expression of a target gene. The expression of a target gene can be manipulated by ASO inhibiting the splicing of an exon, an intron, or a specific splicing site of the target gene, resulting in (e.g., but not limited to): restoring the reading frame of a target defective gene, generating different subtypes of the target gene (such as a dominant negative subtype), skipping a toxic portion of the gene, silencing the gene, and / or altering the structure and function of the gene.

[0119] In some aspects, the technology of the present invention provides an ASO (such as a bipartite ASO) disclosed herein, a composition (such as a pharmaceutical composition) comprising an ASO (such as a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (such as a bipartite ASO) disclosed herein for generating or promoting exon skipping of a target exon. ASO-mediated exon skipping may be a method for manipulating the expression of a target gene. The expression of a target gene can be manipulated by ASO inhibiting the splicing of an exon, an intron, or a specific splicing site of the target gene, resulting in (e.g., but not limited to): restoring the reading frame of a target defective gene, generating different subtypes of the target gene (such as a dominant negative subtype), skipping a toxic portion of the gene, silencing the gene, and / or altering the structure and function of the gene.

[0120] An ASO contains a targeting sequence, which is a single-stranded oligonucleotide that is specific for a target splicing sequence and is substantially complementary thereto and is thus capable of hydrogen bonding with that sequence. A person skilled in the art can readily design the targeting sequence of an ASO to be specific for a suitable target region, and many targeting sequences are well known in the art. For example, precursor mRNA sequences containing suitable splicing sequences can be obtained in publications or annotated, publicly available databases such as the GenBank database operated by NCBI. Many targeting sequences have been incorporated into ASO designs to enhance exon skipping, and some targeting sequences are currently in preclinical or clinical trials. Any of these targeting sequences is suitable for the methods of the technology of the present invention.

[0121] ASOs can be used to regulate exon skipping by blocking (masking) specific sequence motifs (sometimes referred to herein as "splicing sequences") in the precursor mRNA that are essential for exon inclusion by the splicing machinery. An ASO that blocks an abnormal splicing site can restore normal splicing. Alternatively, an ASO that targets some splicing sequences can switch the splicing pattern from a deleterious subtype to a beneficial subtype, or can at least partially convert a non-functional mRNA into a functional mRNA. An example of the latter method is restoring a disrupted reading frame, thereby producing a semi-functional protein rather than a non-functional protein.

[0122] The ASOs of the technology of the present invention (e.g., bipartite ASOs) can be used to block splicing at a target site by directly or indirectly interacting specifically (e.g., binding) with a splicing sequence at the target site. "Splicing sequence" means a sequence that regulates and / or is required for: splicing out a specific intron and / or retaining a specific exon. A splicing sequence can be, for example, a splice donor site (5' splice site), a splice acceptor site (3' splice site), a branch site, an intronic splicing enhancer (ISE), an exonic splicing enhancer (ESE), an intronic splicing silencer, or an exonic splicing silencer.

[0123] In the methods of the technology of the present invention, the ASO (e.g., bis-ASO) used can directly and specifically bind to the target region, which is the target splicing sequence. "Specific binding" means that under the conditions where specific binding is required, the ASO preferentially binds to the target region rather than to non-target sequences. Such conditions can be, for example, physiological conditions in the case of in vivo assays or therapeutic treatments, and for in vitro assays, the conditions under which the assay is performed. Since the mechanism by which the small molecule compounds of the technology of the present invention block splicing (e.g., enhance exon skipping) is unknown for all compounds, it is unknown whether the compounds directly bind to the splicing site or act indirectly (e.g., by binding to another RNA or protein element of the spliceosome). Regardless of the mechanism, the compounds of the technology of the present invention that "specifically" block the target splicing event are compounds that preferentially block a specific splicing event but do not block non-target splicing events under the conditions where specific blocking is required.

[0124] The ASO (e.g., bis-ASO) disclosed herein can have a variety of different backbone chemical compositions, such as morpholino phosphorodiamidate (PMO), 2'-O-methyl, 2'-O-methoxyethyl (MOE), phosphorothioate (PS), 2'-fluororibose (2'-F), 4'-thioribosyl ribose, locked nucleic acid oligonucleotide (LNA), and / or constrained ethyl oligonucleotide (cEt) or peptide nucleic acid, etc., which stabilize the ASO. For example, it can be DNA, RNA, PNA, or LNA, or a chimeric mixture or derivative or modified form thereof. Nucleic acids can be modified at the base moiety, sugar moiety, or phosphate backbone using conventional procedures and modifications. Base modifications include, for example, methylated forms of purines or pyrimidines. Modifications can include other attached groups that will be apparent to those skilled in the art. Examples of oligonucleotide modifications are described throughout this application, for example, in the following "Oligonucleotide Modifications" section.

[0125] The ASO (e.g., bis-ASO) disclosed herein can be constructed using chemical synthesis procedures known in the art. The ASO can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides that are designed to increase the biological stability of the molecule or increase the physical stability of the duplex formed between the antisense and sense nucleic acids, such as phosphorothioate derivatives and acridine-substituted nucleotides.

[0126] Alternatively, an ASO can be produced by an expression vector biological approach, where the nucleic acid has been subcloned in the antisense orientation (i.e., the nucleic acid transcribed from the inserted nucleic acid will have an antisense orientation to the target region). Expression control sequences (such as regulatory sequences) are operably linked to the nucleic acid cloned in the antisense orientation, and these sequences can be selected to direct the expression of the antisense RNA molecule in the target cell. For example, a promoter and / or enhancer or other regulatory sequences can be selected, which can direct the constitutive, tissue-specific or inducible expression of the ASO. Inducible expression of the antisense RNA can be used, for example, regulated by an inducible eukaryotic regulatory system. The antisense expression vector can be in the form of, for example, a recombinant plasmid, phagemid or attenuated virus. Suitable viral vectors include, for example, adeno-associated virus (AAV) or lentiviral vectors. Standard techniques well known in the art can be used to introduce the antisense expression vector into cells.

[0127] The length of the targeting sequence within the ASO can vary, provided that it is capable of selectively binding to the expected splicing sequence within the precursor mRNA molecule. A person skilled in the art can readily determine a satisfactory length. Generally, the length of the targeting sequence of the ASO is from about 10 nt to about 80 nt. Nucleotides of any length within this range (including the endpoints) can be used in the methods of the present invention technology.

[0128] In some embodiments of the present invention technology, the targeting sequence of the ASO comprises a strand that is completely (100%) complementary to the splicing sequence it is designed to inhibit. That is, each consecutive nucleotide in the targeting sequence will hybridize to each nucleotide in the splicing sequence of the target gene. However, 100% sequence identity between the targeting sequence and the target region is not required. Thus, the present invention technology has the advantage of being able to tolerate naturally occurring sequence variations that can be expected due to gene mutations, strain polymorphisms or evolutionary divergence. Alternatively, variants can be artificially generated. Nucleic acid sequences with, for example, small insertions, deletions and single point mutations relative to the target region can be effective in inhibition. The degree of sequence identity can be, for example, 90%, 95%, 98%, 99% or 100%. Of course, such variant ASOs must retain the relevant activity of the ASO from which they are derived (such as the ability to inhibit splicing at the target site). Such variants are sometimes referred to herein as "active variants".

[0129] For further guidance on designing suitable antisense molecules (which block splicing) complementary to regions of precursor mRNA involved in splicing and methods for preparing such molecules and delivering them to cells or subjects, see, for example, US2008 / 0200409 or U.S. Patent Nos. 7,973,015, 7,960,541, 7,902,160, 7,888,012, 7,879,992 or 7,737,110.

[0130] Examples of Oligonucleotides for Dystrophin Gene Splicing Regulation

[0131] In September 2016, the US Food and Drug Administration (FDA) conditionally approved the first DMD antisense drug, Exondys 51, which was developed to skip exon 51 from the mutant DMD. Exondys 51 is an oligonucleotide modified with phosphorodiamidate morpholino oligomers (morpholino or PMO). However, Exondys 51 has still been controversial because there is only weak evidence to support the effectiveness of the drug in both restoring dystrophin to therapeutically beneficial levels and improving clinical outcomes. The FDA had previously rejected another candidate drug for exon 51 skipping in DMD: the 2'-O-methyl-thiophosphate-based oligonucleotide "drisapersen". Although therapeutic agents must ensure the highest possible benefit at the lowest risk dose, no significant improvement in muscle function was demonstrated after treatment with drisapersen, and its use raised concerns about safety.

[0132] Bipartite Antisense Oligonucleotides (Bipartite ASO)

[0133] In some aspects, the technology of the present invention provides bipartite ASO, which comprises: 1) a "targeting" sequence that is fully or substantially complementary to a target region in a target nucleic acid (such as the precursor mRNA of a target gene), and 2) a "decoy" sequence that is operably linked to the 5'-end and / or 3'-end of the targeting sequence and is positioned there. The decoy sequence can mimic the optimal 5' splice site, so it can act as a 5' splice site decoy to interfere with the recognition of the adjacent authentic 5' splice site by U1snRNA. As discussed herein, the presence of the decoy sequence can enhance the efficiency of splicing regulation of the targeting sequence at the target region.

[0134] In various embodiments, the bipartite ASO of the technology of the present invention comprises: 1) a "targeting" sequence that is fully or substantially complementary to a target region in a target nucleic acid (such as the precursor mRNA of a target gene), and 2) a "decoy" sequence that is located immediately upstream of the 5'-end and / or immediately downstream of the 3'-end of the targeting sequence (i.e., the decoy sequence is in the flanking region of the targeting sequence). In some embodiments, the decoy sequence is directly linked to the targeting sequence, and there is no intermediate nucleotide between the decoy sequence and the targeting sequence. In some embodiments, the decoy sequence is located immediately upstream of the 5'-end of the targeting sequence (as shown in Figure 1 A). In other embodiments, the decoy sequence is located immediately downstream of the 3'-end of the targeting sequence.

[0135] When both the 5' end and the 3' end of the targeting sequence are ligated to the bait sequences, the bait sequences ligated to each end can be the same or different, and the ligation methods can be the same or different. For example, two bait sequences can be ligated to the targeting sequence without a linker, or one bait sequence can be ligated without a linker while the other bait sequence is ligated using a linker, or two bait sequences are ligated to the targeting sequence using linkers, but the linkers can be the same or different. In some embodiments, the linker can have 1, 2, 3, 4, or 5 nucleotides.

[0136] In some embodiments, due to the presence of the bait sequence (e.g., with or without the bait sequence), the length of the bait sequence, the sequence of the bait sequence, and / or the position of the bait sequence (e.g., the 5' end and / or the 3' end of the targeting sequence), ASOs containing the same targeting sequence can have different exon skipping effects and / or efficiencies. In certain embodiments, the exon skipping effect and / or efficiency can be quantified by the percentage of exclusion of the target exon in the total transcript of each gene (exclusion %). In certain embodiments, the degree of improvement quantified by the increase in exclusion % can be at least about 2-fold, about 2.18-fold, at least about 2.3-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, and at least about 100-fold, including all values and the ranges between these values, compared to an ASO consisting of the targeting sequence.

[0137] In certain embodiments, compared to an ASO consisting of the targeting sequence, a bipartite ASO containing a bait sequence (e.g., but not limited to the nucleotide sequences of SEQ ID No. 1 to 23 and SEQ ID No. 347 to 353) ligated to the targeting sequence or consisting of the same can achieve comparable efficacy and / or efficiency in generating or promoting exon skipping at a lower dose. In certain embodiments, an ASO containing a bait sequence ligated to the targeting sequence may achieve an exclusion % comparable to that of an ASO consisting only of the targeting sequence (e.g., at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%), and the dose does not exceed about 10%, does not exceed about 25%, does not exceed about 40%, or does not exceed about 50% of the dose of an ASO consisting only of the targeting sequence.

[0138] In certain embodiments of a bipartite ASO comprising the same targeting sequence and decoy sequence, the bipartite ASO having the decoy sequence at the 5' end of the targeting sequence may have a higher exon skipping effect and / or efficiency than the bipartite ASO having the decoy sequence at the 3' end of the targeting sequence. In certain instances, both bipartite ASOs comprising the decoy sequence have a higher exon skipping effect and / or efficiency than an ASO consisting of the same targeting sequence.

[0139] In certain embodiments of a bipartite ASO comprising the same targeting sequence and decoy sequence, the bipartite ASO having the decoy sequence at the 5' end of the targeting sequence may have a lower exon skipping effect and / or efficiency than the bipartite ASO having the decoy sequence at the 3' end of the targeting sequence. In certain instances, both bipartite ASOs comprising the decoy sequence have a higher exon skipping effect and / or efficiency than an ASO consisting of the same targeting sequence.

[0140] In certain embodiments of an ASO comprising the same targeting sequence and decoy sequence, the ASO having the decoy sequence at the 5' end of the targeting sequence is comparable to the ASO having the decoy sequence at the 3' end of the targeting sequence.

[0141] In some embodiments, the bipartite ASO comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID No. 26 to 27, 30 to 31, 34 to 35, 38 to 39, 42 to 43, 46 to 47, 50 to 73, 76 to 90, 93 to 122, 125 to 154, 157 to 186, 189 to 218, 221 to 250, 253 to 282, 285 to 314, 317 to 346, and 354 to 356. In some embodiments, the bipartite ASO comprises or consists of a nucleotide sequence selected from the group consisting of SEQID No. 26, 30, 31, 34, 38, 42, 46, 50, 51, 60, 64, 65, 68, 70, 78, 83, 85, 86, 87, 88, 89, 103, 109, 110, 114, 115, 116, 127, 132, 144, 164, 189, 205, 213, 216, 228, 260, 276, 292, 294, 297, 298, 312, 320, 324, 327, 328, 332, and 354.

[0142] In some embodiments, the target gene is the SMN1 and / or SMN2 gene, and the ASO comprises or consists of a nucleotide sequence selected from the group consisting of: the nucleotide sequences of SEQ ID No. 26 to 27, 30 to 31, 34 to 35, and 354 to 356. In certain embodiments, the ASO comprises or consists of a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 26, 30, 31, and 34. In certain embodiments, the target exon is exon 7 of the SMN1 and / or SMN2 gene.

[0143] In some embodiments, the target gene is the DMD gene, and the ASO comprises or consists of a nucleotide sequence selected from the group consisting of: the nucleotide sequences of SEQ ID No. 38 to 39, 42 to 43, 46 to 47, 50 to 73, 76 to 90, 93 to 122, and 125 to 154. In certain embodiments, the optimal ASO comprises or consists of a nucleotide sequence selected from the group consisting of: the nucleotide sequences of SEQ ID No. 38, 42, 46, 50, 51, 60, 64, 65, 68, 70, 78, 83, 85, 86, 87, 88, 89, 103, 109, 110, 114, 115, 116, 127, 132, and 144.

[0144] In certain embodiments, the target exon is exon 51 of the DMD gene, and the ASO comprises or consists of a nucleotide sequence selected from the group consisting of: the nucleotide sequences of SEQ ID No. 38 to 39, 42 to 43, 46 to 47, 50 to 73, and 76 to 90. In certain embodiments, the optimal ASO comprises or consists of a nucleotide sequence selected from the group consisting of: the nucleotide sequences of SEQ ID No. 38, 42, 46, 50, 51, 60, 64, 65, 68, 70, 78, 83, 85, 86, 87, 88, and 89.

[0145] In certain embodiments, the target exon is exon 53 of the DMD gene, and the ASO comprises or consists of a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 93 to 122. In certain embodiments, the optimal ASO comprises or consists of a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 103, 109, 110, 114, 115, and 116.

[0146] In certain embodiments, the target exon is exon 45 of the DMD gene, and the ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID Nos. 125 to 154 or consists of the same. In certain embodiments, the optimal ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID Nos. 127, 132, and 144 or consists of the same.

[0147] In some embodiments, the target gene is the APP gene, and the ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID Nos. 157 to 186 or consists of the same. In certain embodiments, the optimal ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID No. 164 or consists of the same. In certain embodiments, the target exon is exon 17 of the APP gene.

[0148] In some embodiments, the target gene is the CEP290 gene, and the ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID Nos. 189 to 218 or consists of the same. In certain embodiments, the optimal ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID Nos. 189, 205, 213, and 216 or consists of the same. In certain embodiments, the target exon is exon 41 of the CEP290 gene.

[0149] In some embodiments, the target gene is the HER2 gene, and the ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID Nos. 221 to 250 or consists of the same. In certain embodiments, the optimal ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID No. 228 or consists of the same. In certain embodiments, the target exon is exon 19 of the HER2 gene.

[0150] In some embodiments, the target gene is the ATXN3 gene, and the ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID Nos. 253 to 282 or consists of the same. In certain embodiments, the optimal ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID Nos. 260 and 276 or consists of the same. In certain embodiments, the target exon is exon 10 of the ATXN3 gene.

[0151] In some embodiments, the target gene is the PKM gene, and the ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 285 to 314 or consists of the same. In certain embodiments, the optimal ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 292, 294, 297, 298, and 312 or consists of the same. In certain embodiments, the target exon is exon 10 of the PKM gene.

[0152] In some embodiments, the target gene is the MDM4 gene, and the ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 317 to 346 or consists of the same. In certain embodiments, the optimal ASO comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 320, 324, 327, 328, and 332 or consists of the same. In certain embodiments, the target exon is exon 6 of the MDM4 gene.

[0153] Bait sequence

[0154] In some embodiments, the bait sequence comprises a nucleotide sequence selected from the group consisting of: the nucleotide sequences of SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 347, 348, 349, 350, 351, 352, and 353 or consists of the same. In some embodiments, the length of the bait sequence is 5 to 13 nucleotides (nt). In some embodiments, the length of the bait sequence is 5, 6, 8, 9, 10, 11, 12, or 13 nt.

[0155] In some embodiments, the bait sequence is similar to the optimal 5' splice site and is fully or partially complementary to the single-stranded 5' end of U1snRNA. In some embodiments, the bait sequence is 100% complementary to a part of the single-stranded 5' end of U1 snRNA. In some embodiments, 7-11 nt in the bait sequence is complementary to the 5' end of U1 snRNA.

[0156] In some embodiments, the bait sequence has low sequence complementarity, such as less than about 80%, with the corresponding flanking sequence immediately adjacent to the 5' end or 3' end of the target region in the target nucleic acid (such as mRNA). In some embodiments, the bait sequence is located immediately upstream of the 5' end of the targeting sequence and has low sequence complementarity with the corresponding flanking region immediately downstream of the 3' end of the target region (as Figure 1 shown in A). In some embodiments, the bait sequence is located immediately downstream of the 3' end of the targeting sequence and has low sequence complementarity with the target nucleic acid (such as precursor RNA, and as Figure 1The corresponding flanking region immediately upstream of the 5' end of the target region (as shown in

[0157] A) has low sequence complementarity. In some embodiments, the bait sequence has less than about 80% sequence complementarity with the corresponding region in the target nucleic acid. In some embodiments, the bait sequence has less than about 70% sequence complementarity with the corresponding region in the target nucleic acid. In some embodiments, the bait sequence has less than about 60% sequence complementarity with the corresponding region in the target nucleic acid. In some embodiments, the bait sequence has less than about 50% sequence complementarity with the corresponding region in the target nucleic acid. In some embodiments, the sequence complementarity with the corresponding region in the target nucleic acid is less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5% or 0%. Figure 1 A). In some embodiments, the bait sequence is located upstream of the 5' end of the targeting sequence in the ASO, and in such embodiments, the first nucleotide at the 3' end of the bait sequence does not hybridize to the corresponding flanking nucleotide immediately downstream of the 3' end of the target region in the target nucleic acid (as Figure 1 shown in A). In some embodiments, the bait sequence is located downstream of the 3' end of the targeting sequence in the ASO, and in such embodiments, the first nucleotide at the 5' end of the bait sequence does not hybridize to the corresponding flanking nucleotide immediately upstream of the 5' end of the target region in the target nucleic acid (as

[0158] Targeting sequence

[0159] In various embodiments of the technology of the present invention, the targeting sequence of the ASO is fully or substantially complementary to the target region in a nucleic acid (such as a precursor mRNA). In some embodiments, the targeting sequence of the ASO comprises a strand having exact (100%) complementarity with the target region in the nucleic acid it is designed to inhibit. That is, each consecutive nucleotide in the targeting sequence hybridizes to each corresponding nucleotide in the target region. However, practicing the technology of the present invention may not require 100% sequence identity between the targeting sequence and the target splicing sequence. In other embodiments, the targeting sequence is substantially complementary to the target region; that is, the complementarity of the targeting sequence with the target region in the nucleic acid it is designed to inhibit is more than 70%, more than 75%, more than 80%, more than 90%, more than 95%, more than 98% or more than 99%. The non-complementary positions in the targeting sequence of the ASO can be one or more insertions, deletions, and / or point mutations relative to the target region.

[0160] In some embodiments, the target region is an exon (such as Figure 1as shown in B). In some embodiments, the target region is an intronic region upstream of the exon (such as Figure 1 as shown in B). In some embodiments, the target region is an intronic region downstream of the exon (such as Figure 1 as shown in B). In some embodiments, the targeting sequence binds to the target region, which is the target exon, the flanking intronic sequence upstream of the target exon, the flanking intronic sequence downstream of the target exon, the intron-exon junction upstream of the target exon, or the intron-exon junction downstream of the target exon. In some embodiments, the target exon is exon 7 of the SMN1 gene or the SMN2 gene. In some embodiments, the target exon is exon 7 of the SMN1 gene. In some embodiments, the target exon is exon 7 of the SMN2 gene. In some embodiments, the target exon is exon 51 of the DMD gene. In some embodiments, the target exon is exon 53 of the DMD gene. In some embodiments, the target exon is exon 45 of the DMD gene. In some embodiments, the target exon is exon 17 of the APP gene. In some embodiments, the target exon is exon 41 of the CEP290 gene. In some embodiments, the target exon is exon 19 of the HER2 gene. In some embodiments, the target exon is exon 10 of the ATXN3 gene. In some embodiments, the target exon is exon 10 of the PKM gene. In some embodiments, the target exon is exon 6 of the MDM4 gene.

[0161] In various embodiments, the length of the targeting sequence of the ASO is from about 10 nt to about 80 nt in length. In some embodiments, the length of the targeting sequence of the ASO is from about 10 - 60 nt in length. In some embodiments, the length of the targeting sequence of the ASO is from about 10 - 50 nt in length. In some embodiments, the length of the targeting sequence of the ASO is from about 10 - 40 nt in length. In some embodiments, the length of the targeting sequence of the ASO is from about 12 - 35 nt in length. In some embodiments, the length of the targeting sequence of the ASO is from about 14 - 30 nt in length. In some embodiments, the length of the targeting sequence of the ASO is from about 15 - 25 nt in length. In some embodiments, the length of the targeting sequence of the ASO is from about 18 - 23 nt in length. In some embodiments, the length of the targeting sequence of the ASO is about 20 nt in length. In some embodiments, the targeting sequence of the ASO comprises a nucleotide sequence selected from the group consisting of or consisting of the nucleotide sequences of SEQ ID No.25, 29, 33, 37, 41, 45, 49, 75, 92, 124, 156, 188, 220, 252, 284, and 316.

[0162] In some embodiments, the targeting sequence that hybridizes to the SMN1 and / or SMN2 gene comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 25, 29, and 33 or consists of the same. In certain embodiments, the targeting sequence is exon skipping for exon 7 of the SMN1 and / or SMN2 gene.

[0163] In some embodiments, the targeting sequence that hybridizes to the DMD gene comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 37, 41, 45, 49, 75, 92, and 124 or consists of the same.

[0164] In certain embodiments, the targeting sequence is exon skipping for exon 51 of the DMD gene, and the targeting sequence that hybridizes to the DMD gene comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 37, 41, 45, 49, and 75 or consists of the same.

[0165] In certain embodiments, the targeting sequence is exon skipping for exon 53 of the DMD gene, and the targeting sequence that hybridizes to the DMD gene comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID No. 92 or consists of the same.

[0166] In certain embodiments, the targeting sequence is exon skipping for exon 45 of the DMD gene, and the targeting sequence that hybridizes to the DMD gene comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID No. 124 or consists of the same.

[0167] In some embodiments, the targeting sequence that hybridizes to the APP gene comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID No. 156 or consists of the same. In certain embodiments, the targeting sequence is exon skipping for exon 17 of the APP gene.

[0168] In some embodiments, the targeting sequence that hybridizes to the CEP290 gene comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID No. 188 or consists of the same. In certain embodiments, the targeting sequence is exon skipping for exon 41 of the CEP290 gene.

[0169] In some embodiments, the targeting sequence that hybridizes to the HER2 gene comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID No. 220 or consists of the same. In certain embodiments, the targeting sequence is exon skipping for exon 19 of the HER2 gene.

[0170] In some embodiments, the targeting sequence that hybridizes to the ATXN3 gene comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID No. 259 or consists of the same. In certain embodiments, the targeting sequence is exon skipping for exon 10 of the ATXN3 gene.

[0171] In some embodiments, the targeting sequence that hybridizes to the PKM gene comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID No. 284 or consists of the same. In certain embodiments, the targeting sequence is exon skipping for exon 10 of the PKM gene.

[0172] In some embodiments, the targeting sequence that hybridizes to the MDM4 gene comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID No. 316 or consists of the same. In certain embodiments, the targeting sequence is exon skipping for exon 6 of the MDM4 gene.

[0173] Modification

[0174] In some embodiments, additional flanking nucleotide sequences may be present at one or both ends of the oligonucleotide comprising the bait sequence and the targeting sequence.

[0175] In some embodiments, the ASO further comprises one or more nucleotide modifications. In some embodiments, at least one subunit of the ASO is a non-natural nucleotide analogue having (i) a modified internucleoside linkage, (ii) a modified sugar moiety, (iii) a modified base, or (iv) a combination of the foregoing.

[0176] In some embodiments, the ASO comprises one or more phosphorothioate bonds as modified internucleoside linkages. In some embodiments, 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 internucleoside linkages of the ASO are phosphorothioate bonds. In some embodiments, all internucleoside linkages of the ASO are phosphorothioate bonds.

[0177] In some embodiments, the ASO comprises one or more 2'-O-methoxyethyl sugar moieties as modified sugar moieties. In some embodiments, 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 sugar moieties of the ASO are 2'-O-methoxyethyl sugar moieties. In some embodiments, all sugar moieties of the ASO are 2'-O-methoxyethyl sugar moieties.

[0178] In some embodiments, the ASO comprises one or more 5-methylcytosines in place of cytosine as modified bases. In some embodiments, 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 cytosine bases of the ASO are modified to 5-methylcytosine. In some embodiments, all cytosine bases of the ASO are modified to 5-methylcytosine.

[0179] In some embodiments, the ASO further comprises one or more additional chemical moieties. In some embodiments, one or more additional chemical moieties are covalently linked to one or more termini of the nucleic acid sequence. In some embodiments, the additional chemical moiety is a cell-penetrating peptide.

[0180] In some embodiments, the ASO of the technology of the present invention may comprise a nucleic acid portion bound to a cell-penetrating peptide (CPP) moiety to enhance the transport of the compound into cells. In some embodiments, the CPP moiety is linked to the terminus of the oligonucleotide. In some embodiments, the peptide is capable of penetrating about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the cells (including all integers therebetween) of a given cell culture population and permits macromolecule translocation within multiple tissues in vivo after administration. In some embodiments, the cell-penetrating peptide may be a polyarginine-rich peptide transporter. In some embodiments, the cell-penetrating peptide may be Penetratin or Tat peptide. These peptides are well known in the art and are disclosed in, for example, U.S. Publication No. 2010-0016215A1, which is incorporated herein by reference in its entirety. Exemplary methods of conjugating a peptide to an ASO can be found in PCT Publication WO2012 / 150960, which is incorporated herein by reference in its entirety. In some embodiments, the oligonucleotide and the CPP moiety are bound via a linker. In some embodiments, the oligonucleotide and the CPP moiety are joined by a linker. In some embodiments, the amino acid glycine is used as the linker between the CPP and the oligonucleotide moiety.

[0181] The transport moiety as described above has been shown to greatly enhance the cellular entry of conjugated oligomers relative to the uptake of oligomers in the absence of a conjugated transport moiety. In some embodiments, the CPP is one of the arginine-rich cell-penetrating peptides. Some peptide transporters have been shown to be very effective in delivering compounds containing antisense sequences into primary cells including muscle cells (N.B. Marshall et al. Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing. August 31, 2007. Journal of Immunological Methods. 325:114-126. doi:10.1016 / j.jim.2007.06.009; N. Jearawiriyapaisarn et al. Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice. September 2008. Mol Ther. 16:1624-1629. doi:10.1038 / mt.2008.120; Wu B et al. Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer. September 30, 2008. Proc. Natl. Acad. Sci. USA. 105:14814-14819. doi:10.1073 / pnas.0805676105).

[0182] Analysis for identifying exon skipping effects of bipartite ASO

[0183] Non-limiting examples of determining whether an ASO induces skipping of one or more exons in a transcript of a target gene can be as follows: introducing the ASO to be tested into a suitable cell line (HEK293 cells for SMN1, SMN2, CEP290, and MDM4 genes, HeLa cells for HER2 gene, A549 cells for ATXN3 gene, cells expressing dystrophin (such as human rhabdomyosarcoma cells) for DMD and PKM genes, or a cell line expressing a gene construct that mimics the exon / intron organization of the target region) (e.g., by introducing a plasmid containing an artificial minigene into the cell line), and amplifying the region covering the target exon in the transcript of total RNA from the cell line by RT-PCR, and performing nested PCR or sequence analysis on the PCR amplification product.

[0184] The skipping efficiency can be determined as follows. mRNA of the dystrophin gene is collected from the test cells and amplified by RT-PCR. If we denote "A" as the amplified polynucleotide level of our target mRNA that skips one or more target exons, and if we further denote "B" as the amplified polynucleotide level of our target mRNA where the target exons are not skipped, then using these measurements of "A" and "B", the efficiency is calculated by the following equation:

[0185] Exon skipping rate / efficiency (excluding %) = A / (A + B) × 100

[0186] Exon inclusion rate (inclusion %) = B / (A + B) × 100

[0187] In some embodiments, the bipartite ASO of the technology of the present invention induces skipping of one or more exons in DMD mRNA, wherein the exon skipping efficiency is increased by about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 2-fold or more, about 2.18-fold or more, about 2.3-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, about 6-fold or more, about 7-fold or more, about 8-fold or more, about 9-fold or more, about 10-fold or more, about 11-fold or more, about 12-fold or more, about 13-fold or more, about 14-fold or more, about 15-fold or more, about 16-fold or more, about 17-fold or more, about 18-fold or more, about 19-fold or more, about 20-fold or more, about 25-fold or more, about 30-fold or more, about 40-fold or more, about 50-fold or more, about 60-fold or more, about 70-fold or more, about 80-fold or more, about 90-fold or more, about 100-fold or more compared to an ASO having the same targeting sequence but without the decoy, including all values and the ranges between these values.

[0188] Oligonucleotide Modifications

[0189] Unmodified oligonucleotides may not be optimal in some applications. For example, unmodified oligonucleotides can be readily degraded by, for example, cellular nucleases. Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modification of oligonucleotides can confer improved properties and, for example, can render oligonucleotides more stable to nucleases.

[0190] Since oligonucleotides are polymers of subunits or monomers, many of the modifications described below occur at repetitive positions within the oligonucleotide, such as modifications of bases, sugars, phosphate moieties, or non-bridging oxygens of phosphate moieties. All positions within a given oligonucleotide need not be uniformly modified, and in fact more than one of the foregoing modifications can be incorporated into a single oligonucleotide or even at a single nucleoside within the oligonucleotide.

[0191] In some embodiments, the modification will be present at all subject positions within the oligonucleotide, but this is not the case in many embodiments. For example, the modification can be present only at the 3' or 5' terminal positions, can be present only in the internal region, can be present only in the terminal region, such as positions on the terminal nucleotides or in the last 2, 3, 4, 5, or 10 nucleotides of the oligonucleotide. The modification can be present in the double-stranded region, the single-stranded region, or both. The modification can be present only in the double-stranded region of a double-stranded oligonucleotide, or can be present only in the single-stranded region of a double-stranded oligonucleotide. For example, a phosphorothioate modification at a non-bridging oxygen position can be present only at one or both termini, can be present only in the terminal region, such as positions on the terminal nucleotides or in the last 2, 3, 4, 5, or 10 nucleotides of the strand, or can be present in both the double-stranded and single-stranded regions, particularly at the termini. One or more 5' ends can be phosphorylated.

[0192] The modifications described herein can be the only modification, or the only type of modification included on multiple nucleotides, or the modification can be combined with one or more other modifications described herein. The modifications described herein can also be combined onto the oligonucleotide, such that different nucleotides of the oligonucleotide have different modifications described herein.

[0193] In some embodiments, for example to enhance stability, it may be desirable to include specific nucleobases in the overhang, or in the single-stranded overhang, for example in the 5' or 3' overhang or both, modified nucleotides or nucleotide surrogates. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang will be modified, for example modified by the modifications described herein. Modifications can include, for example, using modifications at the 2' OH group of the ribose, such as using deoxyribonucleotides (such as deoxythymidine) in place of ribonucleotides, and modifications in the phosphate group, such as phosphorothioate modifications. The overhang need not be homologous to the target region.

[0194] Specific modifications are discussed in more detail below.

[0195] Phosphate group

[0196] The phosphate group is a negatively charged species. The charge is evenly distributed over two non-bridging oxygen atoms. However, the phosphate group can be modified by replacing one oxygen with a different substituent. One result of such modification of the RNA phosphate backbone can be an increase in the resistance of the oligoribonucleotide to nucleolytic breakdown. Thus, although not wishing to be bound by theory, in some embodiments it may be desirable to introduce changes that result in an uncharged linker or a charged linker with an asymmetric charge distribution.

[0197] Examples of modified phosphate groups include phosphorothioates, selenophosphates, boranophosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates, and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl), C (i.e., alkyl, aryl, etc.), H, NR2 (R is hydrogen, alkyl, aryl) or (R is alkyl or aryl). The phosphorus atom in the unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygens with one of the above atoms or groups renders the phosphorus atom chiral; in other words, the phosphorus atom in the phosphate group modified in this way is a stereogenic center. The stereogenic phosphorus atom can have an "R" configuration (Rp herein) or an "S" configuration (Sp herein).

[0198] The phosphorodithioate has two sulfur-substituted non-bridging oxygens. The phosphorus center in the phosphorodithioate is achiral, which prevents the formation of oligoribonucleotide diastereoisomers. Thus, although not wishing to be bound by theory, modification of the two non-bridging oxygens (which eliminates the chiral center), such as phosphorodithioate formation, may be desirable as it does not produce a mixture of diastereoisomers. Thus, the non-bridging oxygen can independently be any of S, Se, B, C, H, N or (where R is alkyl or aryl).

[0199] The phosphate linker can also be modified by replacing the bridging oxygen (i.e., the oxygen connecting the phosphate to the nucleoside) with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate), and carbon (bridging methylene phosphonate). The replacement can occur at either or both of the bridging oxygens. When the bridging oxygen is the 3'-oxygen of the nucleoside, replacement with carbon is preferred. When the bridging oxygen is the 5'-oxygen of the nucleoside, replacement with nitrogen is preferred.

[0200] Substitution of the phosphate group

[0201] The phosphate group can be replaced by a non-phosphorus linker. Although not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center for nuclease degradation, replacement thereof with a neutral structural mimic should confer enhanced nuclease stability. Similarly, although not wishing to be bound by theory, in some embodiments, it may be desirable to introduce a modification in which the charged phosphate group is replaced by a neutral moiety.

[0202] Examples of moieties that can replace the phosphate group include methyl phosphonate, hydroxyamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioacetal, acetal, oxime, methylene imino, methylene methyl imino, methylene hydrazine, methylene dimethyl hydrazine, and methylene oxy methyl imino. Preferred substitutions can include methylene carbonyl amino and methylene methyl imino.

[0203] A modified phosphate bond in which at least one of the oxygens attached to the phosphate has been replaced or the phosphate group has been replaced by a non-phosphorus group is also referred to as a "non-phosphodiester backbone bond".

[0204] Substitution of the ribophosphate backbone

[0205] It is also possible to construct scaffolds that mimic oligonucleotides, where the phosphate linkers and riboses are replaced by nuclease-resistant nucleosides or nucleotide surrogates. Without wishing to be bound by theory, it is thought that the lack of a repeating charged backbone reduces binding to proteins that recognize polyanions, such as nucleases. Similarly, without wishing to be bound by theory, in some embodiments, it may be desirable to introduce structural modifications where the bases are linked by a neutral surrogate backbone. Examples include morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleoside surrogates. Additional examples include morpholinos having nucleobases (such as adenine, cytosine, guanine, and thymine) linked to the morpholine ring, and phosphorodiamidate morpholino oligomers (PMOs), which are DNA analogs constructed on a morpholine ring backbone linked by phosphorodiamide bonds.

[0206] Sugar modifications

[0207] Modified RNAs can include modifications of all or some of the sugar groups of ribonucleic acids. For example, the 2'-hydroxyl (OH) can be modified or replaced by many different "oxy" or "deoxy" substituents. Without being bound by theory, enhanced stability is expected since the hydroxyl group can no longer be deprotonated to form a 2'-alkoxide ion. The 2'-alkoxide can catalyze degradation by an intramolecular nucleophilic attack on the linker phosphorus atom. Similarly, without wishing to be bound by theory, some embodiments may require the introduction of alterations that prevent the formation of an alkoxide at the 2' position.

[0208] Examples of "oxy"-2'-hydroxyl modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CH2O) n CH2CH2OR; "locked" nucleic acid (LNA), where the 2'-hydroxyl is linked, for example, by a methylene bridge to the 4'-carbon of the same ribose; O-amine (amine = NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamine) and aminoalkoxy, O(CH2) n amine (e.g., amine = NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamine). Notably, oligonucleotides containing only methoxyethyl (MOE), (OCH2CH2OCH3, a PEG derivative), or 2'-O-methoxyethyl exhibit nuclease stability comparable to that of oligonucleotides modified with robust phosphorothioate modifications.

[0209] "Deoxy" modifications include hydrogen (i.e., deoxyribose, which is particularly relevant to the overhang portions of some dsRNAs); halogen (e.g., fluorine); amino (e.g., N 3 / 4; alkylamino, dialkylamino, heterocyclic group, arylamino, diarylamino, heteroarylamino, diheteroarylamino or amino acid); NH(CH2CH2NH)nCH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclic group, arylamino, diarylamino, heteroarylamino or diheteroarylamino), -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, and these groups may optionally be substituted by, for example, amino functional groups. Preferred substituents are 2'-methoxyethyl, 2'-OCH3, 2'-O-allyl, 2'-C-allyl and 2'-fluoro.

[0210] The sugar moiety may also contain one or more carbons having a stereochemical configuration opposite to that of the corresponding carbon in ribose. Thus, the oligonucleotide may include nucleotides containing, for example, arabinose as the sugar. The monomer may have an α-bond at the position on the sugar, such as an α-nucleoside. The oligonucleotide may also include "abasic" sugars lacking a nucleobase at the C-. These abasic sugars may further contain modifications at one or more positions of the constitutive sugar atoms. The oligonucleotide may also contain one or more sugars in the L-form, such as L-nucleosides.

[0211] Terminal modifications

[0212] The 3'-end and 5'-end of the oligonucleotide may be modified. These modifications may be at the 3'-end, 5'-end or both ends of the molecule. It may include modifying or substituting one or more of the atoms of the entire terminal phosphate or phosphate group. For example, the 3'-end and 5'-end of the oligonucleotide may be bound to other functional molecular entities, such as a labeling moiety, such as a fluorophore (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dye) or a protecting group (based on, for example, sulfur, silicon, boron or ester). The functional molecular entity may be linked to the sugar through a phosphate group and / or a linker. The terminal atom of the linker may be linked to or substitute the connecting atom of the phosphate group or the C-3' or C-5' O, N, S or C group of the sugar. Alternatively, the linker may be linked to or substitute the terminal atom of a nucleotide analogue (e.g., PNA). When the linker / phosphate functional molecular entity-linker / phosphate array is inserted between the two strands of the dsRNA, this array may replace the hairpin RNA loop in the hairpin-type RNA agent.

[0213] Terminal modifications that can be used to modulate activity include modification of the 5'-end with phosphates or phosphate analogs. For example, in a preferred embodiment, the antisense strand of the dsRNA is 5'-phosphorylated or includes a phosphorothioate analog at the 5'-prime terminus. 5'-phosphate modifications include those that are compatible with RISC-mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P-O-5'); 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap (Appp) and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (thiophosphate; (HO)2(S)P-O-5'); 5'-mono-dithiophosphate (dithiophosphate; (HO)(HS)(S)P-O-5'), 5'-phosphorothioate ((HO)2(O)P-S-5'); any additional combination of monophosphate, diphosphate, and triphosphate substituted with oxygen / sulfur (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonate (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonate (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-).

[0214] Terminal modifications can also be used to monitor distribution, and in such embodiments, preferred groups to be added include fluorophores such as fluorescein or Alexa dyes such as Alexa 488. Terminal modifications can also be used to enhance uptake, and modifications suitable for this include cholesterol. Terminal modifications can also be used to crosslink the RNA agent to another moiety; modifications suitable for this include mitomycin C.

[0215] Nucleobase

[0216] Adenine, guanine, cytosine, and uracil are the most common bases in RNA. These bases can be modified or substituted to provide RNAs with improved properties. For example, nuclease-resistant oligoribonucleotides can be prepared using these bases or using synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any of the above modifications. Alternatively, substituted or modified analogs of any of the above bases can be employed, such as the "unusual bases," "modified bases," "unnatural bases," and "universal bases" described herein. Examples include, but are not limited to, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, 8-halo, amino, thiol, thioalkyl, hydroxy, and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkylcytosine-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N4-acetylcytosine, 2-mercaptocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine, or O-alkylated bases.Other purines and pyrimidines include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Concise Encyclopedia Of Polymer Science And Engineering, pages 858 - 859, edited by J.I. Roschwitz, John Wiley & Sons, 1990, and those disclosed in Uwe Englisch et al., Chemically Modified Oligonucleotides as Probes and Inhibitors, 1991 Jun., Angewandte Chemie, International Edition, 30:613 - 629, doi:10.1002 / anie.199106133.

[0217] Cationic group

[0218] Modification of the oligonucleotide may also include attaching one or more cationic groups to the phosphorus atoms of the sugar, base, and / or phosphate or modified phosphate backbone moiety. The cationic group may be attached to any atom capable of substitution on a natural, unusual, or universal base. Preferred positions are those that do not interfere with hybridization, i.e., do not interfere with the hydrogen - bond interactions required for base pairing. The cationic group may be attached, for example, through the C2' position of the sugar or a similar position in a cyclic or acyclic sugar substitute.

[0219] The cationic group may include, for example, a protonated amino group, which is derived from, for example, O - amine (amine = N 3 / 4; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamine); aminoalkoxy, such as O(CH2)n amine (e.g., amine = NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamine); amino (e.g., NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or NH(CH2CH2NH)nCH2CH2 - amine (amine = NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino).

[0220] Placement within the oligonucleotide

[0221] Some modifications may preferably be included at specific positions on the oligonucleotide, such as internal positions of the strand, or on the 5' or 3' end of the oligonucleotide. The preferred positions of modification on the oligonucleotide may confer better properties to the reagent. For example, the preferred positions of specific modifications may confer optimal gene silencing properties, or resistance to increased endonuclease or exonuclease activity.

[0222] One or more nucleotides of the oligonucleotide may have a 2'-5' linkage. One or more nucleotides of the oligonucleotide may have an inverted linkage, such as a 3'-3', 5'-5', 2'-2' or 2'-3' linkage.

[0223] The double-stranded oligonucleotide may include at least one 5'-uridine-adenine-3' (5'-UA-3') dinucleotide, wherein the uridine is a 2'-modified nucleotide; or a terminal 5'-uridine-guanine-3' (5'-UG-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide; or a terminal 5'-cytidine-adenine-3' (5'-CA-3') dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide; or a terminal 5'-uridine-uridine-3' (5'-UU-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide; or a terminal 5'-cytidine-cytidine-3' (5'-CC-3') dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide; or a terminal 5'-cytidine-uridine-3' (5'-CU-3') dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide; or a terminal 5'-uridine-cytidine-3' (5'-UC-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide. Double-stranded oligonucleotides comprising these modifications are particularly stable against endonuclease activity.

[0224] General References

[0225] The oligoribonucleotides and oligoribonucleosides used according to the technology of the present invention can be synthesized by solid-phase synthesis, see, for example, "Oligonucleotide synthesis, a practical approach", edited by M.J. Gait, IRL Press, 1984; "Oligonucleotides and Analogues, A Practical Approach", edited by F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aided methods of oligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter 3, 2'-O-Methyloligoribonucleotide-s:synthesis and applications, Chapter 4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of oligo-2'-deoxyribonucleoside methylphosphonates, and Chapter 7, Oligodeoxynucleotides containing modified bases).Other particularly applicable synthetic procedures, reagents, blocking groups and reaction conditions are described in P. Martin. Ein neuer Zugang zu 2'-O-Alkylribonucleosiden und Eigenschaften deren Oligonucleotide. March 22, 1995. Helv. Chim. Acta. 78:486-504. doi:10.1002 / hlca.19950780219; S. L. Beaucage and R. P. Iyer. Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach. March 20, 1992. Tetrahedron. 48:2223-2311. doi:10.1016 / S0040-4020(01)88752-4; S. L. Beaucage and R. P. Iyer. The synthesis of modified oligonucleotides by the phosphoramidite approach and their applications. July 9, 1993. Tetrahedron. 49:6123-6194. doi:10.1016 / S0040-4020(01)87958-8, or in the references cited therein. Modifications described in WO 00 / 44895, WO 01 / 75164 or WO 02 / 44321 may be used herein. The disclosures of all publications, patents and published patent applications listed herein are hereby incorporated by reference.

[0226] The preparation of phosphite oligoribonucleotides is described in U.S. Patent No. 5,508,270. The preparation of alkylphosphonate oligoribonucleotides is described in U.S. Patent No. 4,469,863. The preparation of phosphoramidite oligoribonucleotides is described in U.S. Patent No. 5,256,775 or U.S. Patent No. 5,366,878. The preparation of phosphotriester oligoribonucleotides is described in U.S. Patent No. 5,023,243. The preparation of boranophosphate oligoribonucleotides is described in U.S. Patent No. 5,130,302 and U.S. Patent No. 5,177,198. The preparation of 3'-deoxy-3'-aminophosphoramidite oligoribonucleotides is described in U.S. Patent No. 5,476,925. 3'-Deoxy-3'-methylenephosphonate oligoribonucleotides are described in Haoyun An et al. Synthesis of novel 3'-C-methylene thymidine and 5-methyluridine / cytidine H-phosphonates and phosphonamidites for new backbone modification of oligonucleotides. March 16, 2001. The Journal of Organic Chemistry. 66(8), pp. 2789-2801. doi:10.1021 / jo001699u. The preparation of sulfur-bridged nucleotides is described in Brian S. Sproat et al. Synthesis of Modified Building Blocks Containing Amino or Thiol Moieties: Application of Modified Oligodeoxyribonucleotides. December 6, 2006. Nucleosides Nucleotides. 7:651-653. doi:10.1080 / 07328318808056302 and Crosstick et al. Tetrahedron Lett. 1989, 30, 4693.

[0227] Modifications to the 2'-sugar moiety can be found in S. Verma et al., MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users, July 1998, Annu. Rev. Biochem. 67:99-134, doi:10.1146 / annurev.biochem.67.1.99 and all references therein. Specific modifications to ribose can be found in the following references: 2'-fluoro (Kawasaki et al., Uniformly modified 2'-deoxy-2'-fluoro-phosphorothioate oligonucleotides as nuclease-resistant antisense compounds with high affinity and specificity for RNA targets, April 1, 1993, J. Med. Chem. 36:831-841, doi:10.1021 / jm00059a007), 2'-MOE (P. Martin, Stereoselektive Synthese von 2'-O-(2-Methoxyethyl)ribonucleosiden: Nachbargruppenbeteiligung der Methoxyethoxy-Gruppe bei der Ribosylierung von Heterocyclen, October 30, 1996, Helv. Chim. Acta. 79:1930-1938, doi:10.1002 / hlca.19960790716), "LNA" (J. Wengel, Synthesis of 3'-C- and 4'-C-Branched Oligodeoxynucleotides and the Development of Locked Nucleic Acid (LNA), December 4, 1998, Acc. Chem. Res. 32:301-310, doi:10.1021 / ar980051p).

[0228] Methylene methylimino-linked oligoribonucleosides (also identified herein as MMI-linked oligoribonucleosides), methylene dimethylhydrazine-linked oligoribonucleosides (also identified herein as MDH-linked oligoribonucleosides), and methylene carbonylamino-linked oligoribonucleosides (also identified herein as amide-3-linked oligoribonucleosides), and methylene aminocarbonyl-linked oligoribonucleosides (also identified herein as amide-4-linked oligoribonucleosides), and mixed backbone compounds having, for example, alternating MMI and PO or PS linkages can be described as in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and published PCT applications PCT / US92 / 04294 and PCT / US92 / 04305 (published as WO 92 / 20822 and WO 92 / 20823, respectively). Acetal and thioacetal-linked oligoribonucleosides can be prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxide-linked oligoribonucleosides can be prepared as described in U.S. Pat. No. 5,223,618. Siloxane substitution is described in James F. Cormier et al. Synthesis of hexanucleotide analogues containing diisopropylsilyl internucleotide linkages. May 25, 1988. Nucleic Acids Res. 16:4583-4594. doi:10.1093 / nar / 16.10.4583. Carbonate substitution is described in J.R. Tittensor. The preparation of nucleoside carbonates. January 1971. Chem. Soc. C. 2656-2662. doi:10.1039 / J39710002656. Carboxymethyl substitution is described in M.D. Edge et al. Synthetic analogues of polynucleotides. Part VIII. Analogues of oligonucleotides containing carboxymethylthymidine. 1991. J. Chem. Soc. Perkin Trans. 1. 1972. doi:10.1039 / P19720001991.The carbamate substitutions are described in E.P. Stirchak et al., Uncharged stereoregular nucleic acid analogs: 2. Morpholinonucleoside oligomers with carbamate internucleoside linkages, Aug. 11, 1989, Nucleic Acids Res. 17, 6129, doi:10.1093 / nar / 17.15.6129.

[0229] Cyclobutyl sugar substitute compounds can be prepared as described in U.S. Patent No. 5,359,044. Pyrrolidine sugar substitutes can be prepared as described in U.S. Patent No. 5,519,134. Morpholino sugar substitutes can be prepared as described in U.S. Patent Nos. 5,142,047 and 5,235,033 and other related patent publications. Peptide nucleic acids (PNA) are known per se and can be prepared according to any of the various procedures mentioned in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5 - 23. It can also be prepared according to U.S. Patent No. 5,539,083.

[0230] Terminal modifications are described in M. Manoharan et al., Oligonucleotide Conjugates as Potential Antisense Drugs with Improved Uptake, Biodistribution, Targeted Delivery, and Mechanism of Action, Jul. 8, 2004, Antisense and Nucleic Acid Drug Development, 12, 103 - 128, doi:10.1089 / 108729002760070849 and the references therein. Nucleobase reference N - 2 substituted purine nucleoside amidites can be prepared as described in U.S. Patent No. 5,459,255. 3 - Deazapurine nucleoside amidites can be prepared as described in U.S. Patent No. 5,457,191. 5,6 - Substituted pyrimidine nucleoside amidites can be prepared as described in U.S. Patent No. 5,614,617. 5 - Propargyl pyrimidine nucleoside amidites can be prepared as described in U.S. Patent No. 5,484,908.

[0231] Manufacture of ASO

[0232] The ASO (e.g., duplex ASO) used in accordance with the technology of the present invention can be conveniently and routinely prepared via well-known solid-phase synthesis techniques. Equipment for such synthesis is sold by several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). A method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066.

[0233] Any other means known in the art for such synthesis can be additionally or alternatively employed. Oligonucleotides are well-known to be prepared using similar techniques, such as phosphorothioates and alkylated derivatives. In one such automated embodiment, diethyl-phosphoramidite is used as the starting material and can be synthesized as described below: S.L. Beaucage et al. Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis. Mar. 9, 2001. Tetrahedron Letters. 22:1859-1862. doi:10.1016 / S0040-4039(01)90461-7.

[0234] The ASO of the technology of the present invention is synthesized in vitro and does not include ASO of biological origin or gene vector constructs designed to direct in vivo synthesis of ASO. The molecules of the technology of the present invention can also be mixed, encapsulated, conjugated, or otherwise combined with mixtures of other molecules, molecular structures, or compounds (e.g., liposomes, receptor-targeting molecules, oral formulations, rectal formulations, topical formulations, or other formulations) for assisting uptake, distribution, and / or absorption.

[0235] Method for improving exon skipping efficacy and / or efficiency of an ASO comprising or consisting of a targeting sequence

[0236] In some aspects, the technology of the present invention provides a method for improving the exon skipping efficacy and / or efficiency of an ASO comprising or consisting of a targeting sequence, which comprises providing one or more bipartite ASOs comprising the targeting sequence and a 5'-splice-site decoy sequence disclosed herein. In certain embodiments, each decoy sequence in one or more bipartite ASOs comprises or consists of a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID No. 1 to 23 and SEQ ID No. 347 to 353. In certain embodiments, at least some of one or more bipartite ASOs have a decoy sequence operably linked to the 5'-end of the targeting sequence. In certain embodiments, at least some of one or more bipartite ASOs have a decoy sequence operably linked to the 3'-end of the targeting sequence. In certain embodiments, at least some of one or more bipartite ASOs have a decoy sequence operably linked to both the 5'-end and the 3'-end of the targeting sequence. In certain embodiments, at least some of one or more bipartite ASOs have a decoy sequence operably linked to only one of the 5'-end and the 3'-end of the targeting sequence.

[0237] In certain embodiments, the method further comprises screening and / or optimizing one or more bipartite ASOs based on their exon skipping efficacy and / or efficiency. In certain embodiments, compared to an ASO having the same targeting sequence but no decoy sequence, the bipartite ASO increases the exon skipping efficiency of the targeting sequence by about 2-fold or higher, about 3-fold or higher, about 4-fold or higher, about 5-fold or higher, about 6-fold or higher, about 7-fold or higher, about 8-fold or higher, about 9-fold or higher, about 10-fold or higher, about 11-fold or higher, about 12-fold or higher, about 13-fold or higher, about 14-fold or higher, about 15-fold or higher, about 16-fold or higher, about 17-fold or higher, about 18-fold or higher, about 19-fold or higher, about 20-fold or higher, about 25-fold or higher, about 30-fold or higher, about 40-fold or higher, about 50-fold or higher, about 60-fold or higher, about 70-fold or higher, about 80-fold or higher, about 90-fold or higher, about 100-fold or higher, including all values and the ranges between these values.

[0238] In certain embodiments, the targeting sequence is capable of hybridizing to a sequence selected from the group consisting of: a target exon, a flanking intron sequence upstream of the target exon, a flanking intron sequence downstream of the target exon, an intron-exon junction upstream of the target exon, and an intron-exon junction downstream of the target exon in a cell or a subject.

[0239] Compositions of bipartite ASOs

[0240] In some aspects, the technology of the present invention provides a composition comprising a bipartite ASO disclosed herein and a pharmaceutically acceptable carrier. In certain embodiments, the composition is a pharmaceutical formulation or composition.

[0241] A vector encoding a bipartite ASO

[0242] In some aspects, the technology of the present invention provides a vector encoding an ASO (such as a bipartite ASO) disclosed herein.

[0243] Use of a bipartite ASO for treating a disease

[0244] In some aspects, the technology of the present invention provides a method for treating a disease and / or its complications in a subject, which comprises administering to the subject an ASO (such as a bipartite ASO) disclosed herein, a composition (pharmaceutical composition) comprising an ASO (such as a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (such as a bipartite ASO) disclosed herein. The ASO (such as a bipartite ASO) may generate or promote exon skipping of a target exon during pre-mRNA splicing. ASO-mediated exon skipping may be a method of manipulating the expression of a target gene. The expression of the target gene can be manipulated by the ASO inhibiting the splicing of an exon, intron, or specific splicing site of the target gene, resulting in (for example, but not limited to): restoring the reading frame of the target defective gene, generating different subtypes of the target gene (such as dominant negative subtypes), skipping the toxic part of the gene, silencing the gene, and / or altering the structure and function of the gene.

[0245] In some aspects, the technology of the present invention provides the use of an ASO (such as a bipartite ASO) disclosed herein, a composition (such as a pharmaceutical composition) comprising an ASO (such as a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (such as a bipartite ASO) disclosed herein for treating a disease and / or its complications. Examples of diseases include but are not limited to those disclosed herein.

[0246] In some aspects, the technology of the present invention provides an ASO (such as a bipartite ASO) disclosed herein, a composition (such as a pharmaceutical composition) comprising an ASO (such as a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (such as a bipartite ASO) disclosed herein for treating a disease and / or its complications. Examples of diseases include but are not limited to those disclosed herein.

[0247] Examples of diseases and / or their complications include but are not limited to diseases and / or their complications that may benefit from exon skipping on one or more target genes. In certain embodiments, the target gene is selected from the group consisting of: SMN1, SMN2, DMD, APP, CEP290, HER2, SCA3, PKM, and MDM4 genes.

[0248] Examples of the disease and / or its complications include, but are not limited to, diseases and / or their complications that can be treated by exon skipping of a target exon. In certain embodiments, the target exon is selected from the group consisting of exon 7 of the endogenous SMN1 and SMN2 genes, exons 45, 51, and 53 of the endogenous DMD gene, exon 17 of the endogenous APP gene, exon 41 of the endogenous CEP290 gene, exon 19 of the endogenous HER2 gene, exon 10 of the SCA3 gene, exon 10 of the endogenous PKM gene, and exon 6 of the endogenous MDM4 gene.

[0249] Examples of the disease and / or its complications include, but are not limited to, diseases and / or their complications that can be treated by inhibiting splicing of an exon, intron, or specific splice site of a target gene.

[0250] Examples of the disease and / or its complications include, but are not limited to, diseases and / or their complications that can be treated by restoring the reading frame of a target defective gene.

[0251] Examples of the disease and / or its complications include, but are not limited to, diseases and / or their complications that can be treated by generating different subtypes (e.g., dominant negative subtypes) of a target gene.

[0252] Examples of the disease and / or its complications include, but are not limited to, diseases and / or their complications that can be treated by skipping the toxic portion of a gene.

[0253] Examples of the disease and / or its complications include, but are not limited to, diseases and / or their complications that can be treated by gene silencing.

[0254] Examples of the disease and / or its complications include, but are not limited to, diseases and / or their complications that can be treated by altering the structure and function of a gene to obtain a beneficial or desired subtype.

[0255] Examples of the disease and / or its complications include, but are not limited to, diseases and / or their complications that can be treated by generating a functional protein encoded by a different subtype of a target gene.

[0256] Examples of diseases and / or their complications can include, but are not limited to, Duchenne muscular dystrophy (DMD), Alzheimer's disease, Joubert syndrome, spinocerebellar ataxia type 3 (SCA3), cancer, such as but not limited to breast cancer, HER2-positive cholangiocarcinoma, colorectal cancer, non-small cell lung cancer, bladder cancer, prostate cancer, lung cancer, cervical cancer, renal cancer, papillary thyroid cancer, colon cancer, colorectal cancer, glioma, ovarian cancer, gastric cancer, hepatoblastoma, fibrolamellar hepatocellular carcinoma, soft tissue sarcoma, osteosarcoma, chronic lymphocytic leukemia, acute myeloid leukemia, mantle cell lymphoma, pediatric Burkitt lymphoma, salivary gland cancer, liver cancer, and melanoma.

[0257] In certain embodiments, an ASO (such as a duplex ASO), a composition comprising an ASO (such as a duplex ASO) (e.g., a pharmaceutical composition), and / or a vector encoding an ASO (such as a duplex ASO) are administered in a therapeutically effective amount.

[0258] Examples of subjects include, but are not limited to, mammals, such as humans.

[0259] In some embodiments, one or more ASOs designed using the methods of one or more of the techniques of the present invention can be used to treat diseases that can be treated by exon skipping. For example, spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by the expansion of a CAG trinucleotide in exon 10 of the ATXN3 gene. ASOs can be used to promote the skipping of exon 9, exon 10, or both to favor a potentially functional or partially functional or non-toxic ATXN2 variant.

[0260] "Treatment" of an individual (e.g., a mammal, such as a human) or a cell is any type of intervention used to attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition or combination therapy, and can be performed prophylactically or after the onset of a pathological event or exposure to a pathogen. Treatment includes any desired effect on the symptoms or pathology of a disease or condition associated with dystrophin, as in some forms of muscular dystrophy, and can include, for example, a minimal change or improvement in one or more measurable markers of the disease or condition being treated. Also included is "prophylactic" treatment, which can be used to reduce the rate of progression of the disease or condition being treated, delay the onset of the disease or condition, or reduce the severity of its onset. "Treatment" or "prevention" does not necessarily indicate the complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

[0261] In some embodiments, treatment with one or more ASOs of the technology of the present invention or treatment with a combination of one or more ASOs of the technology of the present invention and one or more additional therapeutic agents (combination therapy) induces or increases the de novo production of a functional protein encoded by a target gene (e.g., dystrophin in DMD), delays disease progression, alleviates or reduces the symptoms of the disease (e.g., loss of ambulatory ability for DMD, reduced muscle inflammation, reduced muscle damage, improved muscle function, reduced loss of lung function, and / or enhanced muscle regeneration), or any combination thereof, which would be expected to occur in the absence of treatment. In some embodiments, the treatment maintains, delays, or slows disease progression.

[0262] In some embodiments, when the disease is DMD, the treatment maintains ambulatory ability or reduces the loss of ambulatory ability. In some embodiments, the treatment maintains lung function or reduces the loss of lung function. In some embodiments, the treatment maintains or increases the patient's stable walking distance, as measured, for example, by the 6-minute walk test (6MWT). In some embodiments, the treatment maintains, improves, or reduces the time to walk / run 10 meters (i.e., the 10-meter walk / run test). In some embodiments, the treatment maintains, improves, or reduces the time to stand up from a supine position (i.e., the timed get-up-and-go test). In some embodiments, the treatment maintains, improves, or reduces the time to climb four standard stairs (i.e., the four-step climb test). In some embodiments, the treatment maintains, improves, or reduces the patient's muscle inflammation, as measured, for example, by MRI (e.g., MRI of the leg muscles). In some embodiments, the MRI measures changes in the calf muscles. In some embodiments, the MRI measures T2 and / or fat content to identify muscle degeneration. The MRI can identify changes in muscle structure and composition caused by inflammation, edema, muscle damage, and fat infiltration. In some embodiments, muscle strength is measured by the North Star Ambulatory Assessment. In some embodiments, muscle strength is measured by the pediatric outcomes data collection instrument (PODCI).

[0263] In some embodiments, treatment is carried out with one or more ASOs of the technology of the present invention or with a combination of one or more ASOs of the technology of the present invention and one or more additional therapeutic agents (combination therapy).

[0264] In some embodiments, when the disease is DMD, such treatment reduces muscle inflammation, reduces muscle damage, improves muscle function, and / or enhances muscle regeneration. For example, the treatment can stabilize, maintain, improve, or reduce inflammation in a subject. The treatment can also, for example, stabilize, maintain, improve, or reduce muscle damage in a subject. The treatment can, for example, stabilize, maintain, or improve muscle function in a subject. Additionally, for example, the treatment can stabilize, maintain, improve, or enhance muscle regeneration in a subject. In some embodiments, the treatment maintains, improves, or reduces the muscle inflammation that would be expected in the patient in the absence of treatment, as measured, for example, by magnetic resonance imaging (MRI) (such as an MRI of the leg muscles). In some embodiments, treatment with one or more ASOs of the technology of the present invention, or treatment with a combination of one or more ASOs of the technology of the present invention and one or more additional therapeutic agents (combination therapy) increases the production of new dystrophin and slows or reduces the loss of walking ability that would be expected in the absence of treatment. For example, the treatment can stabilize, maintain, improve, or increase the walking ability of a subject (such as the stability of walking). In some embodiments, the treatment maintains or increases the stable walking distance of a patient, as measured, for example, by the 6-minute walk test (6MWT), which is described by Craig M. McDonald et al., "The 6-minute walk test in Duchenne / Becker muscular dystrophy: Longitudinal observations," Muscle Nerve, October 29, 2010, 42:966 - 74, doi:10.1002 / mus.21808, which is incorporated herein by reference). The change in the 6-minute walk distance (6MWD) can be expressed as an absolute value, a percentage change, or a % predicted value change. In some embodiments, the treatment enables a subject to maintain or improve a stable walking distance in the 6MWT, where the subject has a 20% baseline deficit in walking distance relative to healthy peers. The performance of DMD patients in the 6MWT relative to the typical performance of healthy peers can be determined by calculating the % predicted value. For example, the % predicted value of 6MWD for males can be calculated using the following equation: 196.72+(39.81×age)-(1.36×age 2 )+(132.28×height (in meters)). For females, the % predicted value of 6MWD can be calculated using the following equation: 188.61+(51.50×age)-(1.86×age 2) + (86.10 × height (in meters)) (Henricson et al., PloS Curr., 2012, 2nd ed., incorporated herein by reference). In some embodiments, treatment increases the patient's stable walking distance from baseline to greater than 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 50 meters (including all integers therebetween). Muscle function loss in DMD patients may occur in the context of normal childhood growth and development. Indeed, although muscle damage is progressive, younger children with DMD may show an increase in walking distance during the 6MWT over the course of about 1 year. In some embodiments, the 6MWD from DMD patients is compared to that of normal developing control subjects and existing standardized data from age- and sex-matched subjects. In some embodiments, age- and height-based equations fitted to the standardized data may be used to account for normal growth and development. This equation can be used to convert the 6MWD of DMD subjects into a percent predicted (%) value. In some embodiments, analysis of the 6MWD % predicted data represents a method of accounting for normal growth and development and may show that functional gains in the early stage (e.g., less than or equal to 7 years) represent the stable rather than improved ability of DMD patients (Henricson et al., PloS Curr., 2012, 2nd ed., incorporated herein by reference).

[0265] Pharmaceutical formulations (compositions) and delivery

[0266] In some embodiments, the technology of the present invention provides formulations or compositions suitable for the therapeutic delivery of the ASOs described herein. Thus, in some embodiments, the technology of the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of the oligomers described herein formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. Although the oligomers of the technology of the present invention may be administered alone, it is preferred to administer the compounds in the form of a pharmaceutical formulation (composition).

[0267] Methods for delivering nucleic acid molecules are described, for example, in Akhtar et al., Cellular uptake and intracellular fate of antisense oligonucleotides, May 1992, Trends in Cell Bio. 2:139-144, doi:10.1016 / 0962-8924(92)90100-2; and Delivery Strategies for ASO Therapeutics, Akhtar, ed.; and Sullivan et al., PCT WO 94 / 02595. These and other protocols can be used to deliver virtually any nucleic acid molecule, including the isolated oligomers of the technology of the present invention.

[0268] As detailed below, the pharmaceutical compositions of the technology of the present invention can be specifically formulated for administration in solid or liquid form, including those forms suitable for: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., tablets for oral, sublingual, and systemic absorption), pills administered to the tongue, powders, granules, pastes; (2) parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection in the form of, for example, a sterile solution or suspension or a sustained release formulation; (3) topical application, such as to the skin in the form of a cream, ointment, or a controlled release patch or spray; (4) intravaginal or rectal, such as in the form of a pessary, cream, or foam; (5) sublingual; (6) ophthalmic; (7) transdermal; or (8) nasal.

[0269] Some examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffering solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical formulations.

[0270] Additional non-limiting examples of reagents formulated with ASOs of the technology of the present invention include: PEG-conjugated nucleic acids, phospholipid-conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors such as Pluronic P85, which can enhance drug entry into various tissues; biodegradable polymers such as poly(DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (D.F. Emerich et al. Biocompatibility of Poly(DL-Lactide-co-Glycolide) Microspheres Implanted into the Brain. January 1999. Cell Transplant. 8, 47-58. doi:10.1177 / 096368979900800114) Alkermes, Inc., Cambridge, Mass.; and drug-loaded nanoparticles such as drug-loaded nanoparticles made of polybutylcyanoacrylate, which can deliver drugs across the blood-brain barrier and can alter the neuronal uptake mechanism (U. Schroeder et al. Diffusion enhancement of drugs by loaded nanoparticles in vitro. July 1, 1999. Prog Neuropsychopharmacol Biol Psychiatry. 23, 941-949. doi:10.1016 / s0278-5846(99)00037-8).

[0271] The features of the technology of the present invention also lie in the use of a composition comprising surface-modified liposomes, which liposomes contain poly(ethylene glycol) lipids (PEG-modified, branched and unbranched or in a combined form thereof, or long-circulating liposomes or stealth liposomes). The oligomers of the technology of the present invention may also comprise covalently linked PEG molecules of various molecular weights. These formulations provide a method for increasing the amount of drug accumulation in target tissues. Such drug carriers resist opsonization and clearance by the mononuclear phagocyte system (MPS or RES), thereby achieving a longer blood circulation time for the encapsulated drug and enhanced tissue exposure (Danilo D. Lasic et al. The "Stealth" Liposome: A Prototypical Biomaterial. December 1, 1995. Chem. Rev. 95, 2601-2627. doi:10.1021 / cr00040a001; H. Ishiwata et al. Physical-Chemistry Characteristics and Biodistribution of Poly(ethylene glycol)-Coated Liposomes Using Poly(oxyethylene) Cholesteryl Ether. June 1995. Chem. Pharm. Bull. 43, 1005-1011. doi:10.1248 / cpb.43.1005). Such liposomes have been shown to accumulate selectively in tumors, possibly through extravasation and capture in neovascularized target tissues (Danilo D. Lasic et al. Liposomes Revisited. March 3, 1995. Science. 267, 1275-1276. doi:10.1126 / science.7871422; Naoto Oku et al. Real-time analysis of liposomal trafficking in tumor-bearing mice by use of positron emission tomography. August 23, 1995. Biochimica et Biophysica Acta (BBA)-Biomembranes. 1238, 86-90. doi:10.1016 / 0005-2736(95)00106-D).Long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes known to accumulate in tissues of MPS (Y. Liu et al., Cationic liposome-mediated intravenous gene delivery, October 20, 1995, J. Biol. Chem. 42, 24864-24870, doi:10.1074 / jbc.270.42.24864; Choi et al., International PCT Publication No. WO 96 / 10391; Ansell et al., International PCT Publication No. WO 96 / 10390; Holland et al., International PCT Publication No. WO 96 / 10392). Compared to cationic liposomes, long-circulating liposomes can also protect the drug from nuclease degradation to a greater extent, based on their ability to avoid accumulation in metabolically invasive MPS tissues such as the liver and spleen.

[0272] In some embodiments, the techniques of the present invention include preparing oligomeric compositions for delivery as described in U.S. Patent Nos. 6,692,911, 7,163,695, and 7,070,807. In this regard, in one embodiment, the techniques of the present invention provide an oligomer of the present invention in the form of a composition that comprises a copolymer of lysine and histidine (HK) alone (as described in U.S. Patent Nos. 7,163,695, 7,070,807, and 6,692,911) or a combination thereof with PEG (e.g., branched or unbranched PEG or a mixture of both), a combination of PEG and a targeting moiety, or any of the foregoing with a cross-linking agent. In some embodiments, the techniques of the present invention provide an ASO in the form of a composition that comprises polyhistidine modified with gluconic acid or gluconylated polyhistidine / transferrin-polylysine. Those skilled in the art will also recognize that amino acids having properties similar to His and Lys can be substituted in the composition.

[0273] Some embodiments of the oligomers described herein may contain basic functional groups, such as amino or alkylamino groups, and are thus capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. These salts can be prepared in situ during the formulation of a pharmaceutical composition or dosage form, or by separately reacting the purified compound of the technology of the present invention in free base form with a suitable organic or inorganic acid and isolating the salt thus formed during subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate and the like. (See, e.g., S.M. Berge et al., Pharmaceutical Salts, January 1977, J. Pharm. Sci. 66:1-19, doi:10.1002 / jps.2600660104).

[0274] Pharmaceutically acceptable salts of the oligomers of the present invention include, for example, conventional non-toxic salts or quaternary ammonium salts of compounds derived from non-toxic organic or inorganic acids. By way of example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and the like.

[0275] In some embodiments, the oligomers of the technology of the present invention may contain one or more acidic functional groups and are thus capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. These salts can likewise be prepared in situ during the formulation of a pharmaceutical composition or dosage form, or by separately reacting the purified compound in free acid form with a suitable base such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali metal or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines suitable for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like. (See, e.g., S.M. Berge et al., supra).

[0276] Humectants, emulsifiers and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as colorants, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition.

[0277] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

[0278] The formulations of the present invention include formulations suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations may be presented in unit dosage form and may be prepared by any method well known in the pharmaceutical art. The amount of the active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending on the host to be treated and the particular mode of administration. The amount of the active ingredient that may be combined with the carrier material to produce a single dosage form will generally be the amount of the compound that produces a therapeutic effect. Generally, in percentages, this amount will be in the range of about 0.1% to about 99% active ingredient, or about 5% to about 70%, or about 10% to about 30%.

[0279] In some embodiments, the formulations of the present invention include excipients selected from cyclodextrins, celluloses, liposomes, micelle-forming agents (such as bile acids) and polymeric carriers (such as polyesters and polyanhydrides); and the oligomers of the present invention. In some embodiments, the aforementioned formulations enable the oligomers of the present invention to have oral bioavailability.

[0280] The methods for preparing these formulations or compositions include the step of combining the oligomers of the present invention with a carrier and optionally one or more accessory ingredients. Generally, the formulations are prepared by uniformly and closely combining the compounds of the present invention with a liquid carrier or a finely divided solid carrier or both a liquid carrier and a finely divided solid carrier and then shaping the product if necessary.

[0281] The formulations of the technology of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using flavored bases, usually sucrose and gum arabic or tragacanth), powders, granules, or in the form of solutions or suspensions in aqueous or non-aqueous liquids, or in the form of water-in-oil or oil-in-water liquid emulsions, or in the form of elixirs or syrups, or in the form of troches (using inert bases such as gelatin and glycerol, or sucrose and gum arabic) and / or mouthwashes and the like, each containing a predetermined amount of the compound of the technology of the present invention as an active ingredient. The oligomers of the technology of the present invention may also be administered in the form of boluses, pastilles or pastes.

[0282] In the solid dosage forms (capsules, tablets, pills, dragees, powders, granules, lozenges and the like) of the technology of the present invention for oral administration, the active ingredient may be admixed with one or more pharmaceutically acceptable carriers such as sodium citrate or calcium phosphate dibasic and / or any one of the following: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or gum arabic; (3) humectants such as glycerol; (4) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates and sodium carbonate; (5) solution retarders such as paraffin wax; (6) absorption promoters such as quaternary ammonium compounds, and surfactants such as poloxamer and sodium lauryl sulfate; (7) wetting agents such as cetyl alcohol, glycerol monostearate and nonionic surfactants; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid and mixtures thereof; (10) coloring agents; and (11) controlled release agents such as crospovidone or ethyl cellulose. In the case of capsules, tablets and pills, the pharmaceutical composition may also contain buffering agents. Excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like may also be used to use similar types of solid compositions as fillers in soft and hard shell gelatin capsules.

[0283] Lozenges may optionally be compressed or molded with one or more accessory ingredients. Compressed lozenges may be prepared using binders (such as gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (such as sodium carboxymethyl starch or cross-linked sodium carboxymethyl cellulose), surfactants or dispersants. Molded tablets may be produced by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0284] Tablets and other solid dosage forms (such as dragees, capsules, pills, and granules) of the pharmaceutical composition of the present invention technology may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well-known in pharmaceutical formulation technology. It may also be formulated using, for example, hydroxypropyl methylcellulose, other polymer matrices, liposomes, and / or microspheres in different proportions to provide the desired release characteristics so as to provide slow or controlled release of the active ingredient therein. It may be formulated for rapid release, such as by lyophilization. It may be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that is soluble in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be compositions that release the active ingredient solely or preferentially in a delayed manner in a part of the gastrointestinal tract. Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient may also be in the form of microencapsulation, together with one or more of the above excipients, where appropriate.

[0285] Liquid dosage forms for oral administration of the compounds of the present invention technology include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art (such as water or other solvents), solubilizing agents, and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan and their mixtures.

[0286] In addition to the inert diluent, oral compositions may also include adjuvants, such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, coloring agents, fragrances, and preservatives.

[0287] In addition to the active compound, suspensions may also contain suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar, and tragacanth and their mixtures.

[0288] Preparations for rectal or vaginal administration may be presented in the form of suppositories, which may be prepared by mixing one or more compounds of the present invention technology with one or more suitable non-irritating excipients or carriers, which include, for example, cocoa butter, polyethylene glycol, suppository wax, or salicylates, which are solid at room temperature but liquid at body temperature and thus will melt in the rectal or vaginal cavity and release the active compound.

[0289] Formulations or dosage forms for topical or transdermal administration of oligomers as provided herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active oligomers can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants as may be required. In addition to the active compounds of the present technology, ointments, pastes, creams, and gels can contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0290] In addition to the oligomers of the present technology, powders and sprays can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays can additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0291] Transdermal patches have the additional advantage of providing controlled delivery of the oligomers of the present technology to the body. Such dosage forms can be prepared by dissolving or dispersing the oligomers in a suitable medium. Penetration enhancers can also be used to increase the flux of the agent through the skin. In addition to other methods known in the art, the rate of such flux can be controlled by providing a rate controlling membrane or by dispersing the agent in a polymeric matrix or gel.

[0292] Pharmaceutical compositions suitable for parenteral administration can comprise one or more oligomers of the present technology and one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or combinations of sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, which can contain sugars, alcohols, antioxidants, buffers, bacteriostatic agents, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present technology include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0293] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms on the oligomers of the invention can be ensured by various antibacterial and antifungal agents including, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be necessary to include in the composition isotonic agents such as sugars, sodium chloride, and the like. In addition, prolonged absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption such as aluminum monostearate and gelatin.

[0294] In some embodiments, to prolong the action of a drug, it is necessary to slow the absorption of the drug following subcutaneous or intramuscular injection. In addition to other methods known in the art, this can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous material. The rate of drug absorption then depends on its rate of dissolution, which in turn can depend on crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered pharmaceutical forms is achieved by dissolving or suspending the drug in an oily vehicle.

[0295] Injectable depot forms can be prepared by forming microencapsule matrices of the oligomers of the subject invention in biodegradable polymers such as poly(lactic-co-glycolic acid). The rate of oligomer release can be controlled according to the ratio of the oligomer to the polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot forms can also be prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues.

[0296] When the ASOs of the technology of the invention are administered to humans and animals in pharmaceutical form, they can be administered per se or in the form of a pharmaceutical composition containing, for example, from 0.1% to 99% (more preferably from 10% to 30%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0297] As noted above, the formulations or preparations of the technology of the invention can be administered orally, parenterally, topically, or rectally. It is generally administered in a form suitable for each route of administration. For example, it is administered in the form of tablets or capsules, by injection, inhalation, eye wash, ointment, suppository, etc., by injection, infusion, or inhalation; topically by lotion or ointment; and rectally by suppository.

[0298] As used herein, the phrases "parenteral administration" and "administered parenterally" mean a mode of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0299] As used herein, the terms "systemic administration", "systemically administered", "peripheral administration" and "peripherally administered" mean the administration of a compound, drug or other material other than directly to the central nervous system such that it enters the patient's system and thus undergoes metabolism and other similar processes, such as subcutaneous administration.

[0300] Regardless of the chosen route of administration, the ASO of the technology of the present invention and / or the pharmaceutical composition of the technology of the present invention that can be used in a suitable hydrated form can be formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. The actual dosage level of the active ingredient in the pharmaceutical composition of the technology of the present invention can be varied so as to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic effect for a particular patient, composition and mode of administration and that does not impose unacceptable toxicity on the patient.

[0301] The selected dosage level will depend on a variety of factors, including the activity of the particular oligomer or its ester, salt or amide of the technology of the present invention used, the route of administration, the time of administration, the rate of excretion or metabolism of the particular oligomer used, the rate and extent of absorption, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular oligomer, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0302] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the required pharmaceutical composition. For example, the physician or veterinarian can begin administering the compound of the technology of the present invention used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, the suitable daily dosage of the compound of the technology of the present invention will be the amount of the compound that is the lowest dosage effective in producing the therapeutic effect. This effective dosage will generally depend on the factors described above. In general, when used for a specified effect, the oral, intravenous, intraventricular and subcutaneous dosages of the compound of the technology of the present invention for a patient will be in the range of about 0.001 to about 1,000 mcg / g / day, about 0.01 to about 500 mcg / g / day, about 0.1 to about 200 mcg / g / day, about 1 to about 160 mcg / g / day or about 10 to about 150 mcg / g / day.

[0303] When necessary, the effective daily dose of the active compound can be administered in two, three, four, five, six or more sub-doses, which are optionally administered individually in unit dosage forms at appropriate time intervals within a day. In some cases, the dose is administered once a day. In some embodiments, as needed, the dose is administered once or more every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months to maintain the desired expression of functional dystrophin.

[0304] The nucleic acid molecule can be administered to cells by various methods known in the art, including but not limited to: encapsulation in liposomes, by iontophoresis or by incorporation into other vehicles (such as hydrogels, cyclodextrins, biodegradable nanocapsules and bioadhesive microspheres), as described herein and known in the art. In some embodiments, microemulsification techniques can be used to improve the bioavailability of lipophilic (water-insoluble) agents. Examples include Trimetrine (S.K. Dordunoo et al. Preformulation Studies on Solid Dispersions Containing Triamterene or Temazepamin Polyethylene Glycols or Gelucire 44 / 14 for Liquid Filling of Hard Gelatin Capsules. October 2008. Drug Development and Industrial Pharmacy. 17(12), 1685 - 1713. doi:10.3109 / 03639049109057315) and REV 5901 (P.C. Sheen et al. Bioavailability of a Poorly Water-Soluble Drug from Tablet and Solid Dispersion in Humans. July 1991. J. Pharm. Sci. 80(7), 712 - 714. doi:10.1002 / jps.2600800722). Among other benefits, microemulsification provides enhanced bioavailability by preferentially directing absorption through the lymphatic system rather than the circulatory system, thereby bypassing the liver and preventing destruction of the compound in the enterohepatic circulation.

[0305] In some embodiments, the formulation contains micelles formed from oligomers as provided herein and at least one amphiphilic carrier, wherein the average diameter of the micelles is less than about 100 nm. More preferred embodiments provide micelles with an average diameter less than about 50 nm, and even more preferred embodiments provide micelles with an average diameter less than about 30 nm or even less than about 20 nm.

[0306] While all suitable amphiphilic carriers are encompassed, currently preferred carriers are generally those that have a Generally-Recognized-as-Safe (GRAS) status and that can solubilize the compounds of the present technology when the solution is in contact with a complex aqueous phase such as that found in the human gastrointestinal tract and later microemulsify it. Generally, the hydrophilic-lipophilic balance (HLB) value of the amphiphilic components meeting these requirements is 2 - 20, and their structure contains a straight-chain aliphatic hydrocarbon group in the range of C-6 to C-20. Examples are polyethylene glycolated fatty glycerides and polyethylene glycol.

[0307] Examples of amphiphilic carriers include saturated and monounsaturated polyethylene glycolated fatty acid glycerides, such as those obtained from various vegetable oils that are fully or partially hydrogenated. These oils advantageously consist of triglycerides, diglycerides, and monoglycerides of fatty acids, as well as diglycol esters and monoglycol esters of the corresponding fatty acids, where a particularly preferred fatty acid composition includes 4% - 10% capric acid, 3% - 9% caprylic acid, 40% - 50% lauric acid, 14% - 24% myristic acid, 4% - 14% palmitic acid, and 5 - 15% stearic acid. Another useful class of amphiphilic carriers includes sorbitan and / or sorbitol (SPAN series) partially esterified with saturated or monounsaturated fatty acids or the corresponding ethoxylated analogs (TWEEN series).

[0308] Commercially available amphiphilic carriers can be particularly useful, including the Gelucire series, Labrafil, Labrasol, or Lauroglycol (all manufactured and distributed by Gattefosse Corporation, Saint Priest, France), PEG-monooleate, PEG-dioleate, PEG-monolaurate, and dilaurate, lecithin, polysorbate 80, etc. (produced and distributed by many companies in the United States and globally).

[0309] In some embodiments, delivery can be effected by using liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles and the like to introduce the compositions of the technology of the present invention into suitable host cells. In particular, the compositions of the technology of the present invention can be formulated for encapsulation in lipid particles, liposomes, vesicles, nanospheres, nanoparticles or the like for delivery. Such delivery vehicles can be formulated and used using known and conventional techniques.

[0310] Hydrophilic polymers suitable for the technology of the present invention are hydrophilic polymers that are soluble in water, can be covalently linked to vesicle-forming lipids, and are tolerated in vivo without toxic effects (i.e., biocompatible). Suitable polymers include polyethylene glycol (PEG), polylactic acid (also known as poly(lactide)), polyglycolic acid (also known as poly(glycolide)), poly(lactic acid - co - glycolic acid) copolymer, and polyvinyl alcohol. In some embodiments, the molecular weight of the polymer is from about 100 or 120 daltons to about 5,000 or 10,000 daltons, or from about 300 daltons to about 5,000 daltons. In other embodiments, the polymer is polyethylene glycol having a molecular weight of from about 100 to about 5,000 daltons, or a molecular weight of from about 300 to about 5,000 daltons. In some embodiments, the polymer is polyethylene glycol of 750 daltons (PEG(750)). The polymer can also be defined by the number of monomers therein; a preferred embodiment of the technology of the present invention utilizes a polymer having at least about three monomers, and this PEG polymer consists of three monomers (about 150 daltons).

[0311] Other hydrophilic polymers that can be suitable for the technology of the present invention include polyvinylpyrrolidone, poly(methyloxazoline), poly(ethyloxazoline), poly(hydroxypropylmethacrylamide), poly(methacrylamide), poly(dimethylacrylamide), and derived celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0312] In some embodiments, the formulations of the technology of the present invention comprise a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylates and methacrylates, polyethylene polymers, polyglycolides, polysiloxanes, polyurethanes and their copolymers, celluloses, polypropylenes, polyethylenes, polystyrenes, polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho esters), poly(butyric acid), poly(valeric acid), poly(lactide - co - caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates, and their blends, mixtures or copolymers.

[0313] Cyclodextrins are cyclic oligosaccharides composed of 6, 7, or 8 glucose units, denoted by the Greek letters α, β, or γ, respectively. The glucose units are linked by α-1,4-glycosidic bonds. Due to the chair conformation configuration of the sugar units, all secondary hydroxyl groups (at C-2, C-3) are located on one side of the ring, while all primary hydroxyl groups at C-6 are located on the other side. Thus, the outer surface is hydrophilic, making cyclodextrins water-soluble. In contrast, the cavity of cyclodextrin is hydrophobic, as it is lined with the hydrogens of atoms C-3 and C-5 and the ether oxygen. These substrates allow for complexation with a variety of relatively hydrophobic compounds, including, for example, steroid compounds such as 17α-estradiol (see, e.g., Wim van Uden et al. Cyclodextrins as a useful tool for bioconversions in plant cell biotechnology. September 1994. Plant Cell Tissue and Org. Cult. 38:103-113. doi:10.1007 / BF00033867). The complexation proceeds through van der Waals interactions and through hydrogen bond formation. For a general review of the chemical properties of cyclodextrins, see Wenz, Agnew. Chem. Int. Ed. Engl., 33:803-822 (1994).

[0314] The physicochemical properties of cyclodextrin derivatives depend largely on the type and degree of substitution. For example, their solubility in water ranges from insoluble (e.g., triacetyl-β-cyclodextrin) to 147% soluble (w / v) (G-2-β-cyclodextrin). Additionally, they are soluble in a variety of organic solvents. The properties of cyclodextrins enable the control of the solubility of various formulation components by increasing or decreasing their solubility.

[0315] A variety of cyclodextrins and their preparation methods have been described. For example, Parmeter (I) et al. (U.S. Patent No. 3,453,259) and Gramera et al. (U.S. Patent No. 3,459,731) describe neutral cyclodextrins. Other derivatives include cyclodextrins with cationic properties [Parmeter (II), U.S. Patent No. 3,453,257], insoluble crosslinked cyclodextrins (Solms, U.S. Patent No. 3,420,788), and cyclodextrins with anion properties [Parmeter (III), U.S. Patent No. 3,426,011]. Among the cyclodextrin derivatives with anion properties, carboxylic acids, phosphorous acids, phosphinous acids, phosphonic acids, phosphoric acid, thiophosphonic acids, thiosulphinic acid, and sulfonic acids have been attached to the parent cyclodextrin [see Parmeter (III), ibid.]. In addition, Stella et al. (U.S. Patent No. 5,134,127) have described sulfoalkyl ether cyclodextrin derivatives.

[0316] Liposomes consist of at least one lipid bilayer membrane enclosing an aqueous internal compartment. Liposomes can be characterized by membrane type and size. Small unilamellar vesicles (SUVs) have a single membrane and typically have a diameter between 0.02 and 0.05 μm; large unilamellar vesicles (LUVs) are typically greater than 0.05 μm. Oligolamellar large vesicles and multilamellar vesicles have multiple (usually concentric) membrane layers and are typically greater than 0.1 μm. Liposomes with several non-concentric membranes (i.e., several smaller vesicles contained within a larger vesicle) are called multivesicular vesicles.

[0317] One aspect of the technology of the present invention relates to a preparation of liposomes containing oligomers of the technology of the present invention, wherein the liposome membrane is formulated to provide liposomes with increased carrying capacity. Alternatively or additionally, the compounds of the technology of the present invention can be contained within the lipid bilayer of the liposome or adsorbed onto the lipid bilayer of the liposome. The oligomers of the technology of the present invention can aggregate with lipid surfactants and be carried within the internal space of the liposome; in these embodiments, the liposome membrane is formulated to resist the disruptive effects of the agent-surfactant aggregates.

[0318] According to one embodiment of the technology of the present invention, the lipid bilayer of the liposome contains lipids derived from polyethylene glycol (PEG) such that PEG chains extend from the inner surface of the lipid bilayer into the internal space encapsulated by the liposome and from the outer surface of the lipid bilayer into the surrounding environment.

[0319] The active agent contained within the liposomes of the present technology is in dissolved form. Surfactants and aggregates of the active agent, such as emulsions or micelles containing the target active agent, may be encapsulated within the internal space of the liposomes of the present technology. The surfactant serves to disperse and dissolve the active agent and may be selected from any suitable aliphatic, cycloaliphatic or aromatic surfactant, including but not limited to biocompatible lysophosphatidylcholines (LPG) of different chain lengths (e.g., from about C14 to about C20). Polymer-derived lipids, such as PEG-lipids, may also be used to form micelles as they will serve to inhibit micelle / membrane fusion and because addition of the polymer to the surfactant molecule will lower the CMC of the surfactant and aid in micelle formation. Surfactants with a CMC in the micromolar range are preferred; surfactants with higher CMCs may be used to prepare micelles encapsulated within the liposomes of the present technology.

[0320] Liposomes according to the present technology may be prepared by any of a variety of techniques known in the art. See, for example, U.S. Patent No. 4,235,871; published PCT application WO 96 / 14057; New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990), pp. 33-104; Lasic D, Liposomes from physics to applications, Elsevier Science Publishers BV, Amsterdam, 1993. By way of example, liposomes of the present technology may be prepared by diffusing a lipid derivatized with a hydrophilic polymer into preformed liposomes, such as by exposing preformed liposomes to micelles composed of lipid-grafted polymers (lipid concentration corresponding to the final molar percentage of derivatized lipid desired in the liposomes). Liposomes containing hydrophilic polymers may also be formed by homogenization, lipid-field hydration or extrusion techniques known in the art.

[0321] In another exemplary formulation procedure, the active agent is first dispersed by sonication in lysophosphatidylcholine or other low CMC surfactant (including polymer-grafted lipids) that readily dissolves hydrophobic molecules. Subsequently, the resulting micellar suspension of the active agent is used to rehydrate a dry lipid sample containing a suitable molar percentage of polymer-grafted lipid or cholesterol. The lipid and active agent suspension is then formed into liposomes using extrusion techniques known in the art, and the resulting liposomes are separated from the unencapsulated solution by standard column chromatography.

[0322] In one aspect of the technology of the present invention, liposomes are prepared to have a substantially uniform size within a selected size range. An effective sizing method involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes having a selected uniform pore size, the pore size of the membrane corresponding generally to the maximum liposome size produced by extrusion through the membrane. See, for example, U.S. Patent No. 4,737,323 (April 12, 1988). In some embodiments, reagents such as and are used to introduce polynucleotides or proteins into cells.

[0323] The release characteristics of the formulations of the technology of the present invention depend on the encapsulating material, the concentration of the encapsulated drug, and the presence of release modifiers. By way of example, release can be manipulated to be pH-dependent using a pH-sensitive coating that releases only at a low pH such as in the stomach or a higher pH such as in the intestine. An enteric coating can be used to prevent release before passage through the stomach. A multi-layer coating or a mixture of cyanamides encapsulated in different materials can be used to obtain initial release in the stomach followed by subsequent release in the intestine. Release can also be manipulated by including salts or pore formers that can increase water uptake or drug release by diffusion from the capsule. Excipients that modulate drug solubility can also be used to control the release rate. Reagents that enhance matrix degradation or release from the matrix can also be incorporated. It can be added to the drug, added as a separate phase (i.e., as particles), or can be co-dissolved in the polymer phase, depending on the compound. In most embodiments, the amount should be between 0.1% and 30% (w / w polymer). Types of degradation enhancers include inorganic salts such as ammonium sulfate and ammonium chloride; organic acids such as citric acid, benzoic acid, and ascorbic acid; inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide; and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine; and surfactants such as and Pore formers (i.e., water-soluble compounds such as inorganic salts and sugars) that impart microstructure to the matrix are added in particulate form. The range is typically between one percent and thirty percent (w / w polymer).

[0324] Absorption can also be manipulated by changing the residence time of the particles in the digestive tract. This can be achieved, for example, by coating the particles with a mucoadhesive polymer or by selecting a mucoadhesive polymer as the encapsulating material. Examples include polymers having mostly free carboxyl groups such as chitosan, cellulose, and especially polyacrylates (as used herein, polyacrylates refer to polymers including acrylate groups and modified acrylate groups such as cyanoacrylates and methacrylates).

[0325] The oligomers can be formulated to be included within a surgical or medical device or implant, or be adapted to be released by a surgical or medical device or implant. In some aspects, the implant can be coated with or otherwise treated with the oligomers. For example, hydrogels or other polymers, such as biocompatible and / or biodegradable polymers, can be used to coat the implant in conjunction with the compositions of the techniques of the present invention (i.e., the compositions can be adapted to be used with a medical device by using a hydrogel or other polymer). Polymers and copolymers for coating medical devices with agents are well known in the art. Examples of implants include, but are not limited to, stents, drug-eluting stents, sutures, prostheses, vascular catheters, dialysis catheters, vascular grafts, artificial heart valves, pacemakers, implantable cardioverter defibrillators, IV needles, devices for bone fixation and formation (such as pins, screws, plates, and other devices), and artificial tissue matrices for wound healing.

[0326] In addition to the methods provided herein, the oligomers used in accordance with the techniques of the present invention can be formulated for administration in any convenient manner for use in human or veterinary medicine similarly to other pharmaceuticals. The ASOs and their corresponding formulations can be administered alone or in combination with other treatment strategies for treating muscular dystrophy, such as myoblast transplantation, stem cell therapy, administration of aminoglycoside antibiotics, proteasome inhibitors, and upregulation therapies (e.g., utrophin, an autosomal paralog of dystrophin).

[0327] The described routes of administration are intended as a guide only, since a skilled practitioner will be able to readily determine the optimal route of administration and any dosage for any particular animal and condition. A variety of methods have been tried for introducing functional new genetic material into cells in vitro and in vivo (Theodore Friedmann. Progress Toward Human Gene Therapy. 1989. Science. 244:1275-1280. doi:10.1126 / science.266025). These methods include integrating the gene to be expressed into a modified retrovirus (Theodore Friedmann (1989) supra; Steven A. Rosenberg. Immunotherapy and Gene Therapy of Cancer. September 15, 1991. Cancer Research. 51(18), Suppl:5074S-5079S.); integrating it into a non-retroviral vector (such as an adeno-associated virus vector) (M. A. Rosenfeld et al. In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. January 10, 1992. Cell. 68:143-155. doi:10.1016 / 0092-8674(92)90213-V; M. A. Rosenfeld et al. Adenovirus-Mediated Transfer of a Recombinant α1-Antitrypsin Gene to the Lung Epithelium in Vivo. April 19, 1991. Science. 252:431-434. doi:10.1126 / science.2017680); or delivering a transgene linked to a heterologous promoter-enhancer element via liposomes (Theodore Friedmann (1989), supra; Kenneth L. Brigham et al. Rapid Communication: In vivo Transfection of Murine Lungs with a Functioning Prokaryotic Gene using a Liposome Vehicle. October 1989. Am. J. Med. Sci. 298:278-281. doi:10.1097 / 00000441-198910000-00013; Elizabeth G. Nabel et al., Site-Specific Gene Expression in Vivo by Direct Gene Transfer into the Arterial Wall, September 1990, Science, 249:1285-1288, doi:10.1126 / science.211905; Thomas A. Hazinski et al., Localization and Induced Expression of Fusion Genes in the Rat Lung, November 1991, Am. J. Resp. Cell Mol. Biol., 4:206-209, doi:10.1165 / ajrcmb / 4.3.206; and Wang and Huang, pH-sensitive immunoliposomes mediate target-cell-specific delivery and controlled expression of a foreign gene in mouse, November 11, 1987, Proc. Natl. Acad. Sci. USA, 84:7851-7855, doi:10.1073 / pnas.84.22.7851); coupled with ligand-specific, cation-based delivery systems (Wu and Wu, Receptor-mediated gene delivery and expression in vivo, October 1988, J. Biol. Chem., 263:14621-14624, doi:10.1016 / S0021-9258(18)68081-0) or using naked DNA, expression vectors (Elizabeth G. Nabel et al. (1990), supra); Jon A. Wolff et al., Direct Gene Transfer into Mouse Muscle in Vivo, March 23, 1990, Science, 247:1465-1468, doi:10.1126 / science.1690918). Direct injection of the transgene into tissues results in only local expression (M. A. Rosenfeld (1992), supra); M. A. Rosenfeld et al. (1991), supra; Kenneth L. Brigham et al. (1989), supra; Elizabeth G.Nabel (1990), ibid.; and Thomas A. Hazinski et al. (1991), ibid.). The team of Brigham et al. (Rapid Communication: In vivo Transfection of Murine Lungs with a Functioning Prokaryotic Gene using a Liposome Vehicle. October 1989. Am. J. Med. Sci. 298:278 - 281. doi:10.1097 / 00000441 - 198910000 - 00013 and Clinical Research (1991) 39 (abstract)) have reported in vivo transfection only of the lungs of mice after intravenous or intratracheal administration of DNA - liposome complexes. An example of a review article on human gene therapy procedures is W. F. Anderson. Human gene therapy. May 8, 1992. Science. 256:808 - 813. doi:10.1126 / science.1589762.

[0328] Combination therapy

[0329] As used herein, "co - administration" or "co - administering" or "combination therapy" generally refers to the administration of one or more ASOs of the technology of the present invention (e.g., duplex ASOs) together with other medicaments known in the art for treating the same disease or its complications as those treated by the ASO.

[0330] In certain embodiments, the disease is DMD and the other medicaments treat muscular dystrophy or its complications, including but not limited to: corticosteroids (such as cortisol, hydrocortisone, prednisone, prednisolone, deflazacort, triamcinolone, methylprednisolone, dexamethasone, betamethasone, aldosterone, and fludrocortisone); β2-adrenergic agonists (such as albuterol, salbutamol, levalbuterol, terbutaline, pirbuterol, procaterol, clenbuterol, metaproterenol, fenoterol, bitolterol mesylate, ritodrine, isoprenaline, salmeterol, formoterol, bambuterol, and indicaterol); immunosuppressants (such as cyclosporine); anti-fibrotic drugs (such as peginterferon, IL-10, pioglitazone, pentoxifylline, etanercept); exon skipping drugs (such as ASO; for example, ASO targeting exon 51, exon 45, or exon 53, including drisapersen, edeplersen, golodirsen, PRO044, PRO45, PRO051, and PRO053); stop codon skipping drugs (such as gentamycin or other aminoglycoside antibiotics and Ataluren (PTC124)); anabolic steroids (such as oxandrolone); osteoporosis medicaments (such as vitamin D and calcium); constipation medicaments, including laxatives;Cardiomyopathy drugs, including angiotensin-converting enzyme (ACE) inhibitors (such as benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, and trandolapril), diuretics, β-blockers (such as bisoprolol or carvedilol), antiarrhythmic drugs (such as amiodarone); insulin-like growth factor (IGF-1); myostatin inhibitors (such as follistatin, ACE-031, and neutralizing antibodies, including MYO-029); drugs that increase nitric oxide levels and / or nNOS protein levels or activity (such as L-arginine; phosphodiesterase inhibitors, including sildenafil, tadalafil, and pentoxifylline); class II histone deacetylase (HDAC) inhibitors; small molecules that increase the expression of dystrophin-associated proteins (such as SMT C1100); nutritional supplements (such as glutamine, creatine monohydrate, conjugated linoleic acid, alpha-lipoic acid, and beta-hydroxy-beta-methylbutyrate); antihistamines (such as fexofenadine, loratadine, phenindamine, dexchlorpheniramine, terfenadine, cetirizine, etc.); mast cell stabilizers (such as sodium cromoglicate, nedocromil sodium, etc., which can be in the form of aerosols, inhalants, eye drops, etc.); coenzyme Q10 (also known as ubiquinone or ubidecarenone); idebenone or other synthetic derivatives of ubiquinone (such as; ); ω3; resveratrol; phytosterols / stanols; anticoagulants (such as warfarin); and anticholinergic drugs (such as antimuscarinics (such as atropine, benztropine) biperiden, chlorpheniramine (CHLOR-TRIMETON), dicyclomine (dicycloverine), dimenhydrinate diphenhydramine( SOMINEX TM 、 PM, etc.), doxylamine (UNISOM TM ), glycopyrrolate ipratropium orphenadrine, oxitropium oxybutynin( XL), tolterodine( DETRUSITOL), tiotropium trihexyphenidyl, scopolamine, solifenacin, and tropicamide), nicotine antagonists (e.g., ganglionic blockers, including bupropion and hexamethonium, antitussives and ganglionic blockers (such as dextromethorphan), non-depolarizing skeletal muscle relaxants (such as doxacurium and tubocurarine), ganglionic blockers and occasionally adjunctive smoking cessation agents (such as mecamylamine)), which may be in the form of inhalants, nebulized solutions, tablets, and may be administered by rectal, oral, transdermal or parenteral routes). The compositions and methods of the technology of the present invention may also be used in combination with, for example: other gene-based therapeutic methods (such as viral delivery of minidystrophin or microdystrophin, minidystrophin-associated proteins or trans-splicing recombinant AAV vectors); gene editing, including methods involving zinc finger nucleases, transcription activator-like (TAL) type III effector nucleases (TALENs), meganucleases or clustered regularly interspaced short palindromic repeats (CRISPR), or cell-based therapies involving transplantation of various types of precursor cells, such as in vitro manipulated muscle side population cells (undetermined cell lineages) into muscle fibers.The compositions and methods can also be used in conjunction with any of the therapies described in: Pedro Miura et al., Utrophin upregulation for treating Duchenne or Becker muscular dystrophy: how close are we? March 2006, Trends Mol Med 12:122-129, doi:10.1016 / j.molmed.2006.01.002; Susan Jarmin et al., New developments in the use of gene therapy to treat Duchenne muscular dystrophy, December 6, 2013, Expert Opin Biol Ther 14:209-230, doi:10.1517 / 14712598.2014.866087; M.A. Scully et al., Review of Phase II and Phase III clinical trials for Duchenne muscular dystrophy, December 17, 2012, Expert Opin Orphan Drugs 1:33-46, doi:10.1517 / 21678707.2013.746939; R.J. Fairclough et al., Progress in therapy for Duchenne muscular dystrophy, July 29, 2011, Exp Physiol 96:1101-1113, doi:10.1113 / expphysiol.2010.053025; and Michael J. Blankinship et al., Gene Therapy Strategies for Duchenne Muscular Dystrophy Utilizing Recombinant Adeno-associated Virus Vectors, February 2006, Mol Ther 13:241-249, doi:10.1016 / j.ymthe.2005.11.001.

[0331] In some embodiments, one or more ASOs of the technology of the present invention are administered in combination with one or more additional therapeutic agents in a pharmaceutically acceptable dosage form. In accordance with standard pharmaceutical practice, each therapeutic agent in the combination therapies disclosed herein may be administered alone or in the form of a medicament (also referred to herein as a pharmaceutical composition) comprising the therapeutic agent and one or more pharmaceutically acceptable carriers, excipients, and diluents. Each therapeutic agent may be prepared by separately formulating the compound or its pharmaceutically acceptable salt, and both may be administered simultaneously or separately. In addition, the two formulations may be placed in a single package to provide a kit formulation. In some embodiments, the two compounds may be contained in a single formulation. In some embodiments, the therapeutic agents are in the same dosage form, such as the same tablet or pharmaceutical composition. In some embodiments, the therapeutic agents are separate dosage forms with the same mode of administration, such as a kit comprising: a first pharmaceutical composition suitable for parenteral administration comprising an ASO and a pharmaceutically acceptable carrier, and a second pharmaceutical composition suitable for parenteral administration comprising one or more additional therapeutic agents. In some embodiments, the therapeutic agents are separate dosage forms with different modes of administration, such as a kit comprising: a first pharmaceutical composition suitable for parenteral administration comprising an ASO and a pharmaceutically acceptable carrier, a second pharmaceutical composition suitable for oral administration comprising one or more additional therapeutic agents, and optionally, a third pharmaceutical composition suitable for oral administration comprising one or more other therapeutic agents.

[0332] Each therapeutic agent in the combination therapies disclosed herein can be administered simultaneously (i.e., in the same pharmaceutical composition), concurrently (i.e., as separate pharmaceutical compositions administered in any order in sequence), or in any order in sequence. Sequential administration is applicable when the therapeutic agents in the combination therapy are in different dosage forms (e.g., one agent is a tablet or capsule and the other is a sterile liquid) and / or are administered according to different dosing regimens, such as tablets or capsules formulated for daily administration and a composition formulated for parenteral administration (such as once a week, once every two weeks, or once every three weeks). Additionally, in view of the benefits of the technology of the present invention, those skilled in the art will appreciate that when more than one therapeutic agent disclosed herein is administered, the agents need not share the same mode of administration, such as a kit comprising: a first pharmaceutical composition suitable for parenteral administration comprising an ASO and a pharmaceutically acceptable carrier, and a second pharmaceutical composition suitable for oral administration comprising an additional therapeutic agent disclosed herein and a pharmaceutically acceptable carrier. Those skilled in the art will appreciate that co-administration as referred to above in the context of "co-administering" or "co-administration" or "combination therapy" means that a pharmaceutical composition comprising a DMD exon skipping ASO and a pharmaceutical composition comprising an additional therapeutic agent can be administered at the same schedule (i.e., at the same time and on the same day), or at different schedules (i.e., at different but not necessarily distinct schedules). In view of the benefits of the technology of the present invention, other suitable variations of "co-administering", "co-administration", or "combination therapy" will be apparent to those skilled in the art and are part of the meaning of these terms.

[0333] The compositions and methods of the technology of the present invention can also be used in combination with other forms of treatment, which include but are not limited to: physical activity (such as physical therapy, range-of-motion exercise); ambulatory aids, supports, or corrective devices (such as ankle splints, knee-ankle-foot orthosis (KAFO), spinal braces, and wheelchairs); respiratory assistance (such as ventilators); and surgical treatments (such as tendon surgery, scoliosis surgery, pacemaker implantation, and heart transplantation). The choice of specific treatment can vary and will depend on the severity of the pain, the general health of the subject, and the judgment of the treating clinician.

[0334] Kit comprising a bipartite ASO

[0335] In some aspects, the technology of the present invention provides a kit that includes an ASO (such as a bipartite ASO) disclosed herein, a composition (such as a pharmaceutical composition) that includes an ASO (such as a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (such as a bipartite ASO) disclosed herein, for generating or promoting exon skipping of a target exon. ASO-mediated exon skipping may be a method of manipulating the expression of a target gene. The expression of a target gene can be manipulated by ASO inhibiting the splicing of an exon, intron, or specific splicing site of the target gene, resulting in (e.g., but not limited to): restoring the reading frame of a target defective gene, generating different subtypes of the target gene (such as a dominant negative subtype), skipping a toxic portion of a gene, silencing a gene, and / or altering the structure and function of a gene. Examples of target exons and target genes include, but are not limited to, those disclosed herein.

[0336] In some aspects, the technology of the present invention provides a kit that includes an ASO (such as a bipartite ASO) disclosed herein, a composition (such as a pharmaceutical composition) that includes an ASO (such as a bipartite ASO) disclosed herein, and / or a vector encoding an ASO (such as a bipartite ASO) disclosed herein, for treating a disease and / or its complications. Examples of diseases and / or their complications include, but are not limited to, those disclosed herein.

[0337] Examples

[0338] The following examples are provided only for additional description of the technology of the present invention for illustrative purposes and should not be construed as limiting the scope of the technology of the present invention in any way.

[0339] Example 1: Example of a bipartite ASO

[0340] Embodiments of the technology of the present invention include a bipartite ASO that includes a targeting sequence and a decoy sequence (see, for example Figure 1 A and Figure 1B). The targeting sequence binds, with complete or partial complementarity, to the sequence at the target exon, the flanking intronic sequence upstream of the target exon, the flanking intronic sequence downstream of the target exon, the intron-exon junction upstream of the target exon, or the intron-exon junction downstream of the target exon. The decoy sequence, which constitutes all or part of the 11 nt sequence (5'-CAGGTAAGTAT-3'), is completely complementary to the free 5' end of U1 snRNA. Thus, the decoy sequence mimicking the optimal 5' splice site may act as a 5' splice site decoy to interfere with the recognition of the authentic 5' splice site of the target exon by U1 snRNA. In certain embodiments, the length of the decoy sequence can be 12 or 13 nt, as shown in Example 5. Without being bound by any particular theory, the targeting sequence can not only cause exon skipping to some extent by steric hindrance, but also bring the decoy sequence closer to the 5' splice site of the target exon. The decoy sequence may resemble and mimic the optimal 5' splice site, which interferes with the recognition of the authentic 5' splice site by U1 snRNA through potential direct interaction with U1 snRNA. In certain instances, the targeting sequence and the decoy sequence act in concert to induce robust exon skipping. Although both ends of the bipartite ASO can contain the same or different decoy sequences, the bipartite ASOs tested in Examples 2 to 15 contain a decoy at the 5' end or 3' end of the bipartite ASO (e.g., Figure 1 as shown in A). The bipartite ASO exerts its function by binding, through its targeting portion based on Watson-Crick base-pairing, to the target exon, its flanking intronic sequence, or the intron-exon junction (e.g., Figure 1 as shown in B). In certain instances, the length of the optimal decoy sequence can vary between 6 and 11 nt. For the nomenclature and sequences of all tested decoys, see Table 1.

[0341] Unless otherwise specified, in the Examples section, L in the decoy name or ASO name means the decoy is located at the 5' end of the bipartite ASO; and R in the decoy name or ASO name means the decoy is located at the 3' end of the bipartite ASO. For example, L6a means the 6a decoy sequence is located at the 5' end of the bipartite ASO, and R6a means the 6a decoy sequence is located at the 3' end of the bipartite ASO.

[0342] All tested ASOs in Examples 2 to 15 were modified with 2'-O-methoxyethyl (MOE) and had a phosphorothioate (PS) backbone, and all cytosines were 5-methylcytosines. Similar ASOs with other uniform modifications (such as, but not limited to, morpholino phosphorodiamide, and constrained ethyl (cEt)), or mixed modifications, should also work.

[0343] Table 1. 5'-Splice Site Decoy Sequences Tested in Examples 2 to 15

[0344]

[0345]

[0346] Example 2: Testing the Exon Skipping Efficiency of ASO

[0347] HEK293 cells, RD cells, Hela cells, or A549 cells were co-transfected with the ASOs (12.5, 25, 40, or 50 nM) listed in Tables 2 to 12 to test the exon skipping efficiency of ASO in vitro. Each ASO was transfected into the required cells using Lipofectamine 2000, and a separate buffer was used as a negative control. Two days after transfection, the cells were collected, and total RNA samples were isolated for splicing analysis by semi-quantitative fluorescent RT-PCR.

[0348] Some of the ASO targeting sequences listed here were used to demonstrate that the bipartite ASO containing the decoy and the corresponding targeting sequence is much more effective in inhibiting exon splicing than the targeting sequence alone. All ASOs were modified with 2'-O-methoxyethyl (MOE) and had a phosphorothioate (PS) backbone and all 5-methylcytosine (5mC). Etep is the abbreviation of Idepersen. Note that the Idepersen used here was also modified with MOE / PS / 5mC, rather than its original morpholino / phosphorodiamidate modification.

[0349] Example 3: ASO-Induced Exon 7 Skipping of SMN1 and SMN2 Genes

[0350] The 11 nt decoy sequence (e.g., 5'-CAGGTAAGTAT-3') enhanced the effect of three ASO targeting sequences (2203, 1938, and 0120) on exon 7 skipping of the endogenous SMN1 and SMN2 genes in HEK293 cells, with statistical significance, as shown in Figure 2 A to Figure 2 D. Figure 2 A shows a schematic diagram of the target region of the SMN1 / 2 gene. The three ASO targeting sequences bind to their SMN1 / 2 pre-mRNA target sequences with perfect complementarity. The ASO targeting sequence 2203 targets a 20 nt sequence at positions -22 to -3 in intron 6, the ASO targeting sequence 1938 targets a 20 nt sequence at positions 19 to 38 in exon 7, and the ASO targeting sequence 0120 targets a 20 nt sequence at positions 1 to 20 in intron 7. As Figure 2As shown in Figure 2 the middle panel (SMN1) and the right panel (SMN2) of Figure 2 B, when the decoy is linked to the 5'-end (2203-L11) or the 3'-end (2203-R11) of the ASO targeting sequence 2203, the effect of the ASO targeting sequence 2203 on promoting exon 7 skipping increases. Each ASO at 12.5, 25 or 50 nM was transfected into HEK293 cells using Lipofectamine 2000, and the buffer alone was used as a negative control. Two days after transfection, cell samples were collected and total RNA was purified for splicing analysis by semi-quantitative fluorescent RT-PCR (Y. Gao et al. Systematic characterization of short intronic splicing-regulatory elements in SMN2 pre-mRNA. January 8, 2022. Nucleic Acids Research. 50:731-749. doi:10.1093 / nar / gkab1280). The PCR products were digested with DdeI to distinguish their origin (SMN1 or SMN2). The percentage of exon 7 exclusion (% exclusion) in the total transcripts of each gene was calculated. The quantitative presentation of the data (n = 3) is shown in Figure 2 the middle panel (SMN1) and the right panel (SMN2) of Figure 2 C. As shown in Figure 2In the left panel of D (FL: full-length transcript, Δ7: transcript with exon 7 skipped). Data (n = 3) for ASOs containing targeting sequences 2203, 1938, or 0120 are shown as mean ± standard deviation in Figure 2 panel B to Figure 2 the middle panel (SMN1) and right panel (SMN2) of D. *P < 0.05, **P < 0.01 compared to the corresponding ASOs containing only the targeting sequences (e.g., 2203, 1938, or 0120). All ASOs were 2'-O-methoxyethyl modified and had phosphorothioate backbones and all 5-methylcytosines. ASO 2203-L11, 1938-L11, 1938-R11, and 0120-L11 were the best ASOs. For sequence information on the decoy sequences, see Table 1; for sequence information on the ASOs and targeting sequences tested in Example 3, see Table 2. The target sequences (5' to 3') complementary to the ASO targeting sequences 2203 in SMN1 / 2 mRNA are ACUUCCUUUAUUUUCCUUAC (SEQ ID No. 24), the target sequences (5' to 3') complementary to the ASO targeting sequence 1938 in SMN1 / 2 mRNA are AAAGAAGGAAGGUGCUCACA (SEQ ID No. 28), and the target sequences (5' to 3') complementary to the ASO targeting sequence 0120 in SMN1 / 2 mRNA are GUAAGUCUGCCAGCAUUAUG (SEQ ID No. 32).

[0351] As Figure 2 shown in panel B to Figure 2 D, ASOs containing decoy sequence 11 linked to the targeting sequence transfected at a lower concentration achieved comparable exon 7 exclusion % of SMN1 / 2 to ASOs composed of the targeting sequence transfected at a higher concentration. As Figure 2 shown in the left panel of B, 2203-L11 (12.5 nM) achieved comparable exon 7 exclusion % of SMN1 to targeting sequence 2203 (50 nM), and the exon 7 exclusion % of SMN1 for 2203-L11 (12.5 nM) was approximately 2.3 times that of targeting sequence 2203 (25 nM) and 4 times that of targeting sequence 2203 at the same concentration. Similar results were observed in 2203-R11 compared to 2203. Half doses of 2203-R11 (12.5 nM, 25 nM) achieved comparable exon 7 exclusion % of SMN1 to targeting sequence 2203 (25 nM, 50 nM), respectively. 2203-R11 (12.5 nM) demonstrated comparable performance in exon 7 skipping of the SMN2 gene to targeting sequence 2203 (25 nM). As Figure 2As shown in the left panel of C, at the same concentration of 12.5 nM, the exon 7 skipping % of SMN1 of 1938-R11 was approximately 2.18 times that of the skipping % of the targeting sequence 1938, which was comparable to that of the targeting sequence 1938 (25 nM). Figure 2 D shows that 0120-L11 (12.5 nM) achieved an exon 7 skipping % of SMN1 comparable to that of the targeting sequence 0120 (50 nM).

[0352] Three 5-nt bait sequences (AGGTA or 5b, GTAAG or 5d, and AGTAT or 5g) linked to either side of the ASO targeting sequence 1938 were tested for their effects on SMN2 exon 7 splicing. ASO1938-R5b (SEQ ID No. 354: 5'-TGTGAGCACCTTCCTTCTTTAGGTA-3') was relatively strong, showing a skipping effect comparable to that of 1938-R11 ( Figure 2 C), while 1938-R5d (SEQ ID No. 355: 5'-TGTGAGCACCTTCCTTCTTTGTAAG-3') and 1938-R5g (SEQ ID No. 356: 5'-TGTGAGCACCTTCCTTCTTTAGTAT-3') had moderate effects in promoting exon 7 skipping.

[0353] Example 4: ASO-induced exon 51 skipping of the DMD gene

[0354] 11-nt bait sequences (such as 5'-CAGGTAAGTAT-3') improved the effects of several ASO targeting sequences in promoting exon 51 skipping of the endogenous DMD gene, with statistical significance, as shown in Figure 3 A and Figure 3 B. Each ASO or ASO targeting sequence at the specified concentration was transfected into RD cells using Lipofectamine 2000. Two days after transfection, the cells were collected, and total RNA samples were isolated for splicing analysis by semi-quantitative fluorescent RT-PCR. As shown in Figure 3 A, in the embodiments, ASO targeting sequences (148-L11, 155-L11, and 165-L11) with a bait sequence at the 5' end had a greater exon skipping effect than the corresponding ASOs (148, 155, and 165) without a bait sequence, but ASOs (148-R11, 155-R11, and 165-R11) with a bait sequence at the 3' end showed minimal differences from the corresponding ASO targeting sequences without a bait sequence. The ASO targeting sequences 148, 155, and 165 targeted different regions in DMD exon 51 with complete complementarity. As shown in Figure 3As shown in Figure 3 Panel A of Figure 3 Figure 1, and a representative gel of four independent experiments of ASOs containing targeting sequence Etep (Etep, Etep-L11, Etep-R11) is shown in

[0355] Quantification of exon 51 skipping data (n = 4) of ASOs containing targeting sequences 148, 155, 165, or Etep is shown in Figure 3 Panels A to Figure 3 right panel of

[0356] Figure 1B. *P < 0.05 (148-L11 vs. 148); **P < 0.01 (155-L11 vs. 155, 165-L11 vs. 165, or Etep-L11 vs. Etep). ASO 148-L11, 155-L11, 165-L11, Etep-L11, and Etep-R11 are the best ASOs. For sequence information on the decoy sequences, see Table 1; for sequence information on the ASOs and targeting sequences tested in Example 4, see Table 2. The target sequences (5' to 3') complementary to ASO targeting sequence 148 in DMD mRNA are AAUGCCAUCUUCCUUGAUG (SEQ ID No. 36), the target sequences (5' to 3') complementary to ASO targeting sequence 155 in DMD mRNA are AACUAGAAAUGCCAUCUUC (SEQ ID No. 40), the target sequences (5' to 3') complementary to ASO targeting sequence 165 in DMD mRNA are GCCAUCUCCAAACUAGAAA (SEQ ID No. 44), and the target sequences (5' to 3') complementary to ASO targeting sequence Etep in DMD mRNA are CUAGAAAUGCCAUCUUCCUUGAUGUUGGAG (SEQ ID No. 48). The percentage of exon 51 exclusion of DMD induced by Etep-L11 (12.5 nM) is comparable to that of targeting sequence Etep (25 nM).

[0357] Table 2. ASOs and targeting sequences tested in Example 3 and Example 4

[0358]

[0359]

[0360] *: The more "+", the better the exon skipping effect.

[0361] Example 5: ASO-induced exon 51 skipping of the DMD gene, where decoy sequences of different lengths are linked to the 5'-end ("L") of the targeting sequence Etepelson

[0362] Dichotomous ASOs with decoy sequences of different lengths may have varying degrees of influence on the ability of ASO to promote exon skipping in the DMD gene, for example Figure 4 as shown. Twenty-three 5'-splice site decoy sequences with lengths of 6 to 13 nt were tested in the case of the targeting sequence Etepelson (Etep). The tested decoy sequences of 12 nt (L12) and 13 nt (L13) lengths are complementary to the first 12 and 13 nt sequences of the 5'-end of U1 snRNA, respectively. The decoy sequences are located at the 5'-end of the dichotomous ASO. All tested ASOs were modified with MOE and had a PS backbone. 25 nM of each ASO was transfected into RD cells using Lipofectamine 2000, and the separate buffer was used as a negative control. Two days after transfection, the cells were collected, and total RNA samples were isolated for splicing analysis by semi-quantitative fluorescent RT-PCR. All tested dichotomous ASOs showed a stronger effect on exon 51 skipping of the DMD gene than Etepelson alone, with statistical significance. A representative result of three independent experiments is shown in Figure 4 (upper panel, FL: full-length transcript, Δ51: transcript with exon 51 skipped). The quantification of the data (n = 3) is shown in the lower panel of Figure 4 . # P < 0.05 (all relative to Etep); *P < 0.05, **P < 0.01 (all relative to Etep-L11). ASO Etep-L7c, Etep-L8b, Etep-L8c, Etep-L9b, and Etep-L10a are the best ASOs ( Figure 4 ). For sequence information on the decoy sequences, see Table 1; for sequence information on the ASOs and targeting sequences tested in Example 5, see Table 3. The target sequence (5' to 3') in DMD exon 51 is CUAGAAAUGCCAUCUUCCUUGAUGUUGGAG (SEQ ID No. 48).

[0363] Table 3. ASOs and targeting sequences tested in Example 5

[0364]

[0365] Example 6: ASO-induced exon 51 skipping of the DMD gene, where decoy sequences of different lengths were ligated to the 5'-end ("L") of the targeting sequence 000A

[0366] Dichotomous ASOs with decoy sequences of different lengths may have different degrees of influence on the ability of ASOs to promote exon skipping in the DMD gene, for example Figure 5 as shown. Fifteen decoys with lengths from 7 to 11 nt were tested. The decoy sequences were located at the 5'-end of the dichotomous ASOs. The targeting sequence 000A was 21 nt long and targeted positions 50 to 70 of DMD exon 51. All tested ASOs were modified with MOE and had a PS backbone, and all cytosines were 5-methylcytosines. 25 nM of each ASO was transfected into RD cells using Lipofectamine 2000. A separate buffer was used as a negative control. Two days after transfection, the cells were harvested, and total RNA samples were isolated for splicing analysis by semi-quantitative fluorescent RT-PCR. ASO 000A slightly promoted DMD exon 51 skipping. Dichotomous ASOs containing a decoy at the 5'-end ("L") improved the exon skipping effect of 000A with statistical significance. A representative gel of three independent experiments is shown in Figure 5 (upper panel, FL: full-length transcript, Δ51: transcript with exon 51 skipped). Quantification of the data (n = 3) is shown in the lower panel of Figure 5 . *P<0.05, **P<0.01, all relative to 000A. 000A-L7c, 000A-L8c, 000A-L9a, 000A-L9b, 000A-L9c, 000A-L10a, and 000A-L10b were the best ASOs( Figure 5 ). For sequence information on the decoy sequences, see Table 1; for sequence information on the ASOs and targeting sequences tested in Example 6, see Table 4. The target sequence (5' to 3') in DMD exon 51 is AACUGCCAUCUCCAAACUAGA (SEQ ID No. 74).

[0367] Table 4. ASOs and targeting sequences tested in Example 6

[0368]

[0369]

[0370] Example 7: Exon 51 skipping of the DMD gene in the tibialis anterior and gastrocnemius muscles of ASO-induced DMD humanized mice

[0371] Several bipartite ASOs were tested for DMD exon 51 skipping in DMD humanized mice. In the mouse model, a 10,766 nt mouse genomic fragment from the last 9053 nt of DMD intron 50, 233 nt of exon 51 to the first 1480 nt of intron 51 was replaced with the corresponding human genomic fragment (800 nt of intron 50, 233 nt of exon 51, and 800 nt of intron 51). Each humanized male mouse (about 8 weeks old) received 150 mg / kg / injection of ASO via intravenous injection, once every other day. A total of three injections were given. Three days after the last injection, the mice were euthanized and muscle samples were collected for RT-PCR analysis by semi-quantitative fluorescent RT-PCR. As Figure 6 shown, all bipartite ASOs promoted DMD exon 51 skipping with statistical significance compared to the MOE / PS version of the targeting sequence Etep alone. The most potent bipartite ASO Etep-L8c showed more than a 10-fold increase in exon 51 skipping. A representative result of three independent experiments is shown in Figure 6 (upper left panel for tibialis anterior muscle and upper right panel for gastrocnemius muscle, FL: full-length transcript, Δ51: transcript with exon 51 skipped). Quantification of the data (n = 3) is shown in the lower panel of Figure 6 (lower left panel for tibialis anterior muscle and lower right panel for gastrocnemius muscle). *P < 0.05, **P < 0.01, ***P < 0.001, all relative to Etep. For sequence information on the bait sequences, see Table 1; for sequence information on the ASOs and targeting sequences tested in Example 7, see Tables 3 and 4.

[0372] Some ASOs containing a bait increased the exon skipping rate to more than 14-fold compared to ASOs containing the same targeting sequence but without a bait.

[0373] Example 8: ASO-induced exon 53 skipping of the DMD gene with bait sequences of different lengths

[0374] In RD cells, 15 baits with lengths of 7 to 11 nt were tested in the case of the targeting sequence of the MOE / PS version of Vilto. Vilto is a 21-nt PMO approved by the FDA for the treatment of DMD; it promotes DMD exon 53 skipping by targeting the region from position 36 to position 56 in the target exon. 25 nM of each ASO was transfected into RD cells using Lipofectamine 2000. A separate buffer was used as a negative control. Two days after transfection, the cells were collected, and total RNA samples were isolated for splicing analysis by semi-quantitative fluorescent RT-PCR. The bipartite ASOs tested included baits (L7a to L7e, L8a to L8d, L9a to L9c, L10a and L10b, and L11) linked to the 5' (“L”) end of the targeting sequence of Vilto, or baits (R7a to R7e, R8a to R8d, R9a to R9c, R10a and R10b, and R11) linked to the 3' (“R”) end of the targeting sequence of Vilto. As Figure 7 shown, the individual targeting sequence of Vilto slightly promoted DMD exon 53 skipping. Some bipartite ASOs improved the exon skipping effect of Vilto alone, with statistical significance, especially when the bait was placed at the 3' end of the bipartite ASO. A representative result of three independent experiments is shown in Figure 7 (upper left figure for bipartite ASOs containing baits linked to the 5' end of the targeting sequence of Vilto, and upper right figure for bipartite ASOs containing baits linked to the 3' end of the targeting sequence of Vilto, FL: full-length transcript, Δ53: transcript with exon 53 skipped). The quantification of the data (n = 3) is shown in the lower figure of Figure 7 (lower left figure for bipartite ASOs containing baits linked to the 5' end of the targeting sequence of Vilto, and lower right figure for bipartite ASOs containing baits linked to the 3' end of the targeting sequence of Vilto). *P < 0.05, **P < 0.01, ***P < 0.001, all relative to Vilto. ASO Vilto-L9b, Vilto-R7b, Vilto-R7c, Vilto-R8b, Vilto-R8c, and Vilto-R8d were the best ASOs ( Figure 7 ). For sequence information on the bait sequences, see Table 1; for sequence information on the ASOs and targeting sequences tested in Example 8, see Table 5. The target sequence (5' to 3') in DMD exon 53 is GAACACCUUCAGAACCGGAGG (SEQ ID No. 91).

[0375] Table 5. ASOs and targeting sequences tested in Example 8

[0376]

[0377]

[0378] Example 9: ASO-induced partial exon 45 skipping of the DMD gene in the presence of decoy sequences of different lengths

[0379] Fifteen decoys of lengths 7 to 11 nt were tested in the context of targeting sequence 002A. ASO 002A targets the junction of DMD exon 45 and intron 45 and promotes the skipping of the last 32 nt of exon 45 (herein referred to as partial exon skipping), which can restore the reading frame of patients carrying frameshift mutations in the same way as skipping the entire 176-nt exon 45. As Figure 8 shown in A, ASO 002A can activate the cryptic 5' splice site in DMD exon 45 (as indicated by the arrow), which causes the skipping of the last 32 nt of the exon. The small 32-nt portion of DMD exon 45 is designated 45s. Skipping 45s instead of the entire 176-nt exon can benefit patients.

[0380] Twenty-five nM of each ASO was transfected into RD cells using Lipofectamine 2000. Buffer alone was used as a negative control. Two days after transfection, cells were harvested and total RNA samples were isolated for splicing analysis by semi-quantitative fluorescent RT-PCR. The bipartite ASOs tested contained a decoy (L7a to L7e, L8a to L8d, L9a to L9c, L10a and L10b, and L11, Figure 8 B, left column) linked to the 5' (“L”) end of targeting sequence 002A, or a decoy (R7a to R7e, R8a to R8d, R9a to R9c, R10a and R10b, and R11, Figure 8 B, right column) linked to the 3' (“R”) end of targeting sequence 002A. The targeting sequence 002A alone (002A) slightly promoted the skipping of the 32-nt portion of exon 45( Figure 8 B). Some bipartite ASOs improved the partial exon skipping effect of the targeting sequence 002A alone, with statistical significance, especially when the decoy was placed at the 5' end of the bipartite ASO. A representative result of three independent experiments is shown in Figure 8 B (upper left panel for bipartite ASOs containing a decoy linked to the 5' end of targeting sequence 002A, and upper right panel for bipartite ASOs containing a decoy linked to the 3' end of targeting sequence 002A, FL: full-length transcript, Δ45s: transcript with exon 45s skipped). Quantification of the data (n = 3) is shown as mean ± standard deviation in Figure 8In the lower panels of B (left lower panel for the bipartite ASO containing the bait linked to the 5'-end of the targeting sequence 002A, and right lower panel for the bipartite ASO containing the bait linked to the 3'-end of the targeting sequence 002A). *P < 0.05, **P < 0.01, all relative to 002A. ASO 002A-L7c, 002A-L8c, and 002A-R7e are the best ASOs ( Figure 8 B). For sequence information on the bait sequences, see Table 1; and for sequence information on the ASOs and targeting sequences tested in Example 9, see Table 6. The target sequence (5' to 3') at the junction of DMD exon 45 and intron 45 is CAGAAAAAAGAGGUAGGGCGAC (SEQ ID No. 123).

[0381] Table 6. ASOs and targeting sequences tested in Example 9

[0382]

[0383]

[0384] Example 10: Exon 17 skipping of the APP gene induced by ASO in the case of bait sequences of different lengths

[0385] Fifteen bait sequences with lengths from 7 to 11 nt were tested in the case of the targeting sequence 014B. ASO 014B targets exon 17 of the APP gene and promotes exon skipping in the endogenous APP gene. Each 50 nM ASO was transfected into HEK293T cells using Lipofectamine 2000, and the separate buffer was used as a negative control. Two days after transfection, the cells were collected, and total RNA samples were isolated for splicing analysis by semi-quantitative fluorescent RT-PCR.

[0386] The bipartite ASOs tested included baits linked to the 5' ("L") end of the targeting sequence 014B (014B-L7a to 014B-L7e, 014B-L8a to 014B-L8d, 014B-L9a to 014B-L9c, 014B-L10a, 014B-L10b, and 014B-L11, Figure 9 A), or baits linked to the 3' ("R") end of the targeting sequence 014B (014B-R7a to 014B-R7e, 014B-R8a to 014B-R8d, 014B-R9a to 014B-R9c, 014B-R10a, 014B-R10b, and 014B-R11, Figure 9 B).

[0387] Regardless of whether the bait was linked to the 5'-end or 3'-end of the bipartite ASO, most bipartite ASOs improved the exon skipping effect of the individual target sequence 014B (014B) with statistical significance, and the most potent ASO was 014B-L8c. A representative result of three independent experiments of bipartite ASOs containing a bait linked to the 5'-end of the target sequence 014B and bipartite ASOs containing a bait linked to the 3'-end of the target sequence 014B are shown in Figure 9 A and Figure 9 the left panels of Figure 9 A and Figure 9 B, respectively (FL: full-length transcript, Δ17: transcript with exon 17 skipped). Quantification of the data (n = 3) is shown in the right panels of Figure 9 A and Figure 9 B ( Figure 9 the right panel of A is for bipartite ASOs containing a bait linked to the 5'-end of the target sequence 014B, and Figure 9 the right panel of B is for bipartite ASOs containing a bait linked to the 3'-end of the target sequence 014B). *P < 0.05, **P < 0.01, ***P < 0.001 (all relative to 014B). ASO 014B-L8c was the best ASO. For sequence information of the bait, see Table 1; for sequence information of the ASOs and target sequences tested in Example 10, see Table 7, and the target sequence (5' to 3') at the junction of intron 16 and exon 17 of APP is UCAAGGUGUUCUUUGCAG (SEQ ID No. 155).

[0388] Table 7. ASOs and target sequences tested in Example 10

[0389]

[0390] Note: The 014B sequence was derived from doi:10.1016 / j.ymthe.2018.02.029, and the original name was 17-3.

[0391] Example 11: ASO-induced exon 41 skipping of the CEP290 gene with bait sequences of different lengths

[0392] Fifteen bait sequences with lengths from 7 to 11 nt were tested in the case of the target sequence 017B. ASO 017B targeted positions 26 to 45 of CEP290 exon 41 and promoted exon skipping. Each 50 nM ASO was transfected into HEK293 cells using Lipofectamine 2000, and the buffer alone was used as a negative control. Two days after transfection, the cells were harvested and total RNA samples were isolated for splicing analysis by semi-quantitative fluorescent RT-PCR.

[0393] The bis-ASOs tested included baits (017B-L7a to 017B-L7e, 017B-L8a to 017B-L8d, 017B-L9a to 017B-L9c, 017B-L10a and 017B-L10b, and 017B-L11) linked to the 5' (“L”) end of the targeting sequence 017B, Figure 10 A), or baits (017B-R7a to 017B-R7e, 017B-R8a to 017B-R8d, 017B-R9a to 017B-R9c, 017B-R10a and 017B-R10b, and 017B-R11) linked to the 3' (“R”) end of the targeting sequence 017B, Figure 10 B).

[0394] Regardless of whether the bait was linked to the 5' or 3' end of the bis-ASO, most bis-ASOs improved the exon skipping effect of the individual targeting sequence 017B (017B) with statistical significance, and the most potent ASO was 017B-R10a. Representative results of three independent experiments of bis-ASOs containing baits linked to the 5' end of the targeting sequence 017B and bis-ASOs containing baits linked to the 3' end of the targeting sequence 017B are shown respectively in Figure 10 A and Figure 10 the left panels of B (FL: full-length transcript, Δ41: transcript with exon 41 skipped). Quantification of the data (n = 3) is shown in the right panels of Figure 10 A and Figure 10 B ( Figure 10 the right panel of A for bis-ASOs containing baits linked to the 5' end of the targeting sequence 017B, and Figure 10 the right panel of B for bis-ASOs containing baits linked to the 3' end of the targeting sequence 017B). *P < 0.05, **P < 0.01, ***P < 0.001 (all relative to 017B). ASO 017B-L7a, 017B-R7b, 017B-R9a, and 017B-R10a were the best ASOs. For sequence information on the baits, see Table 1; for sequence information on the ASOs and targeting sequences tested in Example 11, see Table 8. The target sequence (5' to 3') in CEP290 exon 41 was AAAGUCUAAUUGAAGAACUC (SEQ ID No. 187).

[0395] Table 8. ASOs and targeting sequences tested in Example 11

[0396]

[0397]

[0398]

[0399] Note: 017B is for self-systematic ASO screening and identification.

[0400] Example 12: ASO-induced exon 19 skipping of the HER2 gene in the presence of decoy sequences of different lengths

[0401] Fifteen decoy sequences of 7 to 11 nt in length were tested in the case of the targeting sequence 024B. ASO 024B targets a sequence covering the last 4 nt of intron 18 and the first 11 nt of exon 19 of the HER2 (also known as ERBB2) gene and moderately promotes exon skipping. 50 nM of each ASO was transfected into HeLa cells using Lipofectamine 2000, and a separate buffer was used as a negative control. Two days after transfection, the cells were harvested, and total RNA samples were isolated for splicing analysis by semi-quantitative fluorescent RT-PCR.

[0402] The bipartite ASOs tested included decoys (024B-L7a to 024B-L7e, 024B-L8a to 024B-L8d, 024B-L9a to 024B-L9c, 024B-L10a and 024B-L10b, and 024B-L11, Figure 11 A) linked to the 5' ("L") end of the targeting sequence 024B, or decoys (024B-R7a to 024B-R7e, 024B-R8a to 024B-R8d, 024B-R9a to 024B-R9c, 024B-R10a and 024B-R10b, and 024B-R11, Figure 11 B) linked to the 3' ("R") end of the targeting sequence 024B.

[0403] Regardless of whether the decoy was linked to the 5' or 3' end of the bipartite ASO, most bipartite ASOs improved the exon skipping effect of the individual targeting sequence 024B (024B) with statistical significance, and the most potent ASO was 024B-L8c. Representative results of three independent experiments of bipartite ASOs containing decoys linked to the 5' end of the targeting sequence 024B and bipartite ASOs containing decoys linked to the 3' end of the targeting sequence 024B are shown respectively in Figure 11 A and Figure 11 the left panels of B (FL: full-length transcript, Δ19: transcript with exon 19 skipped). Quantification of the data (n = 3) is shown as mean ± standard deviation in the right panels of Figure 11 A and Figure 11 B ( Figure 11 the right panel of A is for bipartite ASOs containing decoys linked to the 5' end of the targeting sequence 024B, and Figure 11Right panel of B shows a bipartite ASO containing a bait linked to the 3'-end of the targeting sequence 024B). *P < 0.05, **P < 0.01, ***P < 0.001 (all relative to 024B). ASO 024B-L8c was the best ASO. For sequence information on the bait, see Table 1; for sequence information on the ASOs and targeting sequences tested in Example 12, see Table 9. The target sequence (5' to 3') at the junction of intron 18 and exon 19 of HER2 is CCAGGGCAUCUGGAU (SEQ ID No. 219).

[0404] Table 9. ASOs and targeting sequences tested in Example 12

[0405]

[0406] Note: The 024B sequence is from doi:10.3892 / ijo_00000472, with the original name 2707.

[0407] Example 13: ASO-induced exon 10 skipping of the ATXN3 gene in the presence of bait sequences of different lengths

[0408] Fifteen bait sequences of 7 to 11 nt in length (with the same sequence as VO659 but different chemical modifications) were tested in the case of the targeting sequence 015C. ASO 015C consists of 7 copies of CTG and targets seven CAG repeats, and thus can target the CAG repeats in exon 10 of the ATXN3 (also known as SCA3) gene. ASO 015C promotes exon 10 skipping of the gene. 40 nM of each ASO was transfected into A549 cells using Lipofectamine 2000, and the buffer alone was used as a negative control. Two days after transfection, the cells were collected, and total RNA samples were isolated for splicing analysis by semi-quantitative fluorescent RT-PCR.

[0409] The bipartite ASOs tested included baits linked to the 5' ("L") end of the targeting sequence 015C (015C-L7a to 015C-L7e, 015C-L8a to 015C-L8d, 015C-L9a to 015C-L9c, 015C-L10a and 015C-L10b, and 015C-L11, Figure 12 A), or baits linked to the 3' ("R") end of the targeting sequence 015C (015C-R7a to 015C-R7e, 015C-R8a to 015C-R8d, 015C-R9a to 015C-R9c, 015C-R10a and 015C-R10b, and 015C-R11, Figure 12 B).

[0410] Regardless of whether the decoy was linked to the 5'-end or 3'-end of the bipartite ASO, most bipartite ASOs improved the exon skipping effect of the individual target sequence 015C (015C) with statistical significance, and the most potent ASO was 015C-R8d. Representative results of three independent experiments of bipartite ASOs containing a decoy linked to the 5'-end of the target sequence 015C and bipartite ASOs containing a decoy linked to the 3'-end of the target sequence 015C are shown respectively in Figure 12 A and Figure 12 the left panels of Figure 12 A and Figure 12 B (FL: full-length transcript, Δ10: transcript with exon 10 skipped). Quantification of the data (n = 3) is shown in the right panels of Figure 12 A for bipartite ASOs containing a decoy linked to the 5'-end of the target sequence 015C and Figure 12 the right panel of

[0411] Table 10. ASOs and target sequences tested in Example 13

[0412]

[0413]

[0414] Note: The 015C sequence was derived from doi:10.1016 / j.omtn.2019.07.004, and its original name was VO659.

[0415] Example 14: ASO-induced exon 10 skipping of the PKM gene with decoy sequences of different lengths

[0416] Fifteen decoy sequences of 7 to 11 nt in length were tested in the case of the targeting sequence 027B. ASO 027B targets positions 45 to 62 of exon 10 of PKM and promotes exon 10 skipping and exon 9 inclusion of the endogenous PKM gene. Each ASO at 50 nM was transfected into RD cells using Lipofectamine 2000, and a separate buffer was used as a negative control. Two days after transfection, cells were collected, and total RNA samples were isolated for splicing analysis by semi-quantitative fluorescent RT-PCR. The PCR products were digested with PstI to distinguish their origin (PKM1 or PKM2).

[0417] The decoy ASOs tested included baits (027B-L7a to 027B-L7e, 027B-L8a to 027B-L8d, 027B-L9a to 027B-L9c, 027B-L10a and 027B-L10b, and 027B-L11, Figure 13 A) linked to the 5' (“L”) end of the targeting sequence 027B, or baits (027B-R7a to 027B-R7e, 027B-R8a to 027B-R8d, 027B-R9a to 027B-R9c, 027B-R10a and 027B-R10b, and 027B-R11, Figure 13 B) linked to the 3' (“R”) end of the targeting sequence 027B.

[0418] Regardless of whether the bait was linked to the 5' or 3' end of the decoy ASO, most decoy ASOs improved the exon 10 skipping effect of the individual targeting sequence 027B (027B) with statistical significance. Representative results of three independent experiments of decoy ASOs containing baits linked to the 5' end of the targeting sequence 027B and decoy ASOs containing baits linked to the 3' end of the targeting sequence 027B are shown separately in Figure 13 A and Figure 13 the left panels of B (PKM1: transcripts with exon 10 skipped; PKM2: transcripts with exon 9 skipped; PKMds: both exons skipped). Quantification of the data (n = 3) is shown in the right panels of Figure 13 A and Figure 13 B ( Figure 13 the right panel of A for decoy ASOs containing baits linked to the 5' end of the targeting sequence 027B, and Figure 13Right panel for B (bisected ASO containing a bait linked to the 3'-end of targeting sequence 027B). *P < 0.05, **P < 0.01, ***P < 0.001 (all relative to 027B). ASO 027B-L8c, 027B-L9a, 027B-L10a, 027B-L10b, and 027B-R10a are the best ASOs. For sequence information on the bait, see Table 1; for sequence information on the ASOs and targeting sequences tested in Example 14, see Table 11. The target sequence (5' to 3') in PKM exon 10 is UGAGGAACUCCGCCGCCU (SEQ ID No. 283).

[0419] Table 11. ASOs and targeting sequences tested in Example 14

[0420]

[0421]

[0422] Note: The 027B sequence is from doi:10.1158 / 0008-5472.CAN-20-0948, and its original name was ASO1-cEt / DNA.

[0423] Example 15: ASO-induced exon 6 skipping of the MDM4 gene in the presence of bait sequences of different lengths

[0424] Fifteen bait sequences of 7 to 11 nt in length were tested in the case of targeting sequence 029B. ASO 029B targets a region covering the last 9 nt of exon 6 and the first 16 nt of intron 6 of the MDM4 gene. Alone, ASO 029B moderately promoted exon 6 skipping of the endogenous MDM4 gene. Each 50 nM ASO was transfected into HEK293 cells using Lipofectamine 2000, and the buffer was used as a negative control. Two days after transfection, the cells were harvested, and total RNA samples were isolated for splicing analysis by semi-quantitative fluorescent RT-PCR.

[0425] The bisected ASOs tested included baits (029B-L7a to 029B-L7e, 029B-L8a to 029B-L8d, 029B-L9a to 029B-L9c, 029B-L10a, and 029B-L10b, and 029B-L11) linked to the 5' ("L") end of targeting sequence 029B Figure 14A), or a decoy (029B-R7a to 029B-R7e, 029B-R8a to 029B-R8d, 029B-R9a to 029B-R9c, 029B-R10a and 029B-R10b, and 029B-R11) linked to the 3' ("R") end of the targeting sequence 029B, Figure 14 B).

[0426] Regardless of whether the decoy is linked to the 5' end or the 3' end of the bipartite ASO, all bipartite ASOs except one improved the exon 6 skipping effect of the individual targeting sequence 029B (029B) with statistical significance. Representative results of three independent experiments of bipartite ASOs containing a decoy linked to the 5' end of the targeting sequence 029B and bipartite ASOs containing a decoy linked to the 3' end of the targeting sequence 029B are shown respectively in Figure 14 A and Figure 14 the left panels of B (FL: full-length transcript, Δ6: transcript with exon 6 skipped). Quantification of the data (n = 3) is shown as mean ± standard deviation in Figure 14 A and Figure 14 the right panels of B ( Figure 14 the right panel of A is for bipartite ASOs containing a decoy linked to the 5' end of the targeting sequence 029B, and Figure 14 the right panel of B is for bipartite ASOs containing a decoy linked to the 3' end of the targeting sequence 029B). *P < 0.05, **P < 0.01, ***P < 0.001 (all relative to 029B). ASO 029B-L7d, 029B-L8c, 029B-L9b, 029B-L9c, and 029B-R7a are the best ASOs. For sequence information of the decoys, see Table 1; for sequence information of the ASOs and targeting sequences tested in Example 15, see Table 12. The target sequence (5' to 3') at the MDM4 exon 6-intron 6 junction is CAACUGAAGGUAAAAUCACCACACG (SEQ ID No. 315).

[0427] Table 12. ASOs and targeting sequences tested in Example 15

[0428]

[0429]

[0430] Note: The 029B sequence is from doi:10.1172 / JCI82534, with the original name ASO4.

Claims

1. An oligonucleotide comprising a targeting sequence and a 5'-splice site decoy sequence operably linked to the 5'-end and / or 3'-end of the targeting sequence, or consisting of the targeting sequence and a 5'-splice site decoy sequence operably linked to the 5'-end and / or 3'-end of the targeting sequence: The decoy sequence comprises a nucleotide sequence of 5, 6, 7, 8, 9, 10 or 11 nucleotides complementary to the single-stranded 5'-end of U1 snRNA; The targeting sequence hybridizes to a sequence selected from the group consisting of: a target exon, a flanking intron sequence upstream of the target exon, a flanking intron sequence downstream of the target exon, an intron-exon junction upstream of the target exon, and an intron-exon junction downstream of the target exon; And The decoy sequence operably linked to the 5'-end of the targeting sequence is the same as or different from the decoy sequence operably linked to the 3'-end of the targeting sequence.

2. The oligonucleotide according to claim 1, wherein the decoy sequence comprises one or more nucleotide sequences independently selected from the group consisting of the nucleotide sequences of SEQ ID No. 1 to 23 and SEQ ID No. 347 to 353, or consists of one or more nucleotide sequences independently selected from the group consisting of the nucleotide sequences of SEQ ID No. 1 to 23 and SEQ ID No. 347 to 353.

3. The oligonucleotide according to any one of the preceding claims, wherein the target exon is selected from the group consisting of: exon 7 of the endogenous SMN1 and SMN2 genes, exons 45, 51 and 52 of the endogenous DMD gene, exon 17 of the endogenous APP gene, exon 41 of the endogenous CEP290 gene, exon 19 of the endogenous HER2 gene, exon 10 of the SCA3 gene, exon 10 of the endogenous PKM gene, and exon 6 of the endogenous MDM4 gene.

4. The oligonucleotide according to any one of the preceding claims, wherein the targeting sequence comprises a nucleotide sequence selected from the group consisting of, or consists of, the following nucleotide sequences: The nucleotide sequences of SEQ ID No. 25, 29, 33, 37, 41, 45, 49, 75, 92, 124, 156, 188, 220, 252, 284 and 316.

5. The oligonucleotide according to any one of the preceding claims, wherein the targeting sequence is directly linked to the decoy sequence.

6. The oligonucleotide according to any one of claims 1 to 4, wherein the targeting sequence is linked to the decoy sequence via a linker having 1, 2, 3, 4 or 5 nucleotides.

7. The oligonucleotide according to any one of claims 1 to 6, when the decoy sequence is operably linked to the 5'-end and 3'-end of the targeting sequence, the linker between the targeting sequence and the decoy sequence at the 5'-end of the targeting sequence, and the linker between the targeting sequence and the decoy sequence at the 3'-end of the targeting sequence are the same as or different from each other.

8. The oligonucleotide according to claim 1, comprising a nucleotide sequence selected from the group consisting of, or consisting of, the following nucleotide sequences: The nucleotide sequences of SEQ ID No. 26 to 27, 30 to 31, 34 to 35, 38 to 39, 42 to 43, 46 to 47, 50 to 73, 76 to 90, 93 to 122, 125 to 154, 157 to 186, 189 to 218, 221 to 250, 253 to 282, 285 to 314, 317 to 346, and 354 to 356.

9. The oligonucleotide according to claim 1, which is selected from the group consisting of: the nucleotide sequences of SEQ ID No. 26, 30, 31, 34, 38, 42, 46, 50, 51, 60, 64, 65, 68, 70, 78, 83, 85, 86, 87, 88, 89, 103, 109, 110, 114, 115, 116, 127, 132, 144, 164, 189, 205, 213, 216, 228, 260, 276, 292, 294, 297, 298, 312, 320, 324, 327, 328, 332, and 354.

10. The oligonucleotide according to any one of the preceding claims, which comprises at least one nucleotide analogue, such as, but not limited to, an oligonucleotide modified with 2'-O-methoxyethyl having a phosphodiester or phosphorothioate backbone, and phosphorodiamidate morpholino oligomers.

11. A composition comprising the oligonucleotide according to any one of the preceding claims and a pharmaceutically acceptable carrier.

12. A vector encoding the oligonucleotide according to any one of the preceding claims.

13. A method for generating or promoting exon skipping of a target exon during pre-mRNA splicing, which comprises contacting the pre-mRNA in a cell or a subject with the oligonucleotide according to any one of claims 1 to 10, the composition according to claim 11, and / or the vector according to claim 12 to restore the reading frame or generate a new splicing isoform.

14. The method according to claim 13, wherein ASO-mediated exon skipping manipulates the expression of a target gene by inhibiting the splicing of an exon, an intron, or a specific splicing site of the target gene, resulting in restoring the reading frame of a target defective gene, generating different isoforms (such as dominant negative isoforms) of the target gene, skipping the toxic part of the gene, silencing the gene, and / or altering the structure and function of the gene.

15. A method for improving the exon skipping efficacy and / or efficiency of a target sequence capable of hybridizing with a sequence selected from the group consisting of: a target exon, a flanking intron sequence upstream of the target exon, a flanking intron sequence downstream of the target exon, an intron-exon junction upstream of the target exon, and an intron-exon junction downstream of the target exon in a cell or a subject, the method comprising: obtaining one or more oligonucleotides according to any one of claims 1 to 10 comprising the target sequence and a decoy sequence.

16. The method according to claim 15, further comprising screening the one or more oligonucleotides based on the exon skipping efficacy and / or efficiency of the target exon of the one or more oligonucleotides.

17. The method according to any one of claims 13 to 16, wherein the target gene is selected from the group consisting of SMN1, SMN2, DMD, APP, CEP290, HER2, SCA3, PKM, and MDM4 genes.

18. The method according to any one of claims 13 to 17, wherein the exons of the target gene are selected from the group consisting of exon 7 of the endogenous SMN1 and SMN2 genes, exons 45, 51, and 52 of the endogenous DMD gene, exon 17 of the endogenous APP gene, exon 41 of the endogenous CEP290 gene, exon 19 of the endogenous HER2 gene, exon 10 of the SCA3 gene, exon 10 of the endogenous PKM gene, and exon 6 of the endogenous MDM4 gene.

19. The method according to any one of claims 13 to 18, further comprising delivering to the cell or administering to the subject the oligonucleotide according to any one of claims 1 to 10, the composition according to claim 11, and / or the vector according to claim 12.

20. A method of treating a disease and / or its complication in a subject, comprising administering to the subject the oligonucleotide according to any one of claims 1 to 10, the composition according to claim 11, and / or the vector according to claim 12, wherein the oligonucleotide produces or promotes exon skipping of the target exon.

21. The method according to claim 20, wherein the oligonucleotide inhibits the splicing of an exon, intron, or specific splicing site of a target gene, thereby restoring the reading frame of the target defective gene, producing different subtypes of the target gene (such as a dominant negative subtype), skipping a toxic portion of the gene, silencing the gene, and / or altering the structure and function of the gene.

22. The method according to claim 20 or 21, wherein the disease and / or its complication is selected from the group consisting of diseases and / or their complications that can benefit from exon skipping on one or more genes, the genes being selected from the group consisting of SMN1, SMN2, DMD, APP, CEP290, HER2, SCA3, PKM, and MDM4 genes.

23. The method according to claim 22, wherein the disease and / or its complication is selected from the group consisting of diseases and / or their complications that can benefit from exon skipping of one or more exons, the exons being selected from the group consisting of exon 7 of the endogenous SMN1 and SMN2 genes, exons 45, 51, and 52 of the endogenous DMD gene, exon 17 of the endogenous APP gene, exon 41 of the endogenous CEP290 gene, exon 19 of the endogenous HER2 gene, exon 10 of the SCA3 gene, exon 10 of the endogenous PKM gene, and exon 6 of the endogenous MDM4 gene.

24. The method according to claim 23, wherein the disease and / or its complication is selected from the group consisting of: Duchenne muscular dystrophy, Alzheimer's disease, Joubert syndrome, spinocerebellar ataxia type 3 (SCA3), breast cancer, HER2-positive cholangiocarcinoma, colorectal cancer, non-small cell lung cancer, bladder cancer, prostate cancer, lung cancer, cervical cancer, kidney cancer, papillary thyroid cancer, colon cancer, colorectal cancer, glioma, ovarian cancer, gastric cancer, hepatoblastoma, fibrolamellar hepatocellular carcinoma, soft tissue sarcoma, osteosarcoma, chronic lymphocytic leukemia, acute myeloid leukemia, mantle cell lymphoma, pediatric Burkitt lymphoma, salivary gland cancer, liver cancer or melanoma.

25. The method according to any one of claims 20 to 24, wherein an oligonucleotide according to any one of claims 1 to 10, a composition according to claim 11 and / or a vector according to claim 12 is administered in a therapeutically effective amount.

26. A kit for use in the method according to any one of claims 13 to 25, the kit comprising an oligonucleotide according to any one of claims 1 to 10, a composition according to claim 11 and / or a vector according to claim 12.

Images