Method and composition for regulating splicing of selective intron

Abstract

To provide a method for regulating expression of a target protein or a target RNA by a cell having a selective intron-containing pre mRNA (AIC pre mRNA).SOLUTION: Provided is a method in which AIC pre-mRNA includes selective intron, a first portion of an exon adjacent to 5' splice site of selective intron, and a second portion of an exon adjacent to 3' splice site of selective intron, and includes bringing a cell in contact with a therapeutic agent binding to a targeting region of an AIC pre-mRNA encoding a target protein or a target RNA, which regulates the splicing of selective intron from the AIC pre-mRNA encoding a target protein or a target RNA, by which the level of a processed mRNA encoding a target protein or a target RNA is regulated, and expression of a target protein or a target RNA in the cell is regulated.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 839,572, filed April 26, 2019, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference herein in its entirety. The ASCII copy, created on April 23, 2020, is named 47991-726_601_SL.txt and is 1,023,445 bytes in size. [Background technology]

[0003] Alternative splicing events in the pre-mRNA encoded by a gene can result in non-productive mRNA transcripts, which can lead to reduced protein expression. Therapeutic agents that can target alternative splicing events in the pre-mRNA encoded by a gene can increase the expression level of functional proteins in patients and / or inhibit abnormal protein expression. Such therapeutic agents can be used to treat conditions that would benefit from increased protein expression, or to treat diseases caused by changes in protein expression levels, such as conditions or diseases caused by protein deficiencies. One such example is programmed death-ligand 1 (PD-L1, or CD274), a ligand for programmed cell death-1 (PD-1, CD279).

[0004] PD-1 (CD279) is a crucial immunoregulatory checkpoint molecule that has a profound effect on adaptive immune responses, particularly T cell function (Keir et al., 2008, Ann. Rev. Immunol., 26: 677-704). Its ligands, PD-L1 (CD274) and, to a lesser extent, PD-L2, are expressed on both specialized immune cells and tissues. PD-L1 and PD-L2 interact with PD-1 to transmit inhibitory signals to infiltrating T cells, inducing immune control, anergy, a regulatory phenotype, and control of "aggression." PD-1 and PD-L1 expression are upregulated in inflammatory environments, for example, upon T cell activation (PD-1) or exposure to inflammatory cytokines (PD-L1), constituting an endogenous "brake," or checkpoint. PD-L1 is also upregulated in a wide range of tumors, where its presence suppresses antitumor T cell responses.

[0005] PD-1:PD-L1 interactions are critical mediators of peripheral immune tolerance, i.e., the suppression of T cells that inappropriately attack self-tissues, and have also been shown to regulate established autoimmunity in animal models. Models of inflammatory diseases in which PD-L1 has been shown to be important include the non-obese diabetic (NOD) model of autoimmune diabetes, the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, the collagen-induced arthritis model of rheumatoid arthritis, multiple models of inflammatory bowel disease (ulcerative colitis, Crohn's disease), transplantation and graft-versus-host disease, autoimmune uveitis, and many other settings, suggesting a broad role for the PD-1:PD-L1 pathway in inflammatory diseases (for reviews, see Keir, 2008; Gianchecchi, 2013). In humans, genetic polymorphisms in PD-1 have been associated with systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis, Graves' disease, and multiple sclerosis (Okazaki & Honjo, International Immunology, 2007).

[0006] Certain studies, including those in NOD mice, EAE, contact hypersensitivity, and lupus nephritis, suggest that ectopic or overexpression of PD-L1 can regulate and suppress pathogenic immune responses. Summary of the Invention

[0007] Described herein are methods of modulating expression of a target protein or target RNA by a cell having an alternative intron-containing pre-mRNA (AIC pre-mRNA), the AIC pre-mRNA comprising an alternative intron, a first portion of an exon adjacent to the 5' splice site of the alternative intron, and a second portion of the exon adjacent to the 3' splice site of the alternative intron, comprising contacting the cell with a therapeutic agent that binds to a targeted region of the AIC pre-mRNA encoding the target protein or target RNA, thereby modulating splicing of the alternative intron from the AIC pre-mRNA encoding the target protein or target RNA, thereby modulating the level of processed mRNA encoding the target protein or target RNA and modulating expression of the target protein or target RNA in the cell.

[0008] Described herein are methods of treating a disease or condition in a subject in need thereof by modulating expression of a target protein or target RNA in the cells of the subject, comprising contacting the cells of the subject with a therapeutic agent that modulates splicing of an alternative intron from an alternative intron-containing pre-mRNA (AIC pre-mRNA) encoding the target protein or target RNA, wherein the AIC pre-mRNA comprises an alternative intron, a first portion of an exon adjacent to the 5' splice site of the alternative intron, and a second portion of the exon adjacent to the 3' splice site of the alternative intron, and wherein the therapeutic agent binds to a targeted region of the AIC pre-mRNA encoding the target protein or target RNA, thereby modulating splicing of the alternative intron from the AIC pre-mRNA encoding the target protein or target RNA, thereby modulating the level of processed mRNA encoding the target protein or target RNA and modulating expression of the target protein or target RNA in the cells of the subject.

[0009] In some embodiments, modulating expression of a target protein in a cell comprises increasing expression of the target protein in the cell. In some embodiments, modulating the level of processed mRNA encoding the target protein comprises increasing the level of processed mRNA encoding the target protein. In some embodiments, inclusion of an alternative intron from the AIC pre-mRNA encoding the target protein is increased. In some embodiments, modulating the level of processed mRNA encoding the target protein comprises modulating the level of processed mRNA comprising an alternative intron, a first portion of an exon, and a second portion of an exon.

[0010] In some embodiments, the therapeutic agent is a small molecule. In some embodiments, the therapeutic agent is an antisense oligomer (ASO) complementary to a target region of the AIC pre-mRNA. In some embodiments, the therapeutic agent is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to a target region of the AIC pre-mRNA encoding a target protein. In some embodiments, at least a portion of the target region of the AIC pre-mRNA is within an intron upstream of a first portion of an exon. In some embodiments, at least a portion of the target region of the AIC pre-mRNA is within an intron downstream of a second portion of an exon. In some embodiments, at least a portion of the target region of the AIC pre-mRNA is within an alternative intron. In some embodiments, the target protein produced is In some embodiments, the target RNA produced is a fully functional protein. In some embodiments, the target RNA produced is a fully functional RNA.

[0011] In some embodiments, the target protein is PD-L1 (CD274). In some embodiments, the targeted region of the AIC pre-mRNA to which the Therapeutic Agent binds is located within exon 4 of CD274. In some embodiments, the Therapeutic Agent binds to the targeted region of the CD274 (PD-L1) AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 68, 69, and 71-76. In some embodiments, the Therapeutic Agent modulates splicing of an alternative intron from an exon of the CD274 AIC pre-mRNA, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 68. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of CD274, wherein the AIC pre-mRNA comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99% or 100% sequence identity to any one of SEQ ID NOs: 71-76.

[0012] In some embodiments, the target protein is Rho GTPase-activating protein 23 (ARHGAP23). In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent binds is located within an exon of ARHGAP23. In some embodiments, the therapeutic agent binds to the targeted region of the ARHGAP23 AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 77 and 91. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of ARHGAP23, wherein the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 77. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of ARHGAP23, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 91.

[0013] In some embodiments, the target protein is bromodomain-containing 1 (BRD1). In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent binds is located within an exon of BRD1. In some embodiments, the therapeutic agent binds to the targeted region of the BRD1 AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 78 and 92. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of BRD1, wherein the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 78. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of BRD1, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 92.

[0014] In some embodiments, the target protein is protocadherin-16 (DCHS1). In some embodiments, the targeted region of the AIC pre-mRNA to which the Therapeutic Agent binds is located within an exon of DCHS1. In some embodiments, the Therapeutic Agent binds to the targeted region of the DCHS1 AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 79 and 93. In some embodiments, the Therapeutic Agent modulates splicing of an alternative intron from the AIC pre-mRNA of DCHS1, wherein the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 79. In some embodiments, the Therapeutic Agent The agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of DCHS1, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:93.

[0015] In some embodiments, the target protein is erythrocyte membrane protein band 4.1-like 2 (EPB41L2). In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent binds is located within an exon of EPB41L2. In some embodiments, the therapeutic agent binds to the targeted region of the EPB41L2 AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 80 and 94. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from the EPB41L2 AIC pre-mRNA, wherein the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 80. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of EPB41L2, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 94.

[0016] In some embodiments, the target protein is glutathione peroxidase 8 (GPX8). In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent binds is located within an exon of GPX8. In some embodiments, the therapeutic agent binds to the targeted region of the GPX8 AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 81 and 95. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from the GPX8 AIC pre-mRNA, wherein the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 81. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from an exon of the GPX8 AIC pre-mRNA, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 95.

[0017] In some embodiments, the target protein is human immunodeficiency virus type 1 enhancer-binding protein 3 (HIVEP3). In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent binds is located within an exon of HIVEP3. In some embodiments, the therapeutic agent binds to the targeted region of the HIVEP3 AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 82 and 96. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from the HIVEP3 AIC pre-mRNA, wherein the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 82. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of HIVEP3, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 96.

[0018] In some embodiments, the target protein is inversin (INVS). In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent binds is located within an exon of INVS. In some embodiments, the therapeutic agent binds to the targeted region of the INVS AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 83 and 97. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from the INVS AIC pre-mRNA, wherein the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 83 ... The IC regulates splicing of alternative introns from exons of pre-mRNA, wherein the exons comprise sequences having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:97.

[0019] In some embodiments, the target protein is the dyslexia-associated protein KIAA0319 (KIAA0319). In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent binds is located within an exon of KIAA0319. In some embodiments, the therapeutic agent binds to the targeted region of the KIAA0319 AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 84 and 98. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from the KIAA0319 AIC pre-mRNA, wherein the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 84. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of KIAA0319, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 98.

[0020] In some embodiments, the target protein is NLR family apoptosis inhibitory protein (NAIP). In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent binds is located within an exon of NAIP. In some embodiments, the therapeutic agent binds to the targeted region of the NAIP AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 85 and 99. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from the NAIP AIC pre-mRNA, wherein the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 85. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from an exon of the NAIP AIC pre-mRNA, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 99.

[0021] In some embodiments, the target protein is Patched 2 (PTCH2). In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent binds is located within an exon of PTCH2. In some embodiments, the therapeutic agent binds to PTCH2. Therapeutic agents bind to a targeted region of the AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 86 and 100. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of PTCH2, wherein the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 86. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of PTCH2, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 100.

[0022] In some embodiments, the target protein is protein tyrosine phosphatase receptor type Z1 (PTPRZ1). In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent binds is located within an exon of PTPRZ1. In some embodiments, the therapeutic agent binds to the targeted region of the PTPRZ1 AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 87 and 101. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from the PTPRZ1 AIC pre-mRNA, wherein the alternative intron is a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 87. In some embodiments, the Therapeutic Agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of PTPRZ1, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 101.

[0023] In some embodiments, the target protein is SON. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent binds is located within an exon of SON. In some embodiments, the therapeutic agent binds to the targeted region of the SON AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 88, 89, 102, and 103. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of SON, wherein the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 88 or SEQ ID NO: 89. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of SON, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 102 or SEQ ID NO: 103.

[0024] In some embodiments, the target protein is zinc finger CCHC domain-containing protein 2 (ZCCHC2). In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent binds is located within an exon of ZCCHC2. In some embodiments, the therapeutic agent binds to the targeted region of the ZCCHC2 AIC pre-mRNA, wherein the targeted region is within a sequence selected from SEQ ID NOs: 90 and 104. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of ZCCHC2, wherein the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of ZCCHC2, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 104.

[0025] In some embodiments, the disease or condition is an immune disease or disorder. In some embodiments, the immune disease or disorder is an autoimmune disease or disorder, an inflammatory disease or disorder, a chronic infection, graft-versus-host disease (GVHD), transplant rejection, or a T-cell proliferative disorder. In some embodiments, the immune disease or disorder is an autoimmune disease or disorder or an inflammatory disease or disorder selected from multiple sclerosis, inflammatory bowel disease, autoimmune hepatitis, renal inflammation, rheumatoid arthritis, psoriasis, lupus nephritis, corneal transplant, and uveitis.

[0026] In some embodiments, the disease or condition is caused by a deficiency in the amount or activity of the target protein. In some embodiments, the disease or condition is treated or prevented by increasing the amount or activity of the target protein. In some embodiments, the disease or condition is induced by a loss-of-function mutation in the target protein.

[0027] In some embodiments, a therapeutic agent increases the level of processed mRNA encoding a target protein in a cell. In some embodiments, the level of processed mRNA encoding a target protein in a cell contacted with a therapeutic agent is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about An increase of 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold.

[0028] In some embodiments, a therapeutic agent increases expression of a target protein in a cell. In some embodiments, the level of the target protein in a cell contacted with a therapeutic agent increases by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, or An increase of about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0029] In some embodiments, inclusion of alternative introns from the AIC pre-mRNA encoding the target protein is reduced. In some embodiments, the therapeutic agent reduces the level of processed mRNA encoding the target protein in a cell. In some embodiments, the level of processed mRNA encoding the target protein in a cell contacted with the therapeutic agent is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 1.1 to about 10-fold, about 1.1 to about 11-fold, about 1.1 to about 12-fold, about 1.1 to about 13-fold, about 1.1 to about 14-fold, about 1.1 to about 15-fold, about 1.1 to about 16-fold, about 1.1 to about 17-fold, about 1.1 to about 18-fold, about 1.1 to about 19-fold, about 1.1 to about 20-fold, about 1.1 to about 21-fold, about 1.1 to about 22-fold, about 1.1 to about 23-fold, about 1.1 to about 24-fold, about 1.1 to about 25-fold, about 1.1 to about 26-fold, about 1.1 to about 27-fold, about 1.1 to about 28-fold, about 1.1 to about 29-fold, about 1.1 to about 30-fold, about 1.1 to about 31-fold, about 1.1 to about 32-fold, about 1.1 to about 33-fold, about 1.1 to about about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold decrease.

[0030] In some embodiments, a therapeutic agent reduces expression of a target protein in a cell. In some embodiments, the level of the target protein in a cell contacted with a therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, or It is reduced by about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0031] In some embodiments, the therapeutic agent comprises a backbone modification comprising a phosphorothioate or phosphorodiamidate linkage. In some embodiments, the therapeutic agent comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety. In some embodiments, the therapeutic agent comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the therapeutic agent comprises a modified sugar moiety comprising 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 nucleobases, 8-15 nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 The nucleic acid sequence may consist of nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

[0032] In some embodiments, the method further comprises assessing the mRNA level or expression level of the target protein. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a fetus, embryo, or child. In some embodiments, the one or more cells are ex vivo or in a tissue or organ ex vivo.

[0033] In some embodiments, the therapeutic agent is administered to the subject by intraventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, implantation, or intravenous injection.

[0034] In some embodiments, splicing of an alternative intron from an AIC pre-mRNA encoding a target protein or target RNA produces a processed mRNA with a premature stop codon (PTC). In some embodiments, splicing of an alternative intron from an AIC pre-mRNA encoding a target protein or target RNA produces a processed mRNA with a premature stop codon (PTC) that does not encode a functional target protein or that does not encode a functional RNA. In some embodiments, splicing of an alternative intron from an AIC pre-mRNA encoding a target protein or target RNA produces a processed mRNA with a premature stop codon (PTC) that encodes a non-functional target protein or non-functional target RNA. In some embodiments, splicing of an alternative intron from an AIC pre-mRNA encoding a target protein or target RNA produces a processed mRNA that has a lower translation efficiency or expression efficiency for producing the target protein compared to a corresponding processed mRNA that contains the alternative intron but is otherwise identical. In some embodiments, splicing of an alternative intron from an AIC pre-mRNA encoding a target protein or target RNA produces a processed mRNA that undergoes nonsense-mediated decay (NMD). In some embodiments, splicing of an alternative intron from an AIC pre-mRNA encoding a target protein or target RNA produces a processed mRNA that undergoes nonsense-mediated decay (NMD).

[0035] Provided herein are therapeutic agents for use in the methods described herein. Provided herein are pharmaceutical compositions comprising a therapeutic agent described herein and a pharmaceutically acceptable excipient.

[0036] Provided herein is a method of treating a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 48 by intraventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal injection, subretinal injection, topical application, implantation, or intravenous injection.

[0037] Provided herein are methods for modulating expression of a target protein or target RNA by a cell to treat a disease or condition in a subject in need thereof, the disease or condition being associated with an abnormal protein or abnormal RNA in the subject. a composition comprising a therapeutic agent for treating a disease or condition, wherein the aberrant protein or aberrant RNA is abnormal in amount or activity in a subject, and the therapeutic agent modulates splicing of an alternative intron-containing pre-mRNA (AIC pre-mRNA) encoding a target protein or target RNA, wherein the target protein is (a) the aberrant protein; (b) a protein that functionally activates or deactivates a cell signaling mechanism, thereby altering cellular activity associated with a disease or condition; (c) a protein that functionally enhances or replaces the aberrant protein in the subject; or (d) a protein that functionally reduces or inhibits the aberrant protein in the subject; and the target RNA is (a) the aberrant RNA; (b) a protein that functionally activates or deactivates a cell signaling mechanism, thereby altering cellular activity associated with a disease or condition. (c) an RNA that activates or deactivates to alter cellular activity associated with a disease or condition; (c) an RNA that functionally enhances or replaces an abnormal RNA in a subject; or (d) an RNA that functionally reduces or inhibits an abnormal RNA in a subject; and the AIC pre-mRNA is a composition that includes an alternative intron, a first portion of an exon adjacent to the 5' splice site of the alternative intron, and a second portion of the exon adjacent to the 3' splice site of the alternative intron, thereby regulating the splicing of the alternative intron from the AIC pre-mRNA encoding a target protein or target RNA, thereby regulating the production or activity of the target protein or target RNA in a subject.

[0038] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0039] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0040] [Figure 1] A schematic representation of the generation of different splice variants is shown. [Figure 2A] Measurements of different mRNA isoforms in different cells and conditions are shown. [Figure 2B] Measurements of different mRNA isoforms in different cells and conditions are shown. [Figure 2C] Measurements of different mRNA isoforms in different cells and conditions are shown. [Figure 3] A schematic diagram of the ASO walk is shown. [Figure 4] AC show measurements of mRNA isoform and protein expression in cells transfected with different ASOs. [Figure 5] A to D show measurements of ASO efficacy. [Figure 6A] Measures of ASO efficacy are shown. [Figure 6B] Measures of ASO efficacy are shown. [Figure 6C] Measures of ASO efficacy are shown. DETAILED DESCRIPTION OF THE INVENTION

[0041] array The present application includes nucleotide sequences SEQ ID NOs: 1-3651 listed in Tables 1-3. The nucleotide sequences set forth as SEQ ID NOs: 1-67 in Table 1 are examples of antisense oligomers (ASOs) useful in the methods described herein. The nucleotide sequence set forth as SEQ ID NO: 68 in Table 2 is an example of a sequence that may be targeted by an ASO by the methods described herein. The nucleotide sequence set forth as SEQ ID NO: 69 in Table 2 is an example of a sequence that may be targeted by an ASO by the methods described herein. The nucleotide sequence set forth in Table 2 as SEQ ID NO: 70 is the CD274 amino acid sequence. The nucleotide sequence set forth in Table 2 as SEQ ID NO: 71 is the CD274 genomic sequence. Table 2 as SEQ ID NOs: 72-76 are pre-mRNA sequences. The nucleotide sequences set forth in Table 2 as SEQ ID NOs: 77-90 are alternative introns targeted in the genes listed in the table. The nucleotide sequences set forth in Table 2 as SEQ ID NOs: 91-104 are exons targeted in the genes listed in the table. The nucleotide sequences set forth in Table 2 as SEQ ID NOs: 105-3651 are examples of antisense oligomers (ASOs) useful in the methods described herein. Uppercase letters represent exon sequences, and lowercase letters represent intron sequences.

[0042] Detailed Description of the Invention Splicing and alternative splice sites Intervening sequences, or introns, are removed by a large, highly dynamic RNA-protein complex called the spliceosome, which orchestrates complex interactions between the primary transcript, small nuclear RNAs (snRNAs), and numerous proteins. The spliceosome assembles in an orderly fashion on each intron, starting with recognition of the 5' splice site (5'ss) by U1 snRNA or the 3' splice site (3'ss) by the U2 pathway, which is responsible for binding of the U2 accessory factor (U2AF) to the 3'ss region, facilitating U2 binding to the branch point sequence (BPS). U2AF is a stable heterodimer composed of a 65-kD subunit (U2AF65) encoded by U2AF2, which binds to the polypyrimidine tract (PPT), and a 35-kD subunit (U2AF35) encoded by U2AF1, which interacts with a highly conserved AG dinucleotide in the 3'ss, stabilizing U2AF65 binding. In addition to the BPS / PPT unit and 3'ss / 5'ss, accurate splicing requires auxiliary sequences or structures known as intronic or exon splicing enhancers or silencers that activate or repress splice site recognition. These elements enable the recognition of true splice sites from the vast excess of cryptic or pseudosites in the genomes of higher eukaryotes, which share the same sequence but exceed the number of true sites by an order of magnitude.

[0043] The decision to splice or not can typically be modeled as a stochastic rather than a deterministic process, as even the most well-defined splicing signals can be misspliced. However, under normal conditions, pre-mRNA splicing proceeds with surprisingly high fidelity. This is due, in part, to the activity of adjacent cis-acting auxiliary exon and intron splicing control elements (ESRs or ISRs). These functional elements are typically classified as either exon or intron splicing enhancers (ESEs or ISEs) or silencers (ESSs or ISSs) based on their ability to stimulate or inhibit splicing, respectively. Although there is now evidence that some auxiliary cis-acting elements may act by influencing the dynamics of spliceosome assembly, for example, by affecting the positioning of the spliceosome complex between the U1 snRNP and the 5'ss, it seems highly likely that many elements function in concert with trans-acting RNA-binding proteins (RBPs). For example, the family of serine- and arginine-rich RBPs (SR proteins) is a family of conserved proteins that play a key role in exon definition. SR proteins promote exon recognition by recruiting components of the pre-spliceosome to adjacent splice sites or by antagonizing the effects of ESSs in their vicinity. The repressive effect of ESSs can be mediated by members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, which can alter the recruitment of core splicing factors to adjacent splice sites. In addition to their role in splicing regulation, silencer elements are also known to function as transcriptional regulators, altering the recruitment of core splicing factors to adjacent splice sites, even though they have the typical spacing of exons. They are suggested to play a role in suppressing pseudoexons, which are sets of decoy intron splice sites that do not have a reading frame. ESEs and ESSs, along with their cognate trans-acting RBPs, represent key components in a set of splicing controls that specify how, where, and when mRNA assembles from its precursor.

[0044] Sequences marking exon-intron boundaries are degenerate signals of varying strength that can occur frequently within human genes. In multi-exon genes, different pairs of splice sites can be joined together in many different combinations to generate a wide variety of transcripts from a single gene. This is commonly referred to as alternative pre-mRNA splicing. While most mRNA isoforms produced by alternative splicing can be exported from the nucleus and translated into functional polypeptides, different mRNA isoforms derived from a single gene can vary greatly in their translation efficiency. These mRNA isoforms that contain a premature termination codon (PTC) at least 50 bp upstream of the exon junction complex are likely to be targeted for degradation by the nonsense-mediated mRNA decay (NMD) pathway. Mutations in conventional (BPS / PPT / 3'ss / 5'ss) and auxiliary splicing motifs can lead to aberrant splicing, such as exon skipping, or cryptic (or spurious) exon inclusion or splice site activation, and can contribute significantly to human morbidity and mortality. Both aberrant and alternative splicing patterns can be influenced by natural DNA variants in exons and introns.

[0045] Cryptic (or false) splice sites have the same splicing recognition sequence as true splice sites but are not used in the splicing reaction. They outnumber true splice sites in the human genome by an order of magnitude and are usually suppressed by a molecular mechanism that is not yet fully understood. Cryptic 5' splice sites have the consensus NNN / GUNNNN or NNN / GCNNNN (where N is any nucleotide and is the exon-intron boundary). Cryptic 3' splice sites have the consensus NAG / N. Activation of these splice sites is positively influenced by surrounding nucleotides that make them more similar to the optimal consensus of the native splice site, i.e., MAG / GURAGU and YAG / G, respectively, where M is C or A, R is G or A, and Y is C or U. Activation of cryptic (or false) splice sites may be influenced or determined by the balance between the intrinsic strength of the aberrant splice sites and their true counterparts, the availability of conventional signals in the vicinity of the mutant splice site, the size of the exon and intron, the nature of the mutation, and by disrupting or creating an ESE, ESS, ISE, or ISS.

[0046] Splice sites and their regulatory sequences can be readily identified by those skilled in the art using suitable publicly available algorithms, for example those listed in Kralovicova, J. and Vorechovsky, I. (2007) Global control of aberrant splice site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition. Nucleic Acids Res., 35, 6399-6413.

[0047] An exon sequence may contain both 5' and 3' alternative splice sites, which drive an RNA-binding protein, such as U2AF, to the alternative splice site within the exon sequence, resulting in the splicing of a portion of the exon from the pre-mRNA and producing a processed mRNA lacking that portion of the exon. The region or sequence between the alternative 5' and 3' splice sites within an exon is referred to as the alternative splice site. A pre-mRNA with 5' and 3' alternative splice sites located within an exon sequence can be referred to as an intron. A pre-mRNA with 5' and 3' alternative splice sites located within an exon sequence can be referred to as an alternative intron-containing pre-mRNA (AIC pre-mRNA). In some embodiments, an agent can bind to an alternative splice site or splicing control sequence to prevent binding of an RNA-binding protein, thereby inhibiting splicing of the alternative intron. In some embodiments, an agent can bind to an alternative splice site or splicing control sequence to promote binding of an RNA-binding protein, thereby enhancing splicing of the alternative intron.

[0048] Provided herein are methods and compositions that can regulate aberrant splicing events at alternative splice sites to regulate the level of functional mRNA encoded by a target gene and the expression level of a protein encoded by the target gene. The methods and compositions of the present invention can be used to regulate the expression level of a target protein in a subject or a subject's cells. For example, the methods and compositions of the present invention can be used to increase or decrease the expression level of a target protein in a subject or a subject's cells (without altering the protein sequence). The methods and compositions of the present invention can be used to regulate the level of processed mRNA encoding a target protein in a subject or a subject's cells. For example, the methods and compositions of the present invention can be used to increase or decrease the level of processed mRNA encoding a target protein in a subject or a subject's cells.

[0049] Provided herein are methods of modulating expression of a target protein or target RNA by a cell having an alternative intron-containing pre-mRNA (AIC pre-mRNA). In one aspect, the AIC pre-mRNA comprises an alternative intron, a first portion of an exon adjacent to the 5' splice site of the alternative intron, and a second portion of the exon adjacent to the 3' splice site of the alternative intron. In one aspect, the method comprises contacting the cell with a therapeutic agent that binds to a targeted region of the AIC pre-mRNA encoding the target protein or target RNA, thereby modulating splicing of the alternative intron from the AIC pre-mRNA encoding the target protein or target RNA, thereby modulating the level of processed mRNA encoding the target protein or target RNA and modulating expression of the target protein or target RNA in the cell.

[0050] Provided herein is a method for treating a disease or condition in a subject in need of such treatment by regulating the expression of a target protein or target RNA in the subject's cells. In one aspect, the method includes contacting the subject's cells with a therapeutic agent that regulates the splicing of an alternative intron from an alternative intron-containing pre-mRNA (AIC pre-mRNA) encoding the target protein or target RNA, wherein the AIC pre-mRNA comprises an alternative intron, a first portion of an exon adjacent to the 5' splice site of the alternative intron, and a second portion of an exon adjacent to the 3' splice site of the alternative intron, and the therapeutic agent binds to the target region of the AIC pre-mRNA encoding the target protein or target RNA, thereby regulating the splicing of the alternative intron from the AIC pre-mRNA encoding the target protein or target RNA, thereby regulating the level of processed mRNA encoding the target protein or target RNA and regulating the expression of the target protein or target RNA in the subject's cells.

[0051] In some embodiments, modulating expression of a target protein in a cell comprises increasing expression of the target protein in the cell. In some embodiments, expression of the target protein in the cell is increased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to unmodulated expression of the target protein. In embodiments, expression of the target protein in the cells is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold compared to unregulated expression of the target protein.

[0052] In some embodiments, modulating the level of processed mRNA encoding the target protein comprises increasing the level of processed mRNA encoding the target protein, in some embodiments, the level of processed mRNA encoding the target protein in the cell is increased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the unmodulated level of processed mRNA encoding the target protein. In some embodiments, the level of processed mRNA encoding the target protein in the cell is about 1.1 to about 10 fold, about 1.5 to about 10 fold, about 2 to about 10 fold, about 3 to about 10 fold, about 4 to about 10 fold, about 1.1 to about 5 fold, about 1.1 to about 6 fold, about 1.1 to about 7 fold, about 1.1 to about 8 fold, about 1.1 to about 9 fold, or about 1.1 to about 10 fold higher than the unregulated level of processed mRNA encoding the target protein. fold, about 2 to about 5 fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold increase.

[0053] In some embodiments, splicing of an alternative intron from an AIC pre-mRNA encoding a target protein is inhibited. In some embodiments, modulating the level of processed mRNA encoding the target protein comprises modulating the level of processed mRNA comprising an alternative intron, a first portion of an exon, and a second portion of an exon. In some embodiments, increasing the level of processed mRNA encoding the target protein comprises increasing the level of processed mRNA comprising an alternative intron, a first portion of an exon, and a second portion of an exon. In some embodiments, decreasing the level of processed mRNA encoding the target protein comprises decreasing the level of processed mRNA comprising an alternative intron, a first portion of an exon, and a second portion of an exon.

[0054] In some embodiments, splicing of an alternative intron from the AIC pre-mRNA encoding the target protein is promoted. In some embodiments, modulating expression of the target protein in a cell comprises reducing expression of the target protein in the cell. In some embodiments, expression of the target protein in the cell is reduced by at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% compared to expression of the unmodulated target protein. In some embodiments, the expression of the target protein in the cells is about 1.1 to about 10 fold, about 1.5 to about 10 fold, about 2 to about 10 fold, about 3 to about 10 fold, about 4 to about 10 fold, about 1.1 to about 5 fold, about 1.1 to about 6 fold, about 1.1 to about 7 fold, about 1.1 to about 8 fold, about 1.1 to about 9 fold, about 2 to about 5 fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, or less than the expression of the unregulated target protein. at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold decrease.

[0055] In some embodiments, modulating the level of processed mRNA encoding the target protein comprises reducing the level of processed mRNA encoding the target protein, in some embodiments, the level of processed mRNA encoding the target protein in the cell is reduced by at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% compared to the level of processed mRNA encoding the unmodulated target protein. In some embodiments, the level of processed mRNA encoding the target protein in the cell is about 1.1 to about 10 fold, about 1.5 to about 10 fold, about 2 to about 10 fold, about 3 to about 10 fold, about 4 to about 10 fold, about 1.1 to about 5 fold, about 1.1 to about 6 fold, about 1.1 to about 7 fold, about 1.1 to about 8 fold, about 1.1 to about 9 fold, or about 1.1 to about 10 fold higher than the unregulated level of processed mRNA encoding the target protein. fold, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times reduced.

[0056] In some embodiments, the therapeutic agent is a small molecule. In some embodiments, the therapeutic agent is an antisense oligomer (ASO) complementary to the target region of the AIC pre-mRNA. In some embodiments, the therapeutic agent is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target region of the AIC pre-mRNA encoding the target protein.

[0057] In some embodiments, at least a portion of the targeted region of the AIC pre-mRNA is within an intron upstream of the first portion of the exon. In some embodiments, at least a portion of the targeted region of the AIC pre-mRNA is within an intron downstream of the second portion of the exon. In some embodiments, at least a portion of the targeted region of the AIC pre-mRNA is within an alternative intron. In some embodiments, at least a portion of the targeted region of the AIC pre-mRNA is within the first portion of the exon. In some embodiments, at least a portion of the targeted region of the AIC pre-mRNA is within the second portion of the exon. In some embodiments, at least a portion of the targeted region of the AIC pre-mRNA overlaps with the junction of the intron upstream of the first portion of the exon and the first portion of the exon. In some embodiments, at least a portion of the targeted region of the AIC pre-mRNA overlaps with the junction of the first portion of the exon and the alternative intron. In some embodiments, at least a portion of the targeted region of the AIC pre-mRNA overlaps with the junction of the alternative intron and the second portion of the exon. In some embodiments, at least a portion of the targeted region of the AIC pre-mRNA overlaps with the junction of the second part of the exon and the intron downstream of the second part of the exon.

[0058] In some embodiments, the method is a method of increasing expression of a target protein by cells of a subject having an AIC pre-mRNA encoding the target protein, wherein the subject has a disease or disorder caused by a deficiency in the amount or activity of the target protein. In some embodiments, the deficiency in the amount of the target protein is caused by haploinsufficiency of the target protein. In some embodiments, the disease or disorder is caused by haploinsufficiency of the target protein. The condition is associated with haploinsufficiency of a protein-encoding gene. In some embodiments, a subject has a first allele that encodes a functional target protein and a second allele that results in no production of the target protein. In some embodiments, a subject has a first allele that encodes a functional target protein and a second allele that results in reduced production of the target protein. In another embodiment, a subject has a first allele that encodes a functional target protein and a second allele that encodes a non-functional target protein. In another embodiment, a subject has a first allele that encodes a functional target protein and a second allele that encodes a partially functional target protein. In any of these embodiments, the ASO binds to the targeted region of the AIC pre-mRNA transcribed from the first allele (encoding the functional target protein), thereby inhibiting splicing of the alternative intron from the AIC pre-mRNA, resulting in increased levels of mature mRNA encoding the functional target protein and increased expression of the target protein in the subject's cells.

[0059] In some embodiments, AIC pre-mRNA transcripts encoding proteins that cause a disease or condition are targeted by the ASOs described herein. In some embodiments, AIC pre-mRNA transcripts encoding proteins that are not the cause of the disease are targeted by the ASOs. For example, a disease that is the result of a mutation or deficiency in a first protein in a particular pathway can be ameliorated by targeting an AIC pre-mRNA encoding a second protein, thereby increasing production of the second protein. In some embodiments, the function of the second protein can compensate for the mutation or deficiency in the first protein (which is the cause of the disease or condition).

[0060] In some embodiments, the AIC pre-mRNA transcript encodes a protein that helps alleviate a disease or condition. For example, the AIC pre-mRNA transcript may encode a signaling protein that may activate or deactivate a disease-related signaling pathway. This pathway may be related to the immune response, causing upregulation of the immune response to, for example, abnormal cell proliferation or combat foreign pathogens. This pathway may be related to the immune response, causing downregulation of the immune response to, for example, reduce inflammation or abnormal immune responses (i.e., autoimmune or allergic reactions).

[0061] In some embodiments, the subject: a. a first mutant allele, i) the target protein is produced at a reduced level compared to production from the wild-type allele; ii) the target protein is produced in a form that is reduced in function compared to the equivalent wild-type protein, or iii) no target protein or functional RNA is produced; and b. A second mutant allele, i) the target protein is produced at a reduced level compared to production from the wild-type allele; ii) the target protein is produced in a form that is reduced in function compared to the equivalent wild-type protein; The AIC pre-mRNA is transcribed from a first allele and a second allele. In these embodiments, the ASO binds to the targeted region of the AIC pre-mRNA transcribed from the first allele and the second allele, thereby inhibiting splicing of the alternative intron from the AIC pre-mRNA, resulting in an increase in the level of mRNA encoding the target protein and increased expression of the target protein or functional RNA in the cells of the subject ... A target protein or functional RNA having increased expression levels resulting from inhibiting a target protein or functional RNA may be in either a reduced (partially functional) form compared to the equivalent wild-type protein, or in a fully functional form compared to the equivalent wild-type protein.

[0062] Disclosed herein is a composition comprising a therapeutic agent for use in a method of modulating expression of a target protein or target RNA by a cell to treat a disease or condition in a subject in need thereof, the disease or condition being associated with an aberrant protein or aberrant RNA in the subject, wherein the aberrant protein or aberrant RNA is abnormal in amount or activity in the subject, and the therapeutic agent modulates splicing of an alternative intron-containing pre-mRNA (AIC pre-mRNA) encoding the target protein or target RNA, wherein the target protein is: (a) Abnormal proteins; (b) proteins that functionally activate or deactivate cell signaling mechanisms, thereby altering cellular activity associated with a disease or condition; (c) a protein that functionally enhances or replaces an abnormal protein in a subject; or (d) a protein that functionally reduces or inhibits the abnormal protein in a subject; The target RNA is: (a) Abnormal RNA; (b) RNAs that functionally activate or deactivate cell signaling mechanisms, thereby altering cellular activity associated with a disease or condition; (c) an RNA that functionally enhances or replaces an abnormal RNA in a subject; or (d) an RNA that functionally reduces or inhibits the abnormal RNA in a subject; The AIC pre-mRNA is a composition that includes an alternative intron, a first portion of an exon adjacent to the 5' splice site of the alternative intron, and a second portion of the exon adjacent to the 3' splice site of the alternative intron, thereby regulating the splicing of the alternative intron from the AIC pre-mRNA encoding a target protein or target RNA, thereby regulating the production or activity of the target protein or target RNA in a subject.

[0063] In some embodiments, the target protein produced is a fully functional protein. In some embodiments, the target RNA produced is a functional RNA. In some embodiments, the target RNA produced is a fully functional RNA. In some embodiments, the target protein is PD-L1 (CD274). In some embodiments, the target RNA is a functional RNA that is transcribed from the CD274 gene and encodes the PD-L1 protein. In some embodiments, the target RNA is a fully functional RNA that is transcribed from the CD274 gene and encodes the PD-L1 protein.

[0064] PD-L1 protein The balance between T cell activation, tolerance, and immunopathology plays a key role in autoimmune diseases, such as systemic lupus erythematosus (SLE), type 1 diabetes, rheumatoid arthritis, Graves' disease, multiple sclerosis (MS), and transplant rejection and graft-versus-host disease (GVHD). Key regulators of T cell responses are programmed death 1 (PD-1; also known as CD279) and its ligands, PD-L1 (CD274) and PD-L2. The interaction between PD-1 and its ligands regulates immune responses by delivering inhibitory signals, controlling T cell activation, limiting effector T cell responses, and protecting tissues from immune-mediated damage.

[0065] The PD-1 protein, first identified as a negative regulator of T cell activation in a mouse model of autoimmunity, is a 288-amino acid (aa) type I transmembrane protein containing one immunoglobulin (Ig) superfamily domain, an immunoreceptor tyrosine-based inhibitory motif (ITIM), and an immunoreceptor tyrosine-based switch motif (ITSM). The PD-1 ligand, PD-L1, is a 290-amino acid type I transmembrane protein encoded by the CD274 gene on human chromosome 9, which contains seven exons. The PD-L1 protein contains IgV-like domains, IgC-like domains, and a short approximately 30-amino acid tail of unknown function. The interaction of PD-L1 with the PD-1 receptor is mediated by the IgV-like domain. The human genomic sequence of the CD274 gene is available in NCBI Gene ID 29126. The CD274 canonical mRNA sequence is available in NCBI Reference Sequence: NM_014143.3.

[0066] PD-L1 is constitutively expressed on both hematopoietic and non-hematopoietic cells, including B cells, T cells, plasmacytoid dendritic cells (pDCs), mesenchymal stem cells, and vascular endothelial cells. PD-L1 expression is upregulated by both type I and type II interferons (IFNs) and is reduced in the absence of MyD88, TRAF6, and MEK. Binding of PD-L1 to the PD-1 receptor results in PD-1 cytoplasmic tyrosine phosphorylation, recruitment of SHP-2, and inhibition of PI3K and Akt activity, ultimately resulting in inhibition of T cell receptor (TCR) signaling, which can be overcome by CD28 costimulation. In the absence of CD28 costimulation, PD-1 ligation reduces the induction of cytokines such as IFN-γ and cell survival proteins such as Bcl-xL (Keir et al., 2008, and Trabattoni, et al., 2009, J. Immunol. 183: 4984-4993, incorporated herein by reference). In addition to PD-1, B7-1 has been identified as a binding partner of PD-L1. The interaction between B7-1 and PD-L1 occurs via the IgV-like domain and induces an inhibitory signal in T cells, thereby limiting T cell responses.

[0067] PD1 and PD-L1 prevent the initiation and progression of autoimmunity and play a role in peripheral T cell tolerance (Keir et al., 2008, and Trabattoni et al., 2009). For example, the role of PD-1:PD-L1 interactions in autoimmunity has been demonstrated in PD-1-deficient mice. PD-1:PD-L1 interactions play a role in both positive and negative T cell selection in the thymus, consistent with a role in central tolerance induction. Negative T cell regulation by PD-1:PD-L1 also plays a role in peripheral tolerance by inhibiting autoreactive T cell responses and mediating regulatory T cell responses. As an example, loss of PD-1 or PD-L1 has been reported to result in rapid or exacerbated diabetes in a mouse model of autoimmune T cell-mediated diabetes. Administration of anti-PD-1 or anti-PD-L1 mAbs during induction of experimental autoimmune encephalomyelitis (EAE) in mouse models led to accelerated disease onset and severity (summarized by Keir et al., 2008). NOD mice (Li et al. al, 2015, Diabetes 64:529-540;Wang et al, 2008, Diabetes 57:1861-69), EAE(Hirata Studies of PD-L1 in immune responses to PD-L1 in patients with inflammatory bowel disease (Ritprajak et al., 2010, J. Immunology 184:4918-4925), contact hypersensitivity (Ritprajak et al., 2005, J. Immunology 174:1888-1897), contact hypersensitivity (Ritprajak et al., 2010, J. Immunology 184:4918-4925), and lupus nephritis (Ding et al., 2006, Clinical Immunology 118:258-67) have demonstrated that ectopic expression or overexpression of PD-L1 can control and suppress pathogenic immune responses (each incorporated herein by reference). In addition to these ectopic tissue-based overexpression studies, numerous additional reports have evaluated the effects of soluble PD-L1-Fc or PD-L1-Ig fusions, demonstrating the efficacy of exogenous / ectopic expression. Further evidence of the immunoregulatory consequences of PD-L1 administration is provided.

[0068] In addition to its role in autoimmunity, the interaction between PD-L1 and PD-1 regulates organ transplantation and graft-versus-host disease (GVHD). Both PD-1 and PD-L1 are upregulated on alloreactive T cells in transplant recipients and may regulate alloreactive immune responses. PD-1 is upregulated after the onset of GVHD, and PD-L1 is expressed on most cells in GVHD target organs. Importantly, administration of a PD-L1-blocking antibody accelerated graft rejection in heart, cornea, and skin transplant models. The central role of PD-L1 in graft tolerance has been demonstrated in transplant models using CD274- / - mice as transplant donors or recipients and treated with CTLA-4-Ig to induce tolerance. PD-L1 expression in grafts protected from local pathology and in the recipient immune system was required for the induction and maintenance of transplant tolerance. A potential mechanism by which PD-L1 may attenuate graft rejection is through the induction of T cell apoptosis. In particular, the use of PD-1 and PD-L1 blocking antibodies in transplantation models suggested a PD-1-independent role for PD-L1 in promoting tolerance via the induction of alloreactive T cell apoptosis (Keir, et al., 2008). In some embodiments, the methods and compositions of the invention increase levels of PD-L1, thereby resulting in effects on T cell function, including, for example, pathogenic T cell apoptosis, anergy, and / or exhaustion. In some embodiments, the effect(s) of the methods and compositions of the invention on T cell function are assessed by any suitable method described in the art, e.g., Keir, et al., 2008.

[0069] The methods and compositions of the present invention can be used to increase PD-L1 expression in a subject. Increasing PD-L1 expression in target tissues of an autoimmune disease can result in the suppression and / or control of the autoimmune response involved in the autoimmune disease. In some embodiments, autoimmune responses, e.g., autoreactive T cell responses, are suppressed and / or controlled by the interaction of PD-1 and PD-L1 on activated CD4+ and CD8+ T cells, e.g., killer CD8+ T cells. Overexpression of PD-L1 can inhibit pathogenic CD4+ T cells in vivo while maintaining T cell receptor antigen specificity. H 1 or T H 17 Effector phenotype to T cell regulatory phenotype, e.g., FOXP3+ regulatory T cells (T REG ) phenotype (Amarnath et al., 2011, Science Translational Medicine 3(111):1-13, incorporated herein by reference). In some embodiments, the methods and compositions of the invention are used to increase PD-L1 expression and induce pathogenic CD4+ T cells, e.g., T H 1 or T HIn some embodiments, a switch to a Tr1 regulatory phenotype is induced. Tr1 cells express IL-10 but are classically derived from exposure of Th1 cells (expressing the Tbet transcription factor) upon exposure to IL-27 and other tolerizing stimuli (potentially PD-L1). In some embodiments, the T cell regulatory phenotype is stably maintained over time. In some embodiments, long-term stability of T cell regulatory status is at least about 30 days, at least about 35 days, at least about 40 days, at least about 45 days, at least about 50 days, at least about 55 days, at least about 60 days, at least about 65 days, at least about 70 days, at least about 75 days, at least about 80 days, at least about 85 days, at least about 90 days, at least about 95 days, at least about 100 days, or at least about 110 days. for at least about 120 days, about 30 to about 90 days, about 40 to about 90 days, about 50 to about 90 days, about 60 to about 90 days, about 70 to about 90 days, about 30 to about 100 days, about 40 to about 100 days, about 50 to about 100 days, about 60 to about 100 days, about 70 to about 100 days, about 80 to about 100 days, about 30 to about 120 days, about 40 to about 120 days, about 50 to about 120 days, about 60 to about 120 days, about 70 to about 120 days, about 80 to approximately 120 days, approximately 30 to approximately 120 days, approximately 40 to approximately 120 days, approximately 50 to approximately 120 days, approximately 60 to approximately 120 days, approximately 70 to approximately 120 days, approximately 80 to approximately 120 days, approximately 30 to approximately 150 days, approximately 40 to approximately 150 days, approximately 50 to approximately 150 days, approximately 60 to approximately 150 days, approximately 70 to approximately 150 days, approximately 80 to approximately 150 days, approximately 30 to approximately 150 days, approximately 40 to approximately 150 days, approximately 50 to approximately 150 days, about 60 to about 150 days, about 70 to about 150 days, about 80 to about 150 days, about 30 to about 200 days, about 40 to about 200 days, about 50 to about 200 days, about 60 to about 200 days, about 70 to about 200 days, about 80 to about 200 days, about 30 to about 200 days, about 40 to about 200 days, about 50 to about 200 days, about 60 to about 200 days, about 70 to about 200 days, or about 80 to about 200 days REG indicated by the presence of a phenotype.

[0070] In some embodiments, the presence of a regulatory T cell phenotype is determined by T REG In some embodiments, the presence of a regulatory T cell phenotype is determined based on expression of associated markers or secreted cytokines. In some embodiments, the presence of a regulatory T cell phenotype is determined based on expression of one or more T cells selected from CD4, CD25, CD39, CD73, CD45RO, CD121a (IL-1R1), CD121b (IL-1R2), CD127low, CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD357 (GITR / AITR), FOXP3, FR4(m), GARP, Helios, LAP / TGFβ; and TIGIT. REG expression of associated markers; and / or expression of one or more T cells selected from IL-10, IL-35, and TGFβ REG In some embodiments, the expression of T REGThe presence of a phenotype is determined based on the expression of FOXP3. Expression of T cell markers or cytokine secretion can be assessed by any method known to those skilled in the art and described in the literature, for example, Amarnath et al., 2011, such as ELISA, Western blot assay, flow cytometry assay, or any suitable multiplex assay. Commercially available assays are available for measuring the production of cytokines, chemokines, cytokine receptors, and activation markers as required by the methods of the present invention. For example, a multiplex assay for detecting human cytokines and chemokines can be created using the BD™ Cytometric Bead Array Flex Set system (BD Biosciences, San Jose, CA).

[0071] Alternative intron-containing pre-mRNA (AIC pre-mRNA) In some embodiments, the methods of the invention utilize the presence in cells of an alternative intron-containing pre-mRNA (AIC pre-mRNA) that is transcribed from the CD274 gene and encodes the PD-L1 protein. Splicing of the identified CD274 AIC pre-mRNA species to produce the mature, fully spliced ​​CD274 mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits splicing of the alternative intron. The resulting mature CD274 mRNA is transported to the cytoplasm and translated, thereby increasing the amount of PD-L1 protein in the patient's cells and alleviating symptoms of an immune disorder or any disease or disorder associated with a deficiency in the amount or activity of PD-L1 protein or caused by a loss-of-function mutation in the CD274 gene.

[0072] Table 1 provides a non-limiting list of CD274 ASO sequences useful for increasing PD-L1 production by targeting regions of the CD274 AIC pre-mRNA. In some embodiments, other ASOs useful for these purposes are identified, for example, using the methods described herein. [Table 1-1] [Table 1-2] [Table 1-3]

[0073] In some embodiments, the targeted region of the CD274 AIC pre-mRNA is in exon 4. CD274 intron numbering as used herein corresponds to the mRNA sequence of NM_014143.3. In some embodiments, hybridization of an ASO to the targeted region of the AIC pre-mRNA results in inhibition of splicing at the alternative splice site (5' splice site or 3' splice site) in the alternative intron of exon 4, subsequently increasing PD-L1 protein production. It is understood that intron numbering may be altered with reference to different CD274 isoform sequences. One of skill in the art can determine the corresponding intron number in any CD274 isoform based on the intron sequences provided herein or using the intron numbering provided with reference to the mRNA sequence of NM_014143.3. One of skill in the art can also determine the sequence of adjacent exons in any CD274 isoform to target using the methods of the invention based on the intron sequences provided herein, or using the intron numbers provided with reference to the mRNA sequence of NM_014143.3. In some embodiments, the methods and compositions of the invention are used to increase expression of any known CD274 isoform.

[0074] In some embodiments, the methods of the present invention utilize the presence in cells of an alternative intron-containing pre-mRNA (AIC pre-mRNA) that is transcribed from the ARHGAP23 gene and encodes the Rho GTPase-activating protein 23 protein. Splicing of the identified ARHGAP23 AIC pre-mRNA species to produce mature, fully spliced ​​ARHGAP23 mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits splicing of the alternative intron. The mature ARHGAP23 mRNA is transported to the cytoplasm and translated, thereby increasing the amount of Rho GTPase activating protein 23 protein in the patient's cells and alleviating the symptoms of hormonal disorders or any disease or disorder associated with a deficiency in Rho GTPase activating protein 23 protein amount or activity or induced by a loss-of-function mutation in the ARHGAP23 gene.

[0075] In some embodiments, the methods of the present invention utilize the presence in a cell of an alternative intron-containing pre-mRNA (AIC pre-mRNA) that is transcribed from the BRD1 gene and encodes the mature, fully spliced ​​BRD1 protein. Splicing of the identified BRD1 AIC pre-mRNA species to produce mature BRD1 mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits alternative intron splicing. The resulting mature BRD1 mRNA is transported to the cytoplasm and translated, thereby increasing the amount of bromodomain-containing 1 protein in the patient's cells and alleviating symptoms of immune disorders, or any disease or disorder associated with a deficiency in the amount or activity of bromodomain-containing 1 protein or caused by a loss-of-function mutation in the BRD1 gene.

[0076] In some embodiments, the methods of the present invention utilize the presence in cells of an alternative intron-containing pre-mRNA (AIC pre-mRNA) that is transcribed from the DCHS1 gene and encodes the protocadherin-16 protein. Splicing of the identified DCHS1 AIC pre-mRNA species to produce mature, fully spliced ​​DCHS1 mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits splicing of the alternative intron. The resulting mature DCHS1 mRNA is transported to the cytoplasm and translated, thereby increasing the amount of protocadherin-16 protein in the patient's cells and alleviating symptoms of cardiac dysfunction or any disease or disorder associated with a deficiency in the amount or activity of protocadherin-16 protein or induced by a loss-of-function mutation in the DCHS1 gene.

[0077] In some embodiments, the methods of the present invention utilize the presence in cells of an alternative intron-containing pre-mRNA (AIC pre-mRNA) that is transcribed from the EPB41L2 gene and encodes band 4.1-like protein 2 protein. Splicing of the identified EPB41L2 AIC pre-mRNA species to produce mature, fully spliced ​​EPB41L2 mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits splicing of the alternative intron. The resulting mature EPB41L2 mRNA is transported to the cytoplasm and translated, thereby increasing the amount of band 4.1-like protein 2 protein in the patient's cells and alleviating symptoms of liver-related disorders, or any disease or disorder associated with a deficiency in the amount or activity of band 4.1-like protein 2 protein or induced by a loss-of-function mutation in the EPB41L2 gene.

[0078] In some embodiments, the methods of the present invention utilize the presence in cells of an alternative intron-containing pre-mRNA (AIC pre-mRNA) that is transcribed from the GPX8 gene and encodes glutathione peroxidase 8 protein. Splicing of the identified GPX8 AIC pre-mRNA species to produce mature, fully spliced ​​GPX8 mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits splicing of the alternative intron. The resulting mature GPX8 mRNA is transported to the cytoplasm and translated, thereby increasing the amount of glutathione peroxidase 8 protein in the patient's cells and alleviating symptoms of liver-related disorders, or any disease or disorder associated with a deficiency in the amount or activity of glutathione peroxidase 8 protein or induced by a loss-of-function mutation in the GPX8 gene.

[0079] In some embodiments, the methods of the present invention utilize the presence in cells of an alternative intron-containing pre-mRNA (AIC pre-mRNA) that is transcribed from the HIVEP3 gene and encodes the human immunodeficiency virus type I enhancer-binding protein 3 protein. Splicing of the identified HIVEP3 AIC pre-mRNA species to produce mature, fully spliced ​​HIVEP3 mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits splicing of the alternative intron. The resulting mature HIVEP3 mRNA is transported to the cytoplasm and translated, thereby increasing the amount of human immunodeficiency virus type I enhancer-binding protein 3 protein in the patient's cells and alleviating symptoms of immune disorders or any disease or disorder associated with a deficiency in the amount or activity of human immunodeficiency virus type I enhancer-binding protein 3 protein or induced by a loss-of-function mutation in the HIVEP3 gene.

[0080] In some embodiments, the methods of the invention utilize the presence in cells of an alternative intron-containing pre-mRNA (AIC pre-mRNA) that is transcribed from the INVS gene and encodes an inversin protein. Splicing of the identified INVS AIC pre-mRNA species to produce a mature, fully spliced ​​INVS mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits splicing of the alternative intron. The resulting mature INVS mRNA is transported to the cytoplasm and translated, thereby increasing the amount of inversin protein in the patient's cells and alleviating symptoms of an immune disorder or any disease or disorder associated with a deficiency in inversin protein amount or activity or induced by a loss-of-function mutation in the INVS gene.

[0081] In some embodiments, the methods of the present invention utilize the presence in cells of an alternative intron-containing pre-mRNA (AIC pre-mRNA) transcribed from the KIAA0319 gene and encoding the dyslexia-associated protein KIAA0319 protein. Splicing of the identified KIAA0319 AIC pre-mRNA species to produce a mature, fully spliced ​​KIAA0319 mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits the splicing of the alternative intron. The resulting mature KIAA0319 mRNA is transported to the cytoplasm and translated, thereby increasing the amount of dyslexia-associated protein KIAA0319 protein in the patient's cells and alleviating the symptoms of developmental disorders, or any disease or disorder associated with a deficiency in the amount or activity of the dyslexia-associated protein KIAA0319 protein or caused by a loss-of-function mutation in the KIAA0319 gene.

[0082] In some embodiments, the methods of the present invention utilize the presence in cells of an alternative intron-containing pre-mRNA (AIC pre-mRNA) that is transcribed from the NAIP gene and encodes an NLR family apoptosis inhibitor protein. Splicing of the identified NAIP AIC pre-mRNA species to produce mature, fully spliced ​​NAIP mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits the splicing of the alternative intron. The resulting mature NAIP mRNA is transported to the cytoplasm and translated, thereby increasing the amount of NLR family apoptosis inhibitor protein in the patient's cells and alleviating the symptoms of muscle-related disorders, or any disease or disorder associated with a deficiency in the amount or activity of an NLR family apoptosis inhibitor protein or induced by a loss-of-function mutation in the NAIP gene.

[0083] In some embodiments, the methods of the present invention involve selective intron expression in a cell that is transcribed from the PTCH2 gene and encodes the protein Patched homolog 2 protein. This method utilizes the presence of an alternative intron-containing pre-mRNA (AIC pre-mRNA). Splicing of the identified PTCH2 AIC pre-mRNA species to produce mature, fully spliced ​​PTCH2 mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits alternative intron splicing. The resulting mature PTCH2 mRNA is transported to the cytoplasm and translated, thereby increasing the amount of Patched homolog 2 protein in the patient's cells and alleviating symptoms of immune disorders, or any disease or disorder associated with a deficiency in the amount or activity of Patched homolog 2 protein or caused by a loss-of-function mutation in the PTCH2 gene.

[0084] In some embodiments, the methods of the present invention utilize the presence in cells of an alternative intron-containing pre-mRNA (AIC pre-mRNA) that is transcribed from the PTPRZ1 gene and encodes the protein tyrosine phosphatase receptor type Z1 protein. Splicing of the identified PTPRZ1 AIC pre-mRNA species to produce a mature, fully spliced ​​PTPRZ1 mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits the splicing of the alternative intron. The resulting mature PTPRZ1 mRNA is transported to the cytoplasm and translated, thereby increasing the amount of protein tyrosine phosphatase receptor type Z1 protein in the patient's cells and alleviating the symptoms of psychiatric disorders, or any disease or disorder associated with a deficiency in the amount or activity of protein tyrosine phosphatase receptor type Z1 or caused by a loss-of-function mutation in the PTPRZ1 gene.

[0085] In some embodiments, the methods of the present invention utilize the presence in cells of an alternative intron-containing pre-mRNA (AIC pre-mRNA) that is transcribed from the SON gene and encodes a SON protein. Splicing of the identified SON AIC pre-mRNA species to produce a mature, fully spliced ​​SON mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits splicing of the alternative intron. The resulting mature SON mRNA is transported to the cytoplasm and translated, thereby increasing the amount of SON protein in the patient's cells and alleviating symptoms of an immune disorder or any disease or disorder associated with a deficiency in SON protein amount or activity or caused by a loss-of-function mutation in the SON gene.

[0086] In some embodiments, the methods of the present invention utilize the presence in cells of an alternative intron-containing pre-mRNA (AIC pre-mRNA) that is transcribed from the ZCCHC2 gene and encodes zinc finger CCHC domain-containing protein 2 protein. Splicing of the identified ZCCHC2 AIC pre-mRNA species to produce mature, fully spliced ​​ZCCHC2 mRNA is induced using a therapeutic agent or antisense oligomer (ASO) that inhibits splicing of the alternative intron. The resulting mature ZCCHC2 mRNA is transported to the cytoplasm and translated, thereby increasing the amount of zinc finger CCHC domain-containing protein 2 protein in the patient's cells and alleviating symptoms of immune disorders or any disease or disorder associated with a deficiency in the amount or activity of zinc finger CCHC domain-containing protein 2 protein or induced by a loss-of-function mutation in the ZCCHC2 gene.

[0087] Table 3 provides a non-limiting list of ASO sequences useful for increasing production of proteins encoded by ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, PD-L1, PTCH2, PTPRZ1, SON, ZCCHC2 by targeting regions of the AIC pre-mRNA. In some embodiments, other ASOs useful for these purposes are identified, for example, using the methods described herein.

[0088] Protein expression In some embodiments, the methods described herein are used to increase protein production in a subject in need thereof. In some embodiments, the methods described herein are used to increase functional protein production in a subject in need thereof. As used herein, the term "functional" refers to the amount of protein activity or function required to eliminate any one or more symptoms of the condition being treated, e.g., a psychiatric disorder caused by a genetic defect in the BRD1 gene. In some embodiments, the methods are used to increase production of a fully functional protein or RNA. In some embodiments, the methods are used to increase production of a partially functional protein or RNA. As used herein, the term "partially functional" refers to any amount of protein activity or function that is less than the amount of activity or function required to eliminate or prevent any one or more symptoms of a disease or condition. In some embodiments, a partially functional protein or RNA has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 90%, or at least 95% less activity than a fully functional protein or RNA. In some embodiments, the target protein and the RIC pre-mRNA are encoded by a gene selected from ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, PD-L1, PTCH2, PTPRZ1, SON, and ZCCHC2.

[0089] In some embodiments, the methods described herein are used to increase PD-L1 protein production in a subject in need thereof. In some embodiments, the methods described herein are used to increase functional PD-L1 protein production in a subject in need thereof. As used herein, the term "functional" refers to the amount of PD-L1 protein activity or function required to eliminate any one or more symptoms of the condition being treated, e.g., an immune disorder caused by a genetic defect in PD-L1. In some embodiments, the method is used to increase the production of fully functional PD-L1 protein or RNA. In some embodiments, the method is used to increase the production of partially functional PD-L1 protein or RNA. In some embodiments, the method is a method of increasing target protein expression by cells of a subject having AIC pre-mRNA encoding the target protein, wherein the subject has an immune disease or disorder caused by a deficiency in the amount or activity of the target protein. In some embodiments, the deficiency in the amount of the target protein is caused by haploinsufficiency of the target protein. In some embodiments, the disease or disorder is associated with haploinsufficiency of the gene encoding the target protein. In such embodiments, the subject has a first allele that encodes a functional target protein and a second allele that results in no production of the target protein. In such embodiments, the subject has a first allele that encodes a functional target protein and a second allele that results in the target protein being produced at a reduced level. In another such embodiment, the subject has a first allele that encodes a functional target protein and a second allele that encodes a non-functional target protein. In another such embodiment, the subject has a first allele that encodes a functional target protein and a second allele that encodes a partially functional target protein.In any of these embodiments, the ASO is transcribed from both alleles, i.e., a first allele (encoding a functional target protein) and a second allele (in which the target protein is not produced, the target protein is produced at reduced levels, or the target protein is non-functional). The RIC pre-mRNA binds to a targeted region of the selected AIC pre-mRNA, thereby inhibiting splicing of the alternative intron from the AIC pre-mRNA, resulting in increased levels of mature mRNA from the first allele encoding a functional target protein and increased expression of the target protein in cells of the subject. In some embodiments, the target protein and the RIC pre-mRNA are encoded by a gene selected from ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, CD274, PTCH2, PTPRZ1, SON, and ZCCHC2.

[0090] In some embodiments, the method is a method of increasing expression of PD-L1 protein by cells of a subject having AIC pre-mRNA encoding PD-L1 protein, wherein the subject has an immune disease or disorder caused by a deficiency in the amount or activity of PD-L1 protein. In some embodiments, the deficiency in the amount of PD-L1 protein is caused by haploinsufficiency of PD-L1 protein. In some embodiments, the disease or disorder is associated with haploinsufficiency of the gene encoding PD-L1 protein. In such embodiments, the subject has a first allele that encodes a functional PD-L1 protein and a second allele that results in no PD-L1 protein being produced. In such embodiments, the subject has a first allele that encodes a functional PD-L1 protein and a second allele that results in a reduced level of PD-L1 protein. In another such embodiment, the subject has a first allele that encodes a functional PD-L1 protein and a second allele that encodes a non-functional PD-L1 protein. In another such embodiment, the subject has a first allele that encodes a functional PD-L1 protein and a second allele that encodes a partially functional PD-L1 protein. In any of these embodiments, the ASO binds to a targeted region of the AIC pre-mRNA transcribed from the first allele (which encodes the functional PD-L1 protein), thereby inhibiting splicing of an alternative intron from the AIC pre-mRNA, resulting in increased levels of mature mRNA encoding the functional PD-L1 protein and increased expression of the PD-L1 protein in the subject's cells.

[0091] In some embodiments, the subject has a first allele that encodes a functional target protein and a second allele that encodes a partially functional target protein. The subject has a second allele encoding the target protein, and the ASO binds to a targeted region of the AIC pre-mRNA transcribed from the first allele (encoding the functional target protein) or to a targeted region of the AIC pre-mRNA transcribed from the second allele (encoding the partially functional target protein), thereby inhibiting splicing of an alternative intron from the AIC pre-mRNA, resulting in increased levels of mature mRNA encoding the target protein and increased expression of the functional or partially functional target protein in the subject's cells. In some embodiments, the target protein and RIC pre-mRNA are encoded by a gene selected from ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, PD-L1, PTCH2, PTPRZ1, SON, and ZCCHC2.

[0092] In some embodiments, the subject has a first allele that encodes a functional PD-L1 protein and a second allele that encodes a partially functional PD-L1 protein, and the ASO binds to a targeted region of AIC pre-mRNA transcribed from the first allele (encoding the functional PD-L1 protein) or to a targeted region of AIC pre-mRNA transcribed from the second allele (encoding the partially functional PD-L1 protein), thereby inhibiting splicing of an alternative intron from the AIC pre-mRNA, resulting in an increase in the level of mature mRNA encoding PD-L1 protein and increased expression of functional or partially functional PD-L1 protein in the subject's cells. .

[0093] In a related embodiment, the method is a method of increasing expression of a protein or functional RNA using an ASO. In some embodiments, the target protein and RIC pre-mRNA are encoded by a gene selected from ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, CD274, PTCH2, PTPRZ1, SON, and ZCCHC2. In some embodiments, the ASO is used to increase expression of PD-L1 protein in cells of a subject having AIC pre-mRNA encoding the PD-L1 protein, and the subject has a deficiency in the amount or function of PD-L1 protein.

[0094] In some embodiments, AIC pre-mRNA transcripts encoding proteins that cause a disease or condition are targeted by the ASOs described herein. In some embodiments, AIC pre-mRNA transcripts encoding proteins that are not the cause of the disease are targeted by the ASOs. For example, a disease that is the result of a mutation or deficiency in a first protein in a particular pathway can be ameliorated by targeting an AIC pre-mRNA encoding a second protein, thereby increasing production of the second protein. In some embodiments, the function of the second protein can compensate for the mutation or deficiency in the first protein (which is the cause of the disease or condition).

[0095] In some embodiments, the subject: a. a first mutant allele, i) the protein is produced at a reduced level compared to production from the wild-type allele; ii) the protein is produced in a form that is reduced in function compared to the equivalent wild-type protein; or iii) no protein or functional RNA is produced; and b. A second mutant allele, i) the protein is produced at a reduced level compared to production from a wild-type allele; ii) the protein is produced in a form that is reduced in function compared to the equivalent wild-type protein; The AIC pre-mRNA is transcribed from a first allele and a second allele, and the target protein or functional RNA is encoded by a gene selected from ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, CD274, PTCH2, PTPRZ1, SON, and ZCCHC2. In these embodiments, the ASO binds to the targeted region of the AIC pre-mRNA transcribed from the first allele and the second allele, thereby inhibiting splicing of the alternative intron from the AIC pre-mRNA, resulting in increased levels of the protein-encoding mRNA and increased expression of the target protein or target functional RNA in cells of the subject. In these embodiments, the protein or functional RNA having increased expression levels resulting from inhibiting splicing of the alternative intron from the AIC pre-mRNA is either a form that is reduced in function (partially functional) compared to the equivalent wild-type protein, or a form that is functionally intact (fully functional) compared to the equivalent wild-type PD-L1 protein.

[0096] In some embodiments, the level of processed mRNA encoding the target protein is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 10-fold, or about 1.1 to about 2-fold increased compared to the level of processed mRNA encoding the target protein in control cells, e.g., cells not treated with the ASO, or cells treated with an ASO that does not bind to the targeted region of the target AIC pre-mRNA. 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold increase; wherein the target protein is encoded by a gene selected from ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, CD274, PTCH2, PTPRZ1, SON, and ZCCHC2.

[0097] In some embodiments, subjects treated using the methods of the present invention express a partially functional target protein from one allele, where the partially functional target protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, a splicing mutation, or a partial gene deletion. In some embodiments, subjects treated using the methods of the present invention express a non-functional target protein from one allele, where the non-functional target protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, a splicing mutation, or a partial gene deletion in one allele. In some embodiments, subjects treated using the methods of the present invention have a targeted full-gene deletion in one allele. In some embodiments, the target protein is encoded by a gene selected from ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, CD274, PTCH2, PTPRZ1, SON, and ZCCHC2.

[0098] Increased protein expression As described above, in some embodiments, the methods of the present invention are used to increase expression of a target protein. The target protein may be encoded by a gene selected from ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, CD274, PTCH2, PTPRZ1, SON, and ZCCHC2. In these embodiments, an alternative intron pre-mRNA (AIC pre-mRNA) encoding the target protein is present in the nucleus of a cell. A cell having a target AIC pre-mRNA encoding the target protein, the AIC pre-mRNA including an alternative intron, a first portion of an exon adjacent to the 5' splice site of the alternative intron, and a second portion of an exon adjacent to the 3' splice site of the alternative intron, is contacted with an antisense oligomer (ASO) complementary to the target region of the AIC pre-mRNA. Hybridization of the ASO to the target region of the AIC pre-mRNA results in inhibition of splicing of the alternative intron, and subsequently increases production of the target protein.

[0099] The terms "pre-mRNA" and "pre-mRNA transcript" may be used interchangeably and may refer to any pre-mRNA species having at least one intron. In some embodiments, a pre-mRNA or pre-mRNA transcript comprises a 5'-7-methylguanosine cap and / or a poly-A tail. In some embodiments, a pre-mRNA or pre-mRNA transcript comprises both a 5'-7-methylguanosine cap and a poly-A tail. In some embodiments, a pre-mRNA transcript does not comprise a 5'-7-methylguanosine cap and / or a poly-A tail. A pre-mRNA transcript is a non-productive messenger RNA (mRNA) molecule when it is not translated into protein (or transported from the nucleus to the cytoplasm).

[0100] As used herein, an "alternative intron-containing pre-mRNA" ("AIC pre-mRNA") is a pre-mRNA transcript that contains at least one alternative intron. An AIC pre-mRNA contains an alternative intron, a first portion of an exon adjacent to the 5' splice site of the alternative intron, and a second portion of an exon adjacent to the 3' splice site of the alternative intron. The AIC pre-mRNA contains a second portion of the exon that is spliced ​​out, encoding the target protein. An "AIC pre-mRNA encoding a target protein" is understood to encode the target protein when fully spliced. An "alternative intron" is a region or sequence between the 5' and 3' alternative splice sites within an exon of a pre-mRNA transcript that can be spliced ​​out when the pre-mRNA is processed to produce mRNA (the processed mRNA lacks part of the exon).

[0101] In some embodiments, the therapeutic agent or ASO is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the targeted region of the AIC pre-mRNA encoding the target protein. In some embodiments, at least a portion of the targeted region of the target AIC pre-mRNA is within an intron upstream of the first portion of the exon. In some embodiments, at least a portion of the targeted region of the target AIC pre-mRNA is within an intron downstream of the second portion of the exon. In some embodiments, at least a portion of the targeted region of the target AIC pre-mRNA is within an alternative intron. In some embodiments, at least a portion of the targeted region of the target AIC pre-mRNA is within the first portion of the exon. In some embodiments, at least a portion of the targeted region of the target AIC pre-mRNA is within the second portion of the exon. In some embodiments, at least a portion of the targeted region of the target AIC pre-mRNA overlaps the junction of the first portion of the exon and the alternative intron. In some embodiments, at least a portion of the targeted region of the target AIC pre-mRNA overlaps with the junction of the alternative intron and the second portion of the exon. When used to specify the location of a region or sequence, "within" is understood to include the residues at the recited positions. For example, the region from +6 to +100 includes residues at positions +6 and +100. In some embodiments, the target AIC pre-mRNA is encoded by a gene selected from ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, CD274, PTCH2, PTPRZ1, SON, and ZCCHC2.

[0102] In some embodiments, a mature mRNA encoding the target protein is thereby produced. In some embodiments, the target protein produced is a fully functional target protein. In some embodiments, the target RNA produced is a functional target RNA. In some embodiments, the target RNA produced is a fully functional target RNA. The terms "mature mRNA," "fully spliced ​​mRNA," and "functional RNA" are used interchangeably herein to describe a fully processed mRNA encoding a target protein (e.g., an mRNA that has been transported from the nucleus to the cytoplasm and translated into a target protein) or a fully processed functional RNA. The term "productive mRNA" can also be used to describe a fully processed mRNA encoding a target protein.

[0103] As used herein, the term "comprise," or variations thereof, such as "comprises" or "comprising," should be interpreted to indicate the inclusion of recited features (e.g., in the case of ASOs, a defined nucleobase sequence), but not the exclusion of other features. Thus, as used herein, the term "comprising" is inclusive and does not exclude additional, unrecited features (e.g., in the case of ASOs, the presence of additional, unrecited nucleobases).

[0104] In some embodiments of any of the methods and compositions provided herein, "comprising" can be substituted with "consisting essentially of" or "consisting of." The phrase "consisting essentially of" means that the specified As used herein, the term "consisting" is used to refer to the presence of the recited feature (e.g., nucleobase sequence) alone (such that, in the case of an antisense oligomer consisting of the specified nucleobase sequence, the presence of additional, unrecited nucleobases is excluded).

[0105] As used herein, "wild-type sequence" refers to the nucleotide sequence for a target gene in the published reference genome deposited at the NCBI repository of biological and scientific information (operated by the National Center for Biotechnology Information, National Library of Medicine, 8600 Rockville Pike, Bethesda, MD USA 20894). As used herein, "wild-type sequence" refers to the canonical sequence available at NCBI gene ID 29126. Also as used herein, a nucleotide position designated with an "e" indicates that the nucleotide is present in the sequence of an exon (e.g., an exon adjacent to the 5' splice site or an exon adjacent to the 3' splice site).

[0106] The method includes contacting a cell with a therapeutic agent or ASO that is complementary to a region of the pre-mRNA that encodes the target protein, thereby increasing expression of the target. As used herein, "contacting" or administering to a cell refers to any method that brings an ASO into close proximity with a cell so that the ASO and the cell can interact. The cell that is in contact with the ASO takes up or transports the ASO into the cell. The method includes contacting a cell associated with a condition or disease or a cell involved in a condition or disease with any of the ASOs described herein. In some embodiments, the ASO can be further modified or attached (e.g., covalently linked) to another molecule to target the ASO to a cell type, enhance contact between the ASO and a cell associated with a condition or disease or a cell involved in a condition or disease, or enhance uptake of the ASO.

[0107] As used herein, the terms "increasing protein production" or "increasing expression of a target protein" refer to enhancing the amount of protein translated from mRNA in a cell. A "target protein" can be any protein whose expression / production is desired to be increased.

[0108] In some embodiments, contacting a cell expressing a target AIC pre-mRNA with an ASO that is complementary to the targeted region of the target AIC pre-mRNA transcript results in a measurable increase in the amount of the target protein (e.g., target protein) encoded by the pre-mRNA. Methods for measuring or detecting protein production will be apparent to those skilled in the art and include any known method, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0109] In some embodiments, a therapeutic agent, i.e., an ASO, increases expression of a target protein in a cell. In some embodiments, contacting a cell with an ASO that is complementary to a targeted region of a target AIC pre-mRNA transcript increases the amount of target protein produced by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the amount of protein produced by the cell in the absence of the ASO / treatment. In some embodiments, the expression level of the target protein produced by the ASO-contacted cell is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, or about 5 to about 10-fold increased compared to the expression level of the target protein produced by a control compound. fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold. The control compound can be, for example, an oligonucleotide that is not complementary to the targeted region of the AIC pre-mRNA.

[0110] In some embodiments, the therapeutic agent, i.e., ASO, increases the level of processed mRNA encoding a target protein or mature mRNA encoding a target protein in a cell. In some embodiments, contacting a cell with an ASO that is complementary to the target region of the target AIC pre-mRNA transcript increases the level of processed mRNA encoding the target, including mature mRNA encoding the target protein. In some embodiments, the level of processed mRNA encoding the target protein or mature mRNA encoding the target protein increases by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the level of processed mRNA encoding the target protein in a cell in the absence / absence of ASO treatment. In some embodiments, the level of processed mRNA encoding the target protein, or mature mRNA encoding the target protein produced in cells contacted with an ASO, is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 5-fold, or about 1.1 to about 5-fold, compared to the level of processed mRNA or mature RNA in untreated cells, e.g., untreated cells or cells treated with a control compound. The control compound can be, for example, an oligonucleotide that is not complementary to the targeted region of the target AIC pre-mRNA.

[0111] Antisense oligomers (ASOs) One aspect of the present disclosure is methods and compositions comprising therapeutic agents that modulate (e.g., inhibit or enhance) splicing by binding to a targeted region of the AIC pre-mRNA. In some embodiments, the present disclosure includes methods and compositions comprising antisense oligomers (ASOs) that modulate (e.g., inhibit or enhance) splicing by binding to a targeted region of the AIC pre-mRNA. As used herein, the terms "ASO," "antisense oligomer," and "antisense oligonucleotide" are used interchangeably and refer to an oligomer, such as a polynucleotide, that contains nucleobases that hybridize to a target nucleic acid (e.g., CD274 AIC pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (GU). ASOs can have exact sequence complementarity to the target sequence or near complementarity (e.g., sufficient complementarity to bind to the target sequence and enhance splicing at the splice site). ASOs are designed to bind (hybridize) to a target nucleic acid (e.g., a targeted region of a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, when an ASO hybridizes to a site other than the intended (targeted) nucleic acid sequence, it hybridizes to a limited number of sequences that are not the target nucleic acid (to a small number of sites other than the target nucleic acid). The design of the ASO can take into account the nucleic acid sequence of the targeted region of the pre-mRNA transcript, or the occurrence of sufficiently similar nucleic acid sequences elsewhere in the genome or cellular pre-mRNA or transcriptome, thus preventing the ASO from binding to other sites and causing "off-target" effects. The possibilities are limited. Any ASO known in the art can be used to practice the methods described herein, for example, the ASO in PCT Application No. PCT / US2014 / 054151, entitled "Reducing Nonsense-Mediated mRNA Decay," published as WO2015 / 035091.

[0112] In some embodiments, an ASO "specifically hybridizes" or is "specific" for a targeted region of a target nucleic acid or AIC pre-mRNA. Typically, such hybridization occurs at a Tm substantially greater than 37°C, preferably at least 50°C, and typically between 60°C and approximately 90°C. Such hybridization preferably corresponds to stringent hybridization conditions. At a given ionic strength and pH, the Tm is the temperature at which 50% of the target sequence hybridizes to a complementary oligonucleotide.

[0113] Oligomers, such as oligonucleotides, are "complementary" to each other when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides. A double-stranded polynucleotide can be "complementary" to another polynucleotide when hybridization occurs between one strand of a first polynucleotide and one strand of a second polynucleotide. Complementarity (the degree to which a polynucleotide is complementary to another polynucleotide) can be quantified in terms of the proportion (e.g., percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other according to generally accepted base-pairing rules. The sequence of an ASO can be 100% complementary to the sequence of its target nucleic acid with which it hybridizes; however, the sequence of an ASO does not need to be 100% complementary to the sequence of its target nucleic acid with which it hybridizes. In certain embodiments, an ASO can comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity to a target region within the targeted nucleic acid sequence. For example, an ASO in which 18 of 20 nucleobases of an oligomeric compound are complementary to, and therefore specifically hybridize to, a target region exhibits 90 percent complementarity. In this example, the remaining non-complementary nucleobases may be clustered together or may be interspersed with complementary nucleobases and need not be contiguous with each other or with complementary nucleobases. The percent complementarity of an ASO to a region of a target nucleic acid can be determined using the BLAST program (Basic Local Alignment Search Tool). search tools) and the PowerBLAST program known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0114] An ASO does not need to hybridize to all nucleobases in a target sequence, and the nucleobases to which the ASO hybridizes may be contiguous or non-contiguous. The ASO may hybridize across one or more segments of a pre-mRNA transcript such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop or hairpin structure may be formed). In certain embodiments, the ASO hybridizes to non-contiguous nucleobases in the target pre-mRNA transcript. For example, the ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobases to which the ASO does not hybridize.

[0115] The ASOs described herein comprise nucleobases that are complementary to nucleobases present in the target region of the AIC pre-mRNA. The term ASO refers to an oligonucleotide and a target mRNA. Other oligomeric molecules include those that contain nucleobases capable of hybridizing to the complementary nucleobases above but do not contain sugar moieties, such as peptide nucleic acids (PNAs). ASOs can contain naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the foregoing. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and / or modified backbones. In some embodiments, all of the nucleotides in an ASO are modified nucleotides. Chemical modifications of ASOs or components of ASOs compatible with the methods and compositions described herein will be apparent to those of skill in the art and can be found, for example, in U.S. Pat. No. 8,258,109 B2, U.S. Pat. No. 5,656,612, U.S. Patent Publication No. 2012 / 0190728, and Dias and Stein, Mol. Cancer Ther. 2002, 1, 347-355, which are incorporated herein by reference in their entireties.

[0116] The nucleobases of the ASO may be any naturally occurring unmodified nucleobase, such as adenine, guanine, cytosine, thymine, and uracil, or any synthetic or modified nucleobase that resembles an unmodified nucleobase sufficiently to be capable of hydrogen bonding with a nucleobase present on the target pre-mRNA. Examples of modified nucleobases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

[0117] The ASOs described herein also include a backbone structure connecting the components of the oligomer. The terms "backbone structure" and "oligomeric linkage" may be used interchangeably and refer to the connections between the monomers of the ASO. In naturally occurring oligonucleotides, the backbone includes 3'-5' phosphodiester bonds connecting the sugar moieties of the oligomer. The backbone structure or oligomeric linkage of the ASOs described herein may include, but is not limited to, phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. See, e.g., LaPlanche et al., Nucleic Acids Res. 14:9081(1986);Stec et al., J. Am. Chem. See Soc. 106:6077 (1984), Stein et al., Nucleic Acids Res. 16:3209 (1988), Zon et al., Anti Cancer Drug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford, England (1991)); Stec et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990). In some embodiments, the backbone structure of the ASO does not contain phosphorus and contains peptide bonds, such as peptide nucleic acids (PNAs), or linking groups including carbamates, amides, and linear and cyclic hydrocarbon groups. In some embodiments, the backbone modification is a phosphothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.

[0118] In some embodiments, the stereochemistry at each phosphorus internucleotide linkage of the ASO backbone is random. In some embodiments, the stereochemistry at each phosphorus internucleotide linkage of the ASO backbone is controlled and not random. For example, U.S. Patent Application Publication No. 2014 / 0194610, "Methods for the Synthesis of Functionalized Nucleic Acids," incorporated herein by reference, describes controlling the chirality at each phosphorus atom in a nucleic acid oligomer. This document describes methods for independently selecting the handedness of ASOs. In some embodiments, the ASOs used in the methods of the invention, including but not limited to any of the ASOs described herein in Tables 1 or 3, comprise ASOs with non-random phosphorus internucleotide linkages. In some embodiments, the compositions used in the methods of the invention comprise pure diastereomeric ASOs. In some embodiments, the compositions used in the methods of the invention comprise an ASO having a diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

[0119] In some embodiments, the ASO has a non-random mixture of Rp and Sp configurations in its phosphorus internucleotide linkages. For example, it has been suggested that a mixture of Rp and Sp is necessary for antisense oligonucleotides to balance good activity and nuclease stability (Wan, et al., 2014, "Synthesis, biophysical properties and biological activity of second-generation antisense oligonucleotides containing chiral phosphorothioate linkages," Nucleic Acids Research 42(22): 13456-13468, incorporated herein by reference). In some embodiments, the ASOs used in the methods of the invention, including but not limited to any of the ASOs described herein in Tables 1 or 3, comprise about 5-100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, with the remainder Sp, or comprise about 100% Rp.In some embodiments, the ASOs used in the methods of the present invention, including but not limited to any of the ASOs described herein in Tables 1 or 3, are from about 10% to about 100% Rp, about 15% to about 100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp, about 40% to about 100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about 60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, about 95% to about 100% Rp, about 100% to about 100% Rp, about 110% to about 100% Rp, about 120% to about 100% Rp, about 130% to about 100% Rp, about 140% to about 100% Rp, about 150% to about 100% Rp, about 160% to about 100% Rp, about 170% to about 100% Rp, about 180% to about 100% Rp, about 190% to about 100% Rp, about 210% to about 100% Rp, about 220% to about 100% Rp, about 230% to about 5% to about 100% Rp, about 60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, or about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp, with the remainder being Sp.

[0120] In some embodiments, the ASOs used in the methods of the invention, including but not limited to any of the ASOs described herein in Tables 1 or 3, comprise about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, with the remainder being R In some embodiments, the ASOs used in the methods of the present invention, including but not limited to any of the ASOs described herein in Tables 1 or 3, include about 10% to about 100% Sp, about 15% to about 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, about 40% to about 100% Sp, about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about 90% to about 100% Sp, about 95% to about 100% Sp, about 100% to about 100% Sp, about 110% to about 100% Sp, about 120% to about 100% Sp, about 130% to about 100% Sp, about 140% to about 100% Sp, about 150% to about 100% Sp, about 160% to about 100% Sp, about 170% to about 100% Sp, about 180% to about 100% Sp, about 190% to about 100% Sp, about 21% to about 100% Sp, about 22% to about 100% Sp, about 23% to about 100% Sp, about 24% to about 100% Sp, about 25% to about 100% Sp It contains 5% to about 100% Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55% Sp, with the remainder being Rp.

[0121] Any of the ASOs described herein can contain sugar moieties containing ribose or deoxyribose found in naturally occurring nucleotides, or modified sugar moieties or sugar analogs containing a morpholine ring. Non-limiting examples of modified sugar moieties include 2'-substituents, such as 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'-fluoro (2'F); N3'->P5' phosphoramidate, 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and bicyclic-modified sugars. In some embodiments, the sugar moiety modification is selected from 2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety modification is an additional crosslinking bond, such as a locked nucleic acid (LNA). In some embodiments, the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification. In some embodiments, the sugar moiety comprises a 2',4'-constrained 2'O-methyloxyethyl (cMOE) modification. In some embodiments, the sugar moiety comprises a cEt 2',4'-constrained 2'-O-ethyl BNA modification. In some embodiments, the sugar moiety comprises a tricycloDNA (tcDNA) modification. In some embodiments, the sugar moiety comprises an ethylene nucleic acid (ENA) modification. In some embodiments, the sugar moiety comprises an MCE modification. Modifications are known in the art and described in the literature, e.g., Jarver, et al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications," Nucleic Acid Therapeutics 24(1): 37-47, which is incorporated herein by reference for this purpose.

[0122] In some instances, each monomer of an ASO is modified in the same way; for example, each linkage in the backbone of the ASO contains a phosphorothioate linkage, or each ribose sugar moiety contains a 2'O-methyl modification. Such modifications present in each of the monomer components of an ASO are referred to as "uniform modifications." In some instances, a combination of different modifications may be desired; for example, an ASO may contain a combination of phosphorodiamidate linkages and sugar moieties containing morpholine rings (morpholinos). A combination of different modifications to an ASO is referred to as a "mixed modification" or "mixed chemistry."

[0123] In some embodiments, the ASO comprises one or more backbone modifications. In some embodiments, the ASO comprises one or more sugar moiety modifications. In some embodiments, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs described herein, or any component of the ASO (e.g., the nucleobase, sugar moiety, backbone), can be modified to achieve a desired property or activity of the ASO or to reduce an undesirable property or activity of the ASO. For example, one or more components of the ASO or any ASO can be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript, to reduce binding to any non-target sequence, to reduce degradation by cellular nucleases (i.e., RNase H), to improve uptake of the ASO into a cell and / or a cell's nucleus, to alter the pharmacokinetics or pharmacodynamics of the ASO, or to adjust the half-life of the ASO.

[0124] In some embodiments, ASOs are composed of 2'-O-(2-methoxyethyl) (MOE) phosphorothioate modified nucleotides. ASOs composed of such nucleotides are particularly well suited for the methods disclosed herein; oligomers with such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable for oral delivery in some embodiments described herein. See, e.g., Geary et al., J Pharmacol Exp Ther. 2001;296(3):890-7; Geary et al., J Pharmacol Exp Ther. 2001;296(3):890-7; et al., J Pharmacol Exp Ther. 2001;296(3):898-904.

[0125] Methods for synthesizing ASOs will be known to those of skill in the art. Alternatively, or in addition, ASOs can be obtained from commercial sources.

[0126] Unless otherwise specified, the left-hand end of a single-stranded nucleic acid (e.g., pre-mRNA transcript, oligonucleotide, ASO, etc.) sequence is the 5'-end, and the left-hand direction of a single-stranded or double-stranded nucleic acid sequence is referred to as the 5'-direction. Similarly, the right-hand end or direction of a nucleic acid sequence (single-stranded or double-stranded) is the 3'-end or 3'-direction. Generally, a region or sequence 5' to a reference point in a nucleic acid is referred to as "upstream," and a region or sequence 3' to a reference point in a nucleic acid is referred to as "downstream." Generally, the 5'-direction or 5'-end of an mRNA is where the initiation or start codon is located, and the 3'-end or 3'-direction is where the stop codon is located. In some embodiments, nucleotides upstream of a reference point in a nucleic acid can be designated by negative numbers, while nucleotides downstream of the reference point can be designated by positive numbers. For example, a reference point (e.g., an exon-exon junction in an mRNA) can be designated as the "zero" site, and the nucleotide immediately adjacent to and upstream of the reference point is designated as "minus one," e.g., "-1," while the nucleotide immediately adjacent to and downstream of the reference point is designated as "plus one," e.g., "+1."

[0127] In other embodiments, the ASO is complementary to (and binds to) a targeted region of the target AIC pre-mRNA that is downstream (3' direction) of the 5' splice site of the alternative intron in the target AIC pre-mRNA (e.g., in the direction designated by positive numbers relative to the 5' alternative splice site of the alternative intron). In some embodiments, the ASO is complementary to a targeted region of the target AIC pre-mRNA that is within the region of +1 to +100 relative to the 5' splice site of the alternative intron. In some embodiments, the ASO can be complementary to a targeted region of the target AIC pre-mRNA that is within the region between nucleotide +1 to nucleotide +50 relative to the 5' splice site of the alternative intron. In some embodiments, the ASO is complementary to a targeted region within the region +1 to +90, +1 to +80, +1 to +70, +1 to +60, +1 to +50, +1 to +40, +1 to +30, or +1 to +20 relative to the 5' splice site of the alternative intron. In some embodiments, the target AIC pre-mRNA is selected from the group consisting of ARHGAP23, BRD1, DCHS1, EPB41L2, It is encoded by a gene selected from GPX8, HIVEP3, INVS, KIAA0319, NAIP, CD274, PTCH2, PTPRZ1, SON, and ZCCHC2.

[0128] In other embodiments, the ASO is complementary to (and binds to) a targeted region of CD274 AIC pre-mRNA that is downstream (3' direction) of the 5' splice site of an alternative intron in the CD274 AIC pre-mRNA (e.g., in the direction designated by positive numbers relative to the 5' alternative splice site of the alternative intron). In some embodiments, the ASO is complementary to a targeted region of CD274 AIC pre-mRNA that is within the region +1 to +100 relative to the 5' splice site of the alternative intron. In some embodiments, the ASO can be complementary to a targeted region of CD274 AIC pre-mRNA that is within the region between nucleotide +1 to nucleotide +50 relative to the 5' splice site of the alternative intron. In some embodiments, the ASO is complementary to a targeted region within the region +1 to +90, +1 to +80, 16 to +70, +1 to +60, +1 to +50, +1 to +40, +1 to +30, or +1 to +20 relative to the 5' splice site of the alternative intron.

[0129] In some embodiments, the ASO is complementary to a targeted region of the target AIC pre-mRNA that is upstream (5' direction) of the 3' splice site of an alternative intron in the target AIC pre-mRNA (e.g., in a direction designated by a negative number). In some embodiments, the ASO is complementary to a targeted region of the target AIC pre-mRNA that is within a region of -1 to -100 relative to the 3' splice site of the alternative intron. In some embodiments, the ASO is complementary to a targeted region of the target AIC pre-mRNA that is within a region of -1 to -50 relative to the 3' splice site of the alternative intron. In some embodiments, the ASO is complementary to a targeted region that is within a region of -1 to -90, -1 to -80, -1 to -70, -1 to -60, -1 to -50, -1 to -40, or -1 to -30 relative to the 3' splice site of the alternative intron. In some embodiments, the target AIC pre-mRNA is encoded by a gene selected from ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, CD274, PTCH2, PTPRZ1, SON, ZCCHC2.

[0130] In some embodiments, the ASO is complementary to a targeting region of CD274 AIC pre-mRNA that is upstream (5' direction) of the 3' splice site of an alternative intron in the CD274 AIC pre-mRNA (e.g., in a direction designated by a negative number). In some embodiments, the ASO is complementary to a targeting region of CD274 AIC pre-mRNA within a region of -1 to -100 relative to the 3' splice site of the alternative intron. In some embodiments, the ASO is complementary to a targeting region of CD274 AIC pre-mRNA within a region of -1 to -50 relative to the 3' splice site of the alternative intron. In some embodiments, the ASO is complementary to a targeting region within a region of -1 to -90, -1 to -80, -1 to -70, -1 to -60, -1 to -50, -1 to -40, or -1 to -30 relative to the 3' splice site of the alternative intron.

[0131] In some embodiments, the targeted region of the CD274 AIC pre-mRNA is within the region +100 relative to the 5' splice site of the alternative intron to the region -100 relative to the 3' splice site of the alternative intron.

[0132] In some embodiments, the ASO is complementary to a targeted region of the target AIC pre-mRNA that is within (upstream of) the exon adjacent to the 5' splice site of the alternative intron (e.g., the first part of the exon in which the alternative intron is located). In some embodiments, the ASO is complementary to the first part of the exon adjacent to the 5' splice site of the alternative intron. In some embodiments, the ASO is complementary to a targeted region of the target AIC pre-mRNA within a region of -1e to -100e, -1e to -90e, -1e to -80e, -1e to -70e, -1e to -60e, -1e to -50e, -1e to -40e, -1e to -30e, or -1e to -20e relative to the 5' splice site of the alternative intron. In some embodiments, the target AIC pre-mRNA is encoded by a gene selected from ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, CD274, PTCH2, PTPRZ1, SON, or ZCCHC2.

[0133] In some embodiments, the ASO is complementary to a targeted region of the CD274 AIC pre-mRNA that is within (upstream of) the exon adjacent to the 5' splice site of the alternative intron (e.g., the first portion of the exon in which the alternative intron is located). In some embodiments, the ASO is complementary to a targeted region of the CD274 AIC pre-mRNA that is within a region of +2e to -1e in the first portion of the exon adjacent to the 5' splice site of the alternative intron. In some embodiments, the ASO is complementary to a targeted region of the CD274 AIC pre-mRNA that is within a region of -1e to -100e, -1e to -90e, -1e to -80e, -1e to -70e, -1e to -60e, -1e to -50e, -1e to -40e, -1e to -30e, or -1e to -20e relative to the 5' splice site of the alternative intron.

[0134] In some embodiments, the ASO is complementary to a targeted region of the target AIC pre-mRNA that is within (downstream from) the exon adjacent to the 3' splice site of the alternative intron (e.g., the second portion of the exon in which the alternative intron is located). In some embodiments, the ASO is complementary to a targeted region of the target AIC pre-mRNA that is within a region of +1e to -4e in the exon adjacent to the 3' splice site of the alternative intron. In some embodiments, the ASO is complementary to a targeted region of the target AIC pre-mRNA that is within a region of +1e to +100e, +1e to +90e, +1e to +80e, +1e to +70e, +1e to +60e, +1e to +50e, +1e to +40e, +1e to +30e, or +1e to +20e relative to the 3' splice site of the alternative intron. In some embodiments, the target AIC pre-mRNA is encoded by a gene selected from ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, CD274, PTCH2, PTPRZ1, SON, ZCCHC2.

[0135] In some embodiments, the ASO is complementary to a targeted region of the CD274 AIC pre-mRNA that is within (downstream from) the exon adjacent to the 3' splice site of the alternative intron (e.g., the second portion of the exon in which the alternative intron is located). In some embodiments, the ASO is complementary to a targeted region of the CD274 AIC pre-mRNA that is within a region of +1e to -4e in the exon adjacent to the 3' splice site of the alternative intron. In some embodiments, the ASO is complementary to a targeted region of the CD274 AIC pre-mRNA that is within a region of +1e to +100e, +1e to +90e, +1e to +80e, +1e to +70e, +1e to +60e, +1e to +50e, +1e to +40e, +1e to +30e, or +1e to +20e relative to the 3' splice site of the alternative intron.

[0136] In some embodiments, a therapeutic agent or ASO binds to a targeted region of the CD274 AIC pre-mRNA. In some embodiments, the targeted region is within a sequence selected from SEQ ID NOs: 68, 69, and 71-76. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within exon 4 of CD274. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 1-67.

[0137] In some embodiments, the therapeutic agent or ASO binds to a targeted region of the ARHGAP23 AIC pre-mRNA. In some embodiments, the targeted region is within a sequence selected from SEQ ID NOs: 77 and 91. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within an exon of ARHGAP23. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 105-153.

[0138] In some embodiments, the therapeutic agent or ASO binds to a targeted region of the BRD1 AIC pre-mRNA. In some embodiments, the targeted region is within a sequence selected from SEQ ID NOs: 78 and 92. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within an exon of BRD1. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 154-217.

[0139] In some embodiments, the therapeutic agent or ASO binds to a targeted region of the DCHS1 AIC pre-mRNA. In some embodiments, the targeted region is within a sequence selected from SEQ ID NOs: 79 and 93. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within an exon of DCHS1. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 218-334.

[0140] In some embodiments, the therapeutic agent or ASO binds to a targeted region of the EPB41L2 AIC pre-mRNA. In some embodiments, the targeted region is within a sequence selected from SEQ ID NOs: 80 and 94. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within an exon of EPB41L2. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 335-406.

[0141] In some embodiments, the therapeutic agent or ASO binds to a targeted region of the GPX8 AIC pre-mRNA. In some embodiments, the targeted region is within a sequence selected from SEQ ID NOs: 81 and 95. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within an exon of GPX8. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 407-443.

[0142] In some embodiments, the therapeutic agent or ASO binds to a targeted region of the HIVEP3 AIC pre-mRNA. In some embodiments, the targeted region is within a sequence selected from SEQ ID NOs: 82 and 96. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within an exon of HIVEP3. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 444-1027.

[0143] In some embodiments, a therapeutic agent or ASO binds to a targeted region of the INVS AIC pre-mRNA. In some embodiments, the targeted region is within a sequence selected from SEQ ID NOs: 83-97. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within an exon of INVS. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 1028-1112.

[0144] In some embodiments, the therapeutic agent or ASO binds to a targeted region of the KAII0319 AIC pre-mRNA. In some embodiments, the targeted region is SEQ ID NO: 84- 98. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within an exon of KAII0319. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 1013-1212.

[0145] In some embodiments, the therapeutic agent or ASO binds to a targeted region of the NAIP AIC pre-mRNA. In some embodiments, the targeted region is within a sequence selected from SEQ ID NOs: 85-99. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within an exon of NAIP. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 1213-1501.

[0146] In some embodiments, the therapeutic agent or ASO binds to a targeted region of the PTCH2 AIC pre-mRNA. In some embodiments, the targeted region is within a sequence selected from SEQ ID NOs: 86-100. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within an exon of PTCH2. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 1502-1547.

[0147] In some embodiments, the therapeutic agent or ASO binds to a targeted region of the PTPRZ1 AIC pre-mRNA. In some embodiments, the targeted region is within a sequence selected from SEQ ID NOs: 87-101. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within an exon of PTPRZ1. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 1548-2039.

[0148] In some embodiments, the therapeutic agent or ASO binds to a targeted region of the SON AIC pre-mRNA. In some embodiments, the targeted region is within a sequence selected from SEQ ID NOs: 88, 89, 102, and 103. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within an exon of the SON. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 2040-3458.

[0149] In some embodiments, a therapeutic agent or ASO binds to a targeted region of the ZCCHC2 AIC pre-mRNA. In some embodiments, the targeted region is within a sequence selected from SEQ ID NOs: 90 and 104. In some embodiments, the targeted region of the AIC pre-mRNA to which the therapeutic agent or ASO binds is located within an exon of ZCCHC2. In some embodiments, the ASO has a sequence selected from SEQ ID NOs: 3459-3651.

[0150] ASOs can be of any length suitable for specific binding and effective modulation (e.g., inhibition or enhancement) of splicing. In some embodiments, the ASO consists of 8 to 50 nucleobases. For example, the ASO can be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases in length. In some embodiments, the ASO consists of more than 50 nucleobases. In some embodiments, the ASO comprises 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases Base, 11-25 Nucleic Acid Bases, 11-20 Nucleic Acid Bases, 11-15 Nucleic Acid Bases, 12-50 Nucleic Acid Bases, 12-40 Nucleic Acid Bases, 12-35 Nucleic Acid Bases, 12-30 Nucleic Acid Bases, 12-25 Nucleic Acid Bases, 12-20 Nucleic Acid Bases, 12-15 Nucleic Acid Bases, 13-50 Nucleic Acid Bases, 13-40 Nucleic Acid Bases, 13-35 Nucleic Acid Bases, 13-30 Nucleic Acid Bases, 13-25 Nucleic Acid Bases, 13-20 Nucleic Acid Bases, 14-50 Nucleic Acid Bases, 14-40 Nucleic Acid Bases, 14-35 Nucleic Acid Bases The ASO may be 14-30 nucleobases, 14-25 nucleobases, 14-20 nucleobases, 15-50 nucleobases, 15-40 nucleobases, 15-35 nucleobases, 15-30 nucleobases, 15-25 nucleobases, 15-20 nucleobases, 20-50 nucleobases, 20-40 nucleobases, 20-35 nucleobases, 20-30 nucleobases, 20-25 nucleobases, 25-50 nucleobases, 25-40 nucleobases, 25-35 nucleobases, or 25-30 nucleobases in length. In some embodiments, the ASO is 18 nucleotides in length. In some embodiments, the ASO is 15 nucleotides in length. In some embodiments, the ASO is 25 nucleotides in length.

[0151] In some embodiments, two or more ASOs are used that have different chemistries but are complementary to the same targeting region of the AIC pre-mRNA, hi some embodiments, two or more ASOs are used that are complementary to different targeting regions of the AIC pre-mRNA.

[0152] In some embodiments, the ASOs of the invention are chemically linked to one or more moieties or conjugates, such as targeting moieties or other conjugates that enhance the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholesteryl moieties, aliphatic chains, such as dodecanediol or undecyl residues, polyamine or polyethylene glycol chains, or adamantane acetic acid. Oligonucleotides containing lipophilic moieties and methods for their preparation are described in the published literature. In some embodiments, the antisense oligonucleotides are conjugated to moieties, including, but not limited to, abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), lipids, or polyhydrocarbon compounds. The conjugates can be linked to one or more of the nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base, or phosphate group, using, for example, a linker, as understood in the art and described in the literature. The linker can include a bivalent or trivalent branched linker. In some embodiments, the conjugate is attached to the 3' end of the antisense oligonucleotide. Methods for preparing oligonucleotide conjugates are described, for example, in U.S. Patent No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides," which is incorporated herein by reference.

[0153] In some embodiments, the nucleic acid targeted by the ASO is a CD274 AIC pre-mRNA expressed in a cell, e.g., a eukaryotic cell. In some embodiments, the term "cell" can refer to a population of cells. In some embodiments, the cell is in a subject. In some embodiments, the cell is isolated from a subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in a tissue ex vivo. In some embodiments, the cell is in an organ ex vivo. In some embodiments, the cell is a condition- or disease-associated cell or cell line. In some embodiments, the cell is in vitro (e.g., in cell culture).

[0154] In some embodiments, the therapeutic agent or ASO inhibits splicing of an alternative intron from the AIC pre-mRNA encoding the target protein. In some embodiments, the therapeutic agent or ASO inhibits splicing of a processed intron encoding the target protein in a cell. In some embodiments, the level of processed mRNA encoding the target protein in cells contacted with a therapeutic agent or ASO is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1 to about 10-fold, or about 1 to about 10-fold, compared to the level of processed mRNA encoding the target protein in control cells, e.g., cells not contacted with a therapeutic agent or ASO, or cells contacted with a therapeutic agent or ASO that is not complementary to the targeted region of the AIC pre-mRNA. an increase of 0.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0155] In some embodiments, a therapeutic agent or ASO increases expression of a target protein in a cell. In some embodiments, the level of the target protein in a cell contacted with a therapeutic agent increases by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, or about 1.1 to about 8-fold, compared to the level of the target protein in a control cell, e.g., a cell not contacted with a therapeutic agent or ASO, or a cell contacted with a therapeutic agent or ASO that is not complementary to the targeted region of the AIC pre-mRNA. 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold increase.

[0156] In some embodiments, a therapeutic agent or ASO promotes or enhances splicing of an alternative intron from the AIC pre-mRNA encoding the target protein. In some embodiments, a therapeutic agent or ASO reduces the level of processed mRNA encoding the target protein in a cell. In some embodiments, the level of processed mRNA encoding the target protein in a cell contacted with a therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 10-fold, or about 1.5 to about 10-fold, compared to the level of processed mRNA encoding the target protein in a control cell, e.g., a cell not contacted with a therapeutic agent or ASO, or a cell contacted with a therapeutic agent or ASO that is not complementary to the targeted region of the AIC pre-mRNA. The decrease is about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0157] In some embodiments, a therapeutic agent or ASO reduces expression of a target protein in a cell. In some embodiments, the level of the target protein in a cell contacted with a therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9 ... The decrease is about 8-fold, about 3- to about 9-fold, about 4- to about 7-fold, about 4- to about 8-fold, about 4- to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold.

[0158] Diseases and Disorders In some embodiments, the methods and compositions of the present invention are used to treat any immune disease or disorder in a subject in need thereof. In some embodiments, the immune disease or disorder treated using the methods and compositions of the present invention is, for example, an autoimmune disease or disorder, an inflammatory disease or disorder, a chronic infection, graft-versus-host disease (GVHD), transplant rejection, or a T-cell proliferative disorder. In some embodiments, the immune disease or disorder is an autoimmune disease or disorder or an inflammatory disease or disorder selected from multiple sclerosis, inflammatory bowel disease, autoimmune hepatitis, renal inflammation, rheumatoid arthritis, psoriasis, lupus nephritis, corneal transplant, and uveitis. In some embodiments, the immune disorder is a disorder mediated by T cells, B cells, or NK cells.

[0159] Autoimmune diseases or disorders are conditions characterized by cell, tissue, and / or organ damage caused by a subject's immunological reaction against the subject's own cells, tissues, and / or organs. Inflammatory diseases or disorders refer to a subject's condition characterized by inflammation, including, for example, chronic inflammation. Autoimmune diseases may or may not be associated with inflammation. Inflammation may or may not be caused by an autoimmune disease. Therefore, certain disorders may be characterized as both autoimmune disorders and inflammatory disorders. Autoimmune diseases or disorders that can be treated using the methods and compositions of the present invention include, for example, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune adrenal disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune deficiency syndrome (CFIDS), Chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, diabetes mellitus, eosinophilic fascitis, fibromyalgia / fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré, Hashimoto's thyroiditis, Henoch-Schönlein purpura, idiopathic pulmonary fibrosis, idiopathic / autoimmune thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, systemic lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus-related disorders (e.g., pemphigus vulgaris), pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, The cause may be primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-person syndrome, systemic lupus erythematosus (SLE), Sweet's syndrome, Still's disease, lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.

[0160] In some embodiments, the immune disease or disorder treated using the methods and compositions of the present invention is an inflammatory disease or disorder, such as, for example, arthritis (e.g., rheumatoid arthritis, osteoarthritis), pneumonia, inflammation associated with hepatitis (including viral hepatitis), inflammation associated with infectious diseases, inflammatory bowel disease, enteritis, nephritis (e.g., glomerulonephritis, renal fibrosis), gastritis, vasculitis, pancreatitis, peritonitis, bronchitis, myocarditis, encephalitis, inflammation in post-ischemic reperfusion injury (myocardial ischemia-reperfusion injury), inflammation due to immune rejection after tissue and organ transplantation, burns, various skin inflammations (psoriasis, allergic contact dermatitis, lichen planus), inflammation in multiple organ failure, PTCA or PTC. These include inflammation after R surgery, inflammation associated with atherosclerosis, autoimmune thyroiditis, asthma, encephalitis, chronic obstructive pulmonary disease (COPD), allergic diseases, septic shock, pulmonary fibrosis, undiagnosed spondyloarthropathy, undiagnosed arthropathy, spondyloarthropathy (e.g., psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome or reactive arthritis), inflammatory osteolysis, Wilson's disease, and chronic inflammation due to chronic viral or bacterial infection.

[0161] In some embodiments, the immune disease or disorder treated using the methods and compositions of the invention is a chronic inflammatory disease or disorder selected from, for example, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), Graves' disease, Hashimoto's thyroiditis, allergic contact dermatitis, chronic inflammatory dermatoses (e.g., lichen planus), and diabetes mellitis.

[0162] In some embodiments, the immune disease or disorder treated using the methods and compositions of the invention is, for example, toxic shock syndrome, inflammatory bowel disease, transfusion-induced allosensitization, or T-cell-dependent B-cell mediated disease, osteoarthritis (OA), graft-versus-host reaction (GVH reaction), graft-versus-host disease (GVHD), immune rejection following tissue (e.g., skin, cornea, bone) or organ (e.g., liver, heart, lung, kidney, pancreas) transplantation, an immune response (e.g., production of antibodies against the antigen, cell proliferation, cytokine production) caused by foreign or self-antigens, or a disorder caused by abnormal intestinal immunity (e.g., inflammatory bowel disorder, Crohn's disease, ulcerative colitis, and gastrointestinal allergies).

[0163] Autoimmune diseases can affect any tissue or body part, including, but not limited to, the heart, brain, nerves, muscles, skin, eyes, joints, lungs, kidneys, glands (e.g., thyroid), digestive tract, and blood vessels. The autoimmune disease systemic lupus erythematosus can affect the skin, joints, kidneys, heart, nerves, blood vessels, and other tissues. Type 1 diabetes can affect, for example, glands, eyes, kidneys, and muscles. In some embodiments, subjects treated using the methods and compositions of the invention have a predisposition to developing an autoimmune disease at the time of treatment. In some embodiments, subjects treated using the methods and compositions of the invention are treated prophylactically.

[0164] In some embodiments, subjects with autoinflammatory disorders are treated using the methods and compositions of the present invention. Autoinflammatory disorders are characterized by episodes of severe inflammation that result in symptoms such as fever, rash, and swollen joints. In some cases, inflammatory disorders are caused by chronic conditions. For example, nonalcoholic steatohepatitis (NASH) is an inflammatory disorder associated with fatty liver disease. Other inflammatory disorders contemplated for treatment using the methods and compositions of the present invention include, for example, familial Mediterranean fever (FMF), neonatal-onset multisystem inflammatory disease (NOMID), tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), interleukin-1 receptor antagonist deficiency (DIRA), post-streptococcal and autoimmune renal failure, septic shock, systemic inflammatory response syndrome (SIRS), adult respiratory distress syndrome (ARDS), and inflammation due to envenomation.

[0165] Examples of autoimmune disorders that may be treated by the present invention include, for example, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's disease), multiple sclerosis, systemic lupus erythematosus / lupus nephritis, Hashimoto's thyroiditis, autoimmune hepatitis, myasthenia gravis, myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, and rheumatoid arthritis. , scleroderma / systemic sclerosis, Sjögren's syndrome, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, Behcet's disease, systemic lupus erythematosus, multiple sclerosis (systemic sclerosis and progressive systemic sclerosis), scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (nodular Polyarteritis nodosa and microscopic polyangiitis), aortitis syndrome (Takayasu's arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, mixed connective tissue disease, adult-onset Still's disease, allergic granulomatous vasculitis, hypersensitivity vasculitis, Cogan's syndrome, RS3PE, temporal arteritis, polymyalgia rheumatica, fibromyalgia syndrome, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related diseases (e.g., primary sclerosing cholangitis, autoimmune pancreatitis), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cirrhosis, aortitis syndrome, Goodpasture's syndrome, rapidly progressive glomerulonephritis, These include megaloblastic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease (hyperthyroidism), Hashimoto's thyroiditis, autoimmune adrenal insufficiency, primary hypothyroidism, idiopathic Addison's disease (chronic adrenal insufficiency), type 1 diabetes mellitus, chronic discoid lupus erythematosus, localized scleroderma, psoriasis, psoriatic arthritis, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent fetal loss, and inflammatory bowel disease (ulcerative colitis and Crohn's disease).

[0166] In some embodiments, the methods of the invention comprise contacting a cell of a subject with an ASO. In some embodiments, the contacted cell is an immune system cell in any differentiation state. In some embodiments, the cell is a stem cell, a progenitor cell, a dendritic cell, a macrophage, a peritoneal B1 B cell, a memory B cell, a bone marrow (BM)-derived mast cell, a hematopoietic cell, a non-hematopoietic cell, a B cell, or a T cell. In some embodiments, the immune system cell is a T cell. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a killer CD8+ T cell. In some embodiments, the T cell is an activated T cell. In some embodiments, the T cell is a pathogenic CD4+ T cell. H 1 or T H 17 effector cells.

[0167] In some embodiments, the contacted cells in the methods of the invention are non-hematopoietic cells, such as vascular endothelial cells, fibroblastic reticular cells, epithelial cells, pancreatic islet cells, astrocytes, neurons, Schwann cells of the CNS / PNS, hepatocytes, corneal, renal cells, or cells of immune-privileged sites, including placental trophoblasts or ocular retinal pigment epithelial cells or neurons, or other suitable cell types known in the art and identified for targeting in the treatment of immunological disorders. In some embodiments, the contacted cells are of a cell type that expresses a target protein. In some embodiments, the contacted cells are of a cell type that expresses PD-L1.

[0168] In some embodiments of the invention, cells are obtained from a subject in need thereof, modified with an ASO ex vivo to induce target protein expression (e.g., PD-L1), and adoptively transferred into the subject. H 1 cells are obtained from a subject in need thereof, and REG The cells can be induced to transform into PD-L1 cells and adoptively transferred to a patient in need thereof. In some cases, the subject in need of the cells has a transplant-associated autoimmune disease. In some embodiments, GVHD is treated by inducing PD-L1 expression in a target cell population. In some embodiments, immune disorders are treated by the methods of the invention using ex vivo modified cells. In some embodiments, immune disorders are treated by systemic infusion of the therapeutic methods of the invention.

[0169] In some embodiments, the genetic disorder or condition can be an autosomal dominant disorder, an autosomal recessive disorder, an X-linked dominant disorder, an X-linked recessive disorder, a Y-linked disorder, a mitochondrial disease, or a multifactorial or polygenic disorder. Sometimes, genetic diseases can also be characterized as autosomal dominant, autosomal recessive, X-linked dominant, X-linked recessive, Y-linked, mitochondrial, or a multifactorial or polygenic genetic disease. Autosomal dominant disorders, autosomal recessive disorders, X-linked dominant disorders, X-linked recessive disorders, Y-linked disorders, mitochondrial diseases, or a multifactorial or polygenic disorder can be characterized by impaired protein production. Autosomal dominant disorders, autosomal recessive disorders, X-linked dominant disorders, X-linked recessive disorders, Y-linked disorders, mitochondrial diseases, or multifactorial or polygenic disorders may be characterized by defective splicing.Subjects with autosomal dominant disorders, autosomal recessive disorders, X-linked dominant disorders, X-linked recessive disorders, Y-linked disorders, mitochondrial diseases, or multifactorial or polygenic disorders can have genomes that contain copies of genes that include exons that can encode full-length functional forms of proteins when properly transcribed into fully processed mRNA.Subjects with autosomal dominant disorders, autosomal recessive disorders, X-linked dominant disorders, X-linked recessive disorders, Y-linked disorders, mitochondrial diseases, or multifactorial or polygenic disorders can have genomes that contain copies of genes that include a set of exons that can encode full-length functional forms of proteins when properly transcribed into fully processed mRNA. A subject with an autosomal dominant disorder, an autosomal recessive disorder, an X-linked dominant disorder, an X-linked recessive disorder, a Y-linked disorder, a mitochondrial disease, or a multifactorial or polygenic disorder can have a genome that may contain defective copies of genes that may be unable to produce full-length functional forms of proteins.

[0170] Exemplary genetic disorders include achondroplasia, hereditary hemochromatosis, Down syndrome, hereditary spherocytosis, Tay-Sachs disease, Usher syndrome, hereditary fructose intolerance, hemophilia, muscular dystrophy (e.g., Duchenne muscular dystrophy or DMD), polygenic disorders, breast cancer, ovarian cancer, Parkinson's disease, Bardet-Biedl syndrome, Prader-Willi syndrome, diabetes, heart disease, arthritis, motor neuron disease, albinism, cri-cat syndrome, cystic fibrosis, fragile X syndrome, galactosemia, Huntington's disease, and Jackson-Weiss syndrome. , Klinefelter syndrome, Krabbe disease, Langer-Giedion syndrome, Lesch-Nyhan syndrome, Marfan syndrome, myotonic dystrophy, Nail-Patera syndrome, neurofibromatosis, Noonan syndrome, triple X syndrome, osteogenesis imperfecta, Patau syndrome, phenylketonuria, porphyria, retinoblastoma, Rett syndrome, sickle cell disease, Turner syndrome, Usher syndrome, von Pipel-Lindau syndrome, Waardenburg syndrome, Wilson's disease, xeroderma pigmentosum, XXXX syndrome, or YY syndrome.

[0171] Genetic disorders, e.g., achondroplasia, hereditary hemochromatosis, Down syndrome, hereditary spherocytosis, Tay-Sachs disease, Usher syndrome, hereditary fructose intolerance, hemophilia, muscular dystrophies (e.g., Duchenne muscular dystrophy or DMD), polygenic disorders, breast cancer, ovarian cancer, Parkinson's disease, Bardet-Biedl syndrome, Prader-Willi syndrome, diabetes, heart disease, arthritis, motor neuron disease, albinism, cri-clack syndrome, cystic fibrosis, fragile X syndrome, galactosemia, Huntington's disease, Jackson-Weiss syndrome, Klinefelter syndrome, Claudia-Lucirumab Abbe disease, Langer-Giedion syndrome, Lesch-Nyhan syndrome, Marfan syndrome, myotonic dystrophy, Nail-Patera syndrome, neurofibromatosis, Noonan syndrome, triple X syndrome, osteogenesis imperfecta, Patau syndrome, phenylketonuria, porphyria, retinoblastoma, Rett syndrome, sickle cell disease, Turner syndrome, Usher syndrome, von Pipel-Lindau syndrome, Waardenburg syndrome, Wilson disease, xeroderma pigmentosum, XXXX syndrome, or YY syndrome may be characterized by impaired protein production or defective splicing. Genetic disorders, e.g., achondroplasia, hereditary hemochromatosis, Down syndrome, hereditary spherocytosis, Tay-Sachs disease, Usher syndrome, hereditary fructose intolerance, hemophilia, muscular dystrophies (e.g., Duchenne muscular dystrophy or DMD), polygenic disorders, breast cancer, ovarian cancer, Parkinson's disease, Bardet-Biedl syndrome, Prader-Willi syndrome, diabetes, heart disease, arthritis, motor neuron disease, albinism, cri-clack syndrome, cystic fibrosis, fragile X syndrome, galactosemia, Huntington's disease, Jackson-Weiss syndrome, Klinefelter syndrome, Krabbe disease, Langer-Giedion syndrome, Lesch-Nyhan syndrome , Marfan syndrome, myotonic dystrophy, Nail-Patera syndrome, neurofibromatosis, Noonan syndrome, triple X syndrome, osteogenesis imperfecta, Patau syndrome, phenylketonuria, porphyria, retinoblastoma, Rett syndrome, sickle cell disease, Turner syndrome, Usher syndrome, von Pipel-Lindau syndrome, Waardenburg syndrome, Wilson's disease, xeroderma pigmentosum, XXXX syndrome, or YY syndrome, etc., can contain a copy of a gene that includes exons that, when properly transcribed into fully processed mRNA, are capable of encoding a full-length functional form of a protein; can contain a copy of a gene that includes a set of exons that, when properly transcribed into fully processed mRNA, are capable of encoding a full-length functional form of a protein; or can contain a defective copy of a gene that may be unable to produce a full-length functional form of a protein.

[0172] As described above, the genetic disorder or condition may be an autosomal dominant disorder, an autosomal recessive disorder, an X-linked dominant disorder, an X-linked recessive disorder, a Y-linked disorder, a mitochondrial disease, or a multifactorial or polygenic disorder. The genetic disorder or condition may be an autosomal dominant disorder, an autosomal recessive disorder, an X-linked dominant disorder, an X-linked recessive disorder, a Y-linked disorder, a mitochondrial disease, or a multifactorial or polygenic disorder, and may be characterized by impaired production of proteins or defective splicing.

[0173] Exemplary autosomal dominant disorders can include Huntington's disease, neurofibromatosis type 1, neurofibromatosis type 2, Marfan syndrome, hereditary nonpolyposis colorectal cancer, hereditary multiple exostoses, tuberous sclerosis, von Willebrand's disease, or acute intermittent porphyria.

[0174] Pharmaceutical Composition Pharmaceutical compositions or formulations containing the antisense oligomers (ASOs) of the described compositions and for use in any of the described methods can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the published literature. In some embodiments, pharmaceutical compositions or formulations for treating a subject comprise an effective amount of any of the above-described ASOs, or a pharmaceutically acceptable salt, solvate, hydrate, or ester thereof, and a pharmaceutically acceptable diluent. Pharmaceutical formulations of ASOs may further comprise a pharmaceutically acceptable excipient, diluent, or carrier.

[0175] Pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit / risk ratio. Salts can be prepared in situ during the final isolation and purification of the compound, or separately by reacting its free base function with a suitable organic acid. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other documented methodologies, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, and picrin. Representative salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed where appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0176] In some embodiments, the compositions are formulated into any of many possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, softgels, suppositories, and enemas. In some embodiments, the compositions are formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. Suspensions may also contain stabilizers. In some embodiments, pharmaceutical formulations or compositions of the present invention include, but are not limited to, solutions, emulsions, microemulsions, foams, or liposome-containing formulations (e.g., cationic or non-cationic liposomes).

[0177] Pharmaceutical compositions or formulations of the invention may optionally include one or more penetration enhancers, carriers, excipients, or other active or inactive ingredients known to those of skill in the art or described in the published literature. In some embodiments, liposomes also include sterically stabilized liposomes, e.g., liposomes comprising one or more specialized lipids. These specialized lipids result in liposomes with improved circulation longevity. In some embodiments, sterically stabilized liposomes comprise one or more glycolipids or are derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. In some embodiments, a surfactant is included in the pharmaceutical formulation or composition. The use of surfactants in pharmaceuticals, formulations, and emulsions is well known in the art. In some embodiments, the invention uses a penetration enhancer to provide efficient delivery of the ASO, for example, to aid diffusion across cell membranes and / or enhance the permeability of lipophilic drugs. In some embodiments, the penetration enhancer is a surfactant, fatty acid, bile salt, chelating agent, or non-chelating non-surfactant.

[0178] In some embodiments, the pharmaceutical formulation comprises multiple ASOs. In some embodiments, the ASO is administered in combination with another drug or therapeutic agent. In some embodiments, the ASO is administered with one or more agents that can enhance penetration of the target ASO through the blood-brain barrier by any method known in the art. For example, delivery of an agent to motor neurons in muscle tissue by administration of an adenoviral vector is described in U.S. Patent No. 6,632,427, "Adenoviral-vector-mediated gene transfer into medullary motor neurons," which is incorporated herein by reference. Delivery of a vector directly to the brain, for example, the striatum, thalamus, hippocampus, or substantia nigra, is described in U.S. Patent No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system, particularly in the brain," which is incorporated herein by reference.

[0179] In some embodiments, the ASO is linked or conjugated to an agent that provides desirable pharmaceutical or pharmacodynamic properties. In some embodiments, the ASO is linked or conjugated to a substance known in the art to enhance penetration or transport across the blood-brain barrier, such as a steroid or steroid drug. , binds to an antibody against the transferrin receptor. In some embodiments, the ASO is linked to a viral vector, for example, to make an antisense compound more effective or to increase transport across the blood-brain barrier. In some embodiments, osmotic blood-brain barrier disruption is achieved by the addition of sugars, such as mesoerythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, arabinose, D(- ... Infusion of donitol, D(+)arabitol, L(-)arabitol, D(+)fucose, L(-)fucose, D(-)lyxose, L(+)lyxose, and L(-)lyxose, or amino acids such as glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine, is also used. Methods and materials for enhancing blood-brain barrier penetration are described, for example, in U.S. Pat. No. 4,866,042, "Method for the delivery of genetic material across the blood-brain barrier," U.S. Pat. No. 6,294,520, "Material for passage through the blood-brain barrier," and U.S. Pat. No. 6,936,589, "Parenteral delivery systems”.

[0180] In some embodiments, the ASOs of the invention are chemically linked to one or more moieties or conjugates, such as targeting moieties or other conjugates that enhance the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholesteryl moieties, aliphatic chains, such as dodecanediol or undecyl residues, polyamine or polyethylene glycol chains, or adamantane acetic acid. Oligonucleotides containing lipophilic moieties and methods for their preparation are described in the published literature. In some embodiments, the ASOs are conjugated to moieties, including, but not limited to, abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), lipids, or polyhydrocarbon compounds. Conjugates can be linked to one or more of any nucleotides containing the ASO at any of several positions on the sugar, base, or phosphate group, using, for example, linkers, as understood in the art and described in the literature. The linker can include a bivalent or trivalent branched linker. In some embodiments, the conjugate is attached to the 3' end of the ASO. Methods for preparing oligonucleotide conjugates are described, for example, in U.S. Patent No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides," incorporated herein by reference.

[0181] Targeted treatment Any of the compositions provided herein can be administered to an individual. "Individual" can be used interchangeably with "subject" or "patient." An individual can be a mammal, e.g., a human, or an animal such as a non-human primate, rodent, rabbit, rat, mouse, horse, donkey, goat, cat, dog, cow, pig, or sheep. In some embodiments, the individual is a human. In some embodiments, the individual is a fetus, embryo, or child. In some embodiments, the individual is a non-human animal. In other embodiments, the individual can be another eukaryotic organism, such as a plant. In some embodiments, the compositions provided herein are administered to cells ex vivo. In some embodiments, the compositions provided herein are administered to cells ex vivo. The agent is administered to the organ or tissue ex vivo (eg, by perfusion or other means) prior to transplantation of the organ or tissue.

[0182] In some embodiments, the compositions provided herein are administered to an individual as a method of treating a disease or disorder. In some embodiments, the individual has an autoimmune or inflammatory disease, such as any of the diseases described herein. In some embodiments, the individual is at risk of having a disease, such as any of the diseases described herein. In some embodiments, the individual is at increased risk of having a disease or disorder caused by an insufficient amount of a protein or insufficient activity of a protein. If the individual is at "increased risk" of having a disease or disorder caused by an insufficient amount of a protein or insufficient activity of a protein, the method involves preventative or prophylactic treatment. For example, an individual may be at increased risk of having such a disease or disorder due to a family history of the disease. Typically, individuals at increased risk of having such a disease or disorder benefit from prophylactic treatment (e.g., by preventing or delaying the onset or progression of the disease or disorder). In some embodiments, the individual is at increased risk of having a disease or disorder caused by an abnormal amount of a protein or abnormal activity of a protein.

[0183] In some embodiments, subjects treated using the methods and compositions of the invention have received previous treatment for a disease or disorder (e.g., an immune disease or disorder). Previous treatments include, for example, corticosteroids, transplant agents such as cyclophosphamide and azathioprine, targeted biologics including mAbs that block TNF, IL-17, IL-12 / 23, or directed against cytokines including CD20 (rituximab) or CD52 (alemtuzumab), IFN-β, small molecules such as dimethyl fumarate, and JAK or sphingosine-1-phosphate inhibitors, or intravenous immunoglobulin. In some embodiments, the methods of the invention are used concurrently with or subsequent to other treatments.

[0184] Generally, the preferred route for administration of the ASOs of the present invention will vary depending on the cell type to which delivery of the ASO is desired. As known to those skilled in the art and described in the literature, different tissues and organs may be affected by a disorder (e.g., an immune disorder) depending on the disorder and the affected individual. In some embodiments, the ASOs of the present invention are administered to a patient parenterally. In some embodiments, the ASOs of the present invention are administered to a patient by intracerebroventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal injection, subretinal injection, topical application, implantation, or intravenous injection. In some embodiments, a fetus is treated in utero, for example, by administering an ASO composition to the fetus directly or indirectly (e.g., via the mother).

[0185] In some embodiments, subjects with immune disorders affecting the brain or CNS, e.g., autoimmune disorders such as multiple sclerosis, are treated by intrathecal injection, intracerebroventricular injection, subcutaneous administration, or intravenous administration. Subjects with immune disorders affecting the gut, e.g., inflammatory disorders such as inflammatory bowel disease, are treated by oral or subcutaneous administration of the present invention. In some embodiments, inflammatory conditions affecting liver or kidney inflammation are treated by subcutaneous or intravenous administration of the present invention. In some embodiments, joint inflammation, e.g., rheumatoid arthritis or psoriatic arthritis, is treated by direct synovial injection or topical administration of the present invention. In some embodiments, skin inflammation, e.g., in subjects with psoriasis, is treated by topical administration of the present invention. In some embodiments, ocular inflammation, e.g., after corneal transplantation or in uveitis, is treated by topical, intravitreal, subretinal, or implant administration of the present invention. In some embodiments, subjects with lupus are treated by subcutaneous administration of the present invention. In some embodiments, the mode of administration is tissue-specific. i.e., to upregulate target protein levels (e.g., PD-L1) in target tissues, minimizing the potential for suppression of anti-pathogen immunity. In some embodiments, an appropriate mode of administration will be selected by one of skill in the art based on available literature and their knowledge of a given condition.

[0186] In some embodiments, the methods and compositions of the present invention are used in combination with immunosuppressive therapy. Immunosuppressive therapy can include any treatment that suppresses the immune system. Immunosuppressive therapy can help alleviate, minimize, or eliminate transplant rejection in the recipient. For example, immunosuppressive therapy can include immunosuppressive drugs. Immunosuppressive drugs that can be used before, during, and / or after transplantation include, for example, MMF (mycophenolate mofetil (Cellcept)), ATG (antithymocyte globulin), anti-CD154 (CD4OL), anti-CD40 (2C10, ASKP1240, CCFZ533X2201), alemtuzumab (Campath), anti-CD20 (rituximab), anti-IL-6R antibodies (tocilizumab, Actemra), anti-IL-6 antibodies (sarilumab, olokizumab), CTLA4-Ig (abatacept / Orencia), belatacept (L EA29Y), sirolimus (Rapimune), everolimus, tacrolimus (Prograf), daclizumab (Ze-napax), basiliximab (Simulect), infliximab (Remicade), cyclosporine, deoxyspergualin, soluble complement receptor 1, cobra venom factor, compstatin, anti-C5 antibody (eculizumab / Soliris), methylprednisolone, FTY720, everolimus, leflunomide, anti-IL-2R-Ab, rapamycin, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody, and anti-CD122 antibody. In some embodiments, two or more immunosuppressive agents or drugs are used together or sequentially. Immunosuppression can also be achieved using non-drug regimens, including, but not limited to, total body irradiation, thymic irradiation, and total and / or partial splenectomy. These techniques can also be used in conjunction with one or more immunosuppressant drugs, if desired, in conjunction with the methods and compositions of the present invention.

[0187] Methods for identifying additional ASOs that regulate splicing Also within the scope of the present invention are methods for identifying (determining) additional ASOs that modulate (e.g., inhibit or enhance) splicing of the AIC pre-mRNA, specifically alternative introns. In some embodiments, the target AIC pre-mRNA is encoded by a gene selected from ARHGAP23, BRD1, DCHS1, EPB41L2, GPX8, HIVEP3, INVS, KIAA0319, NAIP, CD274, PTCH2, PTPRZ1, SON, and ZCCHC2. In some embodiments, within the scope of the present invention are methods for identifying (determining) additional ASOs that modulate (e.g., inhibit or enhance) splicing of the CD274 AIC pre-mRNA, specifically alternative introns. ASOs that specifically hybridize to different nucleotides within the target region of the pre-mRNA can be screened to identify (determine) ASOs that modulate (e.g., reduce or improve) the rate and / or extent of splicing of the alternative intron. In some embodiments, the ASO may promote the binding of a splicing repressor(s) / silencer. In some embodiments, the ASO may block or interfere with the binding site(s) of the splicing repressor(s) / silencer. Any method known in the art can be used to identify (determine) an ASO that produces a desired effect (e.g., enhanced protein or functional RNA production) when hybridized to a target region of a pre-mRNA. These methods can also be used to enhance the expression of an alternative intron by binding to a target region in an intron upstream of a first portion of an exon adjacent to the 5' splice site of the alternative intron, in an intron downstream of a second portion of an exon adjacent to the 3' splice site of the alternative intron, in the first portion of an exon, in the second portion of an exon, or in an alternative intron. ASOs can be identified that modulate the splicing of alternative introns. Examples of methods that can be used are provided below.

[0188] A round of screening, called an ASO "walk," may be performed using ASOs designed to hybridize to a target region of the pre-mRNA. For example, the ASOs used in the ASO walk can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 5' splice site of the alternative intron (e.g., a portion of the sequence of the target intron or exon located upstream of the alternative intron, or a portion of the sequence of the first portion of the exon adjacent to the 5' splice site of the alternative intron) to approximately 100 nucleotides downstream of the 5' splice site of the alternative intron and / or from approximately 100 nucleotides upstream of the 3' splice site of the alternative intron to approximately 100 nucleotides downstream of the 3' splice site of the alternative intron (e.g., a portion of the sequence of the target intron or exon located downstream of the alternative intron, or a portion of the sequence of the second portion of the exon adjacent to the 3' splice site of the alternative intron). For example, a first ASO 15 nucleotides in length can be designed to specifically hybridize between nucleotides +1 and +15 relative to the 5' splice site of an alternative intron. A second ASO can be designed to specifically hybridize between nucleotides +6 and +20 relative to the 5' splice site of an alternative intron. The ASOs are designed to span the target region of the pre-mRNA. In some embodiments, ASOs can be tiled more closely, for example, every 1, 2, 3, or 4 nucleotides. Furthermore, ASOs can be tiled from 100 nucleotides downstream of the 5' splice site to 100 nucleotides upstream of the 3' splice site.

[0189] One or more ASOs or a control ASO (an ASO with a scrambled sequence, a sequence not expected to hybridize to the target region) are delivered, for example, by transfection, to a disease-related cell line expressing the target pre-mRNA (e.g., the AIC pre-mRNA described elsewhere herein). The splicing-modulating effect (e.g., splicing-inhibiting effect or splicing-inducing effect) of each ASO can be evaluated by methods known in the art, such as reverse transcriptase (RT)-PCR using primers spanning the splice junction. An increase in RT-PCR products produced using primers spanning the splice junction in ASO-treated cells compared to control ASO-treated cells indicates that splicing of the target intron has been inhibited. A decrease or absence of RT-PCR products produced using primers spanning the splice junction in ASO-treated cells compared to control ASO-treated cells indicates that splicing of the target intron has been enhanced. As described in Example 2, RT-PCR can also use primers flanking the splice junction. RT-PCR products may be of different sizes due to the absence or presence of splicing events or altered or additional splice junctions. For example, the presence of smaller RT-PCR products may indicate that additional splicing events have occurred or that splicing events have occurred at different positions. Smaller RT-PCR products may indicate that alternative introns have been spliced ​​out.

[0190] In some embodiments, the splicing efficiency, the ratio of spliced ​​to unspliced ​​pre-mRNA, the rate of splicing, or the extent of splicing can be modulated (e.g., reduced or improved) using the ASOs described herein. The amount of protein or functional RNA encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced protein production). RNA or Any method known in the art for assessing and / or quantifying protein production can be used.

[0191] A second round of screening, called an ASO "micro-walk," may be performed using ASOs designed to hybridize to target regions of the pre-mRNA. The ASOs used in the ASO micro-walk are tiled nucleotide by nucleotide to further refine the nucleotide sequence of the pre-mRNA whose splicing is modulated (e.g., inhibited or enhanced) when hybridized by the ASO.

[0192] The regions defined by ASOs that inhibit or promote splicing of the target intron are investigated in more detail by ASO "micro-walks" with ASOs spaced in 1-nucleotide steps, as well as longer ASOs, typically 18–25 nucleotides.

[0193] As described above for ASO walk, ASO micro-walking is performed by delivering one or more ASOs, or a control ASO (an ASO with a scrambled sequence that is not expected to hybridize to the target region), to a disease-relevant cell line expressing the target pre-mRNA, for example, by transfection. The splicing-inhibiting or splicing-inducing effect of each ASO can be assessed by methods known in the art, such as reverse transcriptase (RT)-PCR using primers spanning the splice junction. An increase in RT-PCR products produced using primers spanning the splice junction in ASO-treated cells compared to control ASO-treated cells indicates that splicing of the target intron has been inhibited. A decrease or absence of RT-PCR products produced using primers spanning the splice junction in ASO-treated cells compared to control ASO-treated cells indicates that splicing of the target intron has been enhanced. As described in Example 2, RT-PCR can also use primers flanking the splice junction. RT-PCR products may be of different sizes due to the absence or presence of splicing events or altered or additional splice junctions. For example, the presence of smaller RT-PCR products may indicate that additional splicing events have occurred or that splicing events have occurred at different positions. Smaller RT-PCR products may indicate that alternative introns have been spliced. In some embodiments, the splicing efficiency, the ratio of unspliced ​​to spliced ​​pre-mRNA, the splicing rate, or the extent of splicing can be modulated (e.g., reduced or improved) using the ASOs described herein. The amount of protein or functional RNA encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced protein production).Any method known in the art for assessing and / or quantifying RNA or protein production can be used, such as qPCR, RT-PCR, Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0194] ASOs that, when hybridized to a region of pre-mRNA, result in modulation of splicing (e.g., inhibition or enhancement of splicing) and modulation of protein production (e.g., increase or decrease of protein production) can be tested in vivo using animal models, such as transgenic mouse models in which a full-length human gene has been knocked in or humanized mouse models of disease. The preferred route for administration of the ASO can vary depending on the disease and / or cell type to which delivery of the ASO is desired. The ASO may be administered, for example, by intracerebroventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal injection, subretinal injection, topical application, implantation, or intravenous injection. After administration, the model animals The efficacy of ASO treatment can be determined by evaluating cells, tissues, and / or organs of a subject, for example, by assessing splicing (efficiency, rate, extent) and protein production by methods known in the art and described herein. Animal models can also be any phenotypic or behavioral indicator of disease or disease severity.

[0195] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby. [Example]

[0196] The present disclosure will be more specifically illustrated by the following examples, however, it should be understood that the present invention is not limited in any way by these examples.

[0197] Example 1: Identification of alternative introns in CD274 transcripts by RNAseq using next-generation sequencing Whole-transcriptome shotgun sequencing can be performed using next-generation sequencing to reveal a snapshot of the transcripts produced by the CD274 gene and identify alternative introns. To this end, polyA+ RNA from the nuclear and cytoplasmic fractions of AST (human astrocyte) cells can be isolated and a cDNA library constructed using Illumina's TruSeq Stranded mRNA Library Preparation Kit. The library can be paired-end sequenced, yielding 100-nucleotide reads that can be mapped to the human genome. Mapped reads were visualized using the UCSC Genome Browser (operated by the UCSC Genome Informatics Group, Center for Biomolecular Science & Engineering, University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA 95064; see, for example, Rosenbloom et al., 2015, "The UCSC Genome Browser database: 2015 update," Nucleic Acids Research 43, Database Issue, doi: 10.1093 / nar / gku1177). Coverage and read counts can be estimated based on peak signal. Based on sequencing reads, exon 4 in CD274 was identified as harboring a potential alternative intron. A schematic diagram of the alternative intron construct is shown in Figure 1. The mRNA sequence encoding the functional protein is labeled 110. Sequence 110 consists of three exons: 101, 103-105, and 107. Introns 102 and 106 are spliced ​​out and are not part of construct 110. However, an alternative form of mRNA, labeled 120, may be produced which results in a premature termination codon (PTC) and therefore does not encode a functional protein.This alternative mRNA is instead composed of 101, 103, 105, and 107, with 104 (part of the exon in construct 110) spliced ​​out as an "alternative intron."

[0198] Example 2: In vitro observation of alternative introns in exon 4. PCR primers were designed with homology to exon 4 and exon 6, and RT-PCR reactions were performed to identify amplicons associated with mRNA transcripts in Huh7 cells. Amplification reactions are performed using total RNA (labeled T) and RNA from fractionated cells corresponding to the nucleus (labeled N) and cytoplasm (labeled C) to generate amplicons of mRNA transcripts. The amplicons are run on a polyacrylamide gel to observe the intensities of different PCR products. Products resulting from the removal of the "alternative intron" (ai) are observed to be lower on the gel and smaller in size than products corresponding to full-length functional mRNA transcripts (labeled can). To visualize the level of ai removal, cells were treated with cycloheximide (CHX) or a DMSO control. CHX, a translation inhibitor, inhibits nonsense-mediated mRNA decay, which normally degrades mRNAs lacking alternative introns, because ai removal leads to the introduction of PTCs. Figure 2A shows a gel and a schematic of the can and ai products. Figure 2B shows the percent abundance of products resulting from ai removal relative to the total (CAN + ai). FIG. 2C shows a gel, schematic diagram, and percent abundance of the products resulting from the removal of ai relative to total in two human cell lines (ARPE-19 and HUVEC) and non-human primate retina (cyno retina).

[0199] Example 3: Design of an ASO-walk targeting exon 4 of CD274 An ASO walk was designed to target exon 4 (SEQ ID NOS: 1-67). The region spanning nucleotides +68 to +253 was targeted using 2'-O-MOE RNA, PS backbone, 18-mer ASOs shifted at 5-nucleotide intervals. Figure 3 shows this ASO walk, with each black box indicating the extent of the ASO and the sequence shown below the black blocks indicating the target sequence in exon 4. Each block spans 18 nucleotides, representing an 18-nucleotide ASO, starting 5 nucleotides apart and representing a 5-nucleotide interval "walk."

[0200] Example 4: Screening of ASO-walk targeting exon 4 of CD274 The ASOs from Example 3 were screened using ARPE-19 cells. The ASOs were transfected into ARPE-19 cells for 24 hours using 80 nM of the ASO shown in Figure 3. RT-PCR products were amplified using primers in exon 4 and exon 6 and run on a polyacrylamide gel. Figure 4A shows a higher band corresponding to the full-length can mRNA and a lower band corresponding to the shorter aim RNA. Figure 4B shows quantification of RT-PCR products plotted as the percentage of alternative introns (ai / (can+ai)*100). ASOs were indicated as potential candidates if the relative abundance of ai was reduced. For example, treatment with ASO34 correlated with a decrease in relative abundance compared to the control (mock). Figure 4C shows Taqman qPCR analysis using RNA from the sample in panel A. The Taqman probe was located at the exon 3-exon 4 junction. As observed, CD247 mRNA expression generally inversely correlates with the relative abundance of ai transcripts. For example, ASO34 shows increased CD247 expression compared to controls.

[0201] Example 5: Measurement of ASO activity Selected ASOs were transfected into Huh7 cells using 80 nM ASO for 21 hours. Multiple assays were used to determine the general activity of the selected ASOs. Figure 5A shows RT-PCR using RNA from Huh7 cells transfected with 80 nM selected ASOs for 21 hours, followed by 3 hours of cycloheximide treatment. Primers were located in exons 4 and 6. Figure 5B shows quantification of RT-PCR products plotted as a percentage of the selected intron (ai / (can+ai)*100). ASO34 resulted in a decrease in the amount of ai product. This is observed in Figure 5B, where the intensity of the band corresponding to ai was significantly higher than that of mock-transfected cells. Figure 5C shows that TaqMan qPCR analysis using RNA from Huh7 cells transfected for 24 hours quantified the activity of 80 nM ASO compared to mock. The TaqMan qPCR probe was positioned over the exon 3-4 junction. The results show a higher relative abundance of full-length can mRNA in ASO34-transfected cells compared to mock-transfected cells. Figure 5D shows the mean fluorescence intensity from flow cytometry analysis of Huh7 cells transfected with 80 nM ASO34 for 5 days, plotted as fold change relative to the control (mock) to quantify PD-L1 protein expression. [Table 2]

[0202] Example 6: Alternative introns in various gene transcripts Whole transcriptome shotgun sequencing can be performed using next-generation sequencing to reveal a snapshot of the transcripts produced by the gene and identify alternative introns. As described in Example 1, cells can be isolated and cDNA libraries can be constructed using Illumina's TruSeq Stranded mRNA Library Preparation Kit. The libraries can be analyzed, and exons in other genes can be identified as having potential alternative introns based on the sequencing reads. ASO walks can be designed to target the identified exons using 2'-O-MOE RNA, PS backbone, and 18-mer ASOs shifted by 5 nucleotide intervals, as in Example 3. The ASOs can then be validated through the exemplary screening method of Example 4, and then used to prevent or promote the inclusion of alternative intron events in mRNA transcripts. The ASOs can be further injected into mouse models to confirm increased activity and expression. Various animal disease models can be used to validate increased expression of the target protein. Table 3 provides some examples of genes that can be targeted using the methods described above. The genes listed in Table 3 are known to comprise alternative introns. In addition to the targeted exons, the chromosomal coordinates of the genes are provided. Table 3 also lists several exemplary diseases that can be treated by increasing gene expression. The sequences and chromosomal coordinates provided in Table 3 are generated from the GRCh38 / hg38 assembly. [Table 3-1] [Table 3-2] [Table 3-3]

[0203] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method for regulating expression of a target protein or target RNA by a cell having an alternative intron-containing pre-mRNA (AIC pre-mRNA), wherein the AIC pre-mRNA comprises an alternative intron, a first portion of an exon adjacent to the 5' splice site of the alternative intron, and a second portion of the exon adjacent to the 3' splice site of the alternative intron, the method comprising contacting the cell with a therapeutic agent that binds to a targeted region of the AIC pre-mRNA encoding the target protein or the target RNA, thereby regulating splicing of the alternative intron from the AIC pre-mRNA encoding the target protein or the target RNA, thereby regulating the level of processed mRNA encoding the target protein or the target RNA and regulating the expression of the target protein or the target RNA in the cell.

2. 1. A method of treating a disease or condition in a subject in need thereof by modulating expression of a target protein or target RNA in the cells of the subject, comprising contacting the cells of the subject with a therapeutic agent that modulates splicing of an alternative intron from an alternative intron-containing pre-mRNA (AIC pre-mRNA) encoding the target protein or the target RNA, wherein the AIC pre-mRNA comprises the alternative intron, a first portion of an exon adjacent to a 5' splice site of the alternative intron, and a second portion of the exon adjacent to a 3' splice site of the alternative intron, and wherein the therapeutic agent binds to a targeted region of the AIC pre-mRNA encoding the target protein or the target RNA, thereby modulating splicing of the alternative intron from the AIC pre-mRNA encoding the target protein or the target RNA, thereby modulating the level of processed mRNA encoding the target protein or the target RNA and modulating the expression of the target protein or the target RNA in the cells of the subject.

3. 3. The method of claim 1 or 2, wherein modulating the expression of the target protein in the cell comprises increasing the expression of the target protein in the cell.

4. 3. The method of claim 1 or 2, wherein regulating the level of processed mRNA encoding the target protein comprises increasing the level of processed mRNA encoding the target protein.

5. 3. The method of claim 1 or 2, wherein inclusion of the alternative intron from the AIC pre-mRNA encoding the target protein is increased.

6. 6. The method of any one of claims 1 to 5, wherein modulating the level of processed mRNA encoding the target protein comprises modulating the level of processed mRNA comprising the alternative intron, the first portion of the exon, and the second portion of the exon.

7. The method of any one of claims 1 to 6, wherein the therapeutic agent is a small molecule.

8. The method of any one of claims 1 to 7, wherein the therapeutic agent is an antisense oligomer (ASO) complementary to the targeted region of the AIC pre-mRNA.

9. The therapeutic agent is a therapeutic agent for targeting the AIC pre-mRNA encoding the target protein.

9. The method of any one of claims 1 to 8, wherein the region is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the region.

10. 10. The method of any one of claims 1 to 9, wherein at least a portion of the targeted region of the AIC pre-mRNA is within the alternative intron.

11. 10. The method of any one of claims 1 to 9, wherein at least a portion of the targeted region of the AIC pre-mRNA is within the first portion of the exon.

12. 10. The method of any one of claims 1 to 9, wherein at least a portion of the targeted region of the AIC pre-mRNA is within the second portion of the exon.

13. The method of any one of claims 1 to 12, wherein the target protein produced is a fully functional protein.

14. The method of any one of claims 1 to 12, wherein the target RNA produced is a functional RNA.

15. The method of any one of claims 1 to 12, wherein the target RNA produced is fully functional RNA.

16. The method of any one of claims 1 to 15, wherein the target protein is PD-L1 (CD274).

17. 17. The method of any one of claims 1 to 16, wherein the targeted region of the AIC pre-mRNA to which the therapeutic agent binds is located within exon 4 of CD274.

18. 17. The method of any one of claims 1-16, wherein the therapeutic agent binds to a targeted region of CD274 AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 68, 69, and 71-76.

19. 17. The method of any of claims 1-16, wherein the therapeutic agent modulates splicing of alternative introns from exons of the AIC pre-mRNA of CD274, and the AIC pre-mRNA comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NOs: 71-76.

20. The method of any one of claims 1 to 15, wherein the target protein is Rho GTPase-activating protein 23 (ARHGAP23).

21. 16. The method of any one of claims 1-15, wherein the therapeutic agent binds to a targeted region of Rho GTPase-activating protein 23 (ARHGAP23) AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 77 and 91.

22. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of ARHGAP23, and the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

77.

23. The therapeutic agent selectively induces ARHGAP23 from the AIC pre-mRNA exon.

16. The method of any one of claims 1 to 15, wherein the method modulates splicing of an exon, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

91.

24. 24. The method of any one of claims 20 to 23, wherein the disease or condition is sclerocystic ovary syndrome or polycystic ovary syndrome.

25. The method of any one of claims 1 to 15, wherein the target protein is BRD1.

26. 16. The method of any one of claims 1 to 15, wherein the therapeutic agent binds to a targeted region of BRD1 AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 78 and 92.

27. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of BRD1, and the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

78.

28. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of BRD1, and the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

92.

29. 29. The method of any one of claims 25 to 28, wherein the disease or condition is schizophrenia or bipolar disorder or adenoid cystic carcinoma.

30. The method of any one of claims 1 to 15, wherein the target protein is protocadherin-16 (DCHS1).

31. 16. The method of any one of claims 1 to 15, wherein the therapeutic agent binds to a targeted region of DCHS1 AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 79 and 93.

32. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of DCHS1, and the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

79.

33. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of DCHS1, and the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

93.

34. 34. The method of any one of claims 30 to 33, wherein the disease or condition is Van Malderghem-Wetzburger-Verloes syndrome or mitral valve prolapse, myxomatous 2 or colorectal cancer or periventricular heterotopia, autosomal recessive or familial mitral valve prolapse.

35. 4. The method of claim 1, wherein the target protein is band 4.1-like protein 2 (EPB41L2).

16. The method according to any one of 1 to 15.

36. 16. The method of any one of claims 1 to 15, wherein the therapeutic agent binds to a targeted region of EPB41L2 AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 80 and 94.

37. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of EPB41L2, and the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

80.

38. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of EPB41L2, and the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

94.

39. 39. The method of any one of claims 35 to 38, wherein the disease or condition is cirrhosis of the liver.

40. The method according to any one of claims 1 to 15, wherein the target protein is glutathione peroxidase 8 (GPX8).

41. 16. The method of any one of claims 1 to 15, wherein the therapeutic agent binds to a targeted region of GPX8 AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 81 and 95.

42. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of GPX8, and the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

81.

43. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of GPX8, and the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

95.

44. 44. The method of any one of claims 40 to 43, wherein the disease or condition is cirrhosis of the liver.

45. The method according to any one of claims 1 to 15, wherein the target protein is human immunodeficiency virus type 1 enhancer-binding protein 3 (HIVEP3).

46. 16. The method of any one of claims 1 to 15, wherein the therapeutic agent binds to a targeted region of HIVEP3 AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 82 and 96.

47. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of HIVEP3, and the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

82.

48. The therapeutic agent selectively targets an exon of the AIC pre-mRNA of HIVEP3.

16. The method of any one of claims 1 to 15, wherein the method modulates splicing of an exon, wherein the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

96.

49. 49. The method of any one of claims 45 to 48, wherein the disease or condition is colorectal cancer.

50. The method of any one of claims 1 to 15, wherein the target protein is inversin (INVS).

51. 16. The method of any one of claims 1 to 15, wherein the therapeutic agent binds to a targeted region of INVS AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 83 and 97.

52. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of INVS, and the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

83.

53. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of INVS, and the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

97.

54. 54. The method of any one of claims 50 to 53, wherein the disease or condition is nephronophthisis 2 or cholestasis or infantile cholestasis or renal dysplasia and retinal dysplasia (disorders).

55. The method of any one of claims 1 to 15, wherein the target protein is the dyslexia-associated protein KIAA0319.

56. 16. The method of any one of claims 1 to 15, wherein the therapeutic agent binds to a targeted region of a dyslexia-associated protein KIAA0319 AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 84 and 98.

57. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of KIAA0319, and the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

84.

58. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of KIAA0319, and the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

98.

59. 59. The method of any one of claims 55 to 58, wherein the disease or condition is dyslexia or developmental dyslexia or dyslexia.

60. The method of any one of claims 1 to 15, wherein the target protein is NLR family inhibitor of apoptosis protein (NAIP).

61. 16. The method of any one of claims 1-15, wherein the therapeutic agent binds to a targeted region of the NAIP AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 85 and 99.

62. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of NAIP, and the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

85.

63. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of NAIP, and the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

99.

64. 64. The method of any one of claims 60 to 63, wherein the disease or condition is spinal muscular atrophy type II or spinal muscular atrophy, infantile chronic or hereditary motor neuropathy proximal type I or juvenile spinal muscular atrophy.

65. The method of any one of claims 1 to 15, wherein the target protein is protein Patched homolog 2 (PTCH2).

66. 16. The method of any one of claims 1-15, wherein the therapeutic agent binds to a targeted region of PTCH2 AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 86 and 100.

67. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of PTCH2, and the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

86.

68. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of PTCH2, and the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

100.

69. 69. The method of any one of claims 65 to 68, wherein the disease or condition is medulloblastoma or pigmented basal cell carcinoma or gastrointestinal stromal sarcoma or oculo-dental-digital syndrome or medullomyoblastoma or basal cell carcinoma or childhood medulloblastoma or macrostomia or desmoplastic medulloblastoma or hydrocephalus or adult medulloblastoma or melanotic medulloblastoma or basal cell nevus syndrome.

70. The method of any one of claims 1 to 15, wherein the target protein is protein tyrosine phosphatase receptor type Z1 (PTPRZ1).

71. 16. The method of any one of claims 1 to 15, wherein the therapeutic agent binds to a targeted region of PTPRZ1 AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 87 and 101.

72. The therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of PTPRZ1, and the alternative intron is at least 80% homologous to SEQ ID NO:

87.

16. The method of any one of claims 1 to 15, wherein the sequence comprises a sequence having 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity.

73. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of PTPRZ1, and the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

101.

74. 74. The method of any one of claims 70 to 73, wherein the disease or condition is schizophrenia or pneumoconiosis or bagasse disease.

75. The method of any one of claims 1 to 15, wherein the target protein is SON.

76. 16. The method of any one of claims 1 to 15, wherein the therapeutic agent binds to a targeted region of SON AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 88, 89, 102, and 103.

77. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of SON, and the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 88 or 89.

78. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of SON, and the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 102 or 103.

79. 79. The method of any one of claims 75 to 78, wherein the disease or condition is malignant neoplasm of the salivary glands or ZTTK syndrome or adenoid cystic carcinoma.

80. The method of any one of claims 1 to 15, wherein the target protein is zinc finger CCHC domain-containing protein 2 (ZCCHC2).

81. 16. The method of any one of claims 1 to 15, wherein the therapeutic agent binds to a targeted region of ZCCHC2 AIC pre-mRNA, and the targeted region is within a sequence selected from SEQ ID NOs: 90 and 104.

82. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from the AIC pre-mRNA of ZCCHC2, and the alternative intron comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

90.

83. 16. The method of any one of claims 1-15, wherein the therapeutic agent modulates splicing of an alternative intron from an exon of the AIC pre-mRNA of ZCCHC2, and the exon comprises a sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:

104.

84. 84. The method of any one of claims 80 to 83, wherein the disease or condition is influenza.

85. The method of any one of claims 2 to 19, wherein the disease or condition is an immune disease or disorder.

86. 86. The method of claim 85, wherein the immune disease or disorder is an autoimmune disease or disorder, an inflammatory disease or disorder, a chronic infection, graft-versus-host disease (GVHD), transplant rejection, or a T-cell proliferative disorder.

87. 87. The method of claim 86, wherein the immune disease or disorder is an autoimmune disease or disorder or an inflammatory disease or disorder selected from multiple sclerosis, inflammatory bowel disease, autoimmune hepatitis, kidney inflammation, rheumatoid arthritis, psoriasis, lupus nephritis, corneal transplant, and uveitis.

88. 88. The method of any one of claims 2 to 87, wherein the disease or condition is caused by a deficiency in the amount or activity of the target protein.

89. 88. The method of any one of claims 2 to 87, wherein the disease or condition is treated or prevented by increasing the amount or activity of the target protein.

90. 88. The method of any one of claims 2 to 87, wherein the disease or condition is induced by a loss-of-function mutation in the target protein.

91. 91. The method of any one of claims 1 to 90, wherein the therapeutic agent increases the level of processed mRNA encoding the target protein in the cell.

92. the level of processed mRNA encoding the target protein in the cells contacted with the therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, or about 2 to about 5-fold higher than the level of processed mRNA encoding the target protein in control cells; , about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold increase.

93. 93. The method of any one of claims 1 to 92, wherein the therapeutic agent increases the expression of the target protein in the cell.

94. The level of the target protein in the cells contacted with the therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold increase.

95. 3. The method of claim 1 or 2, wherein inclusion of the alternative intron from the AIC pre-mRNA encoding the target protein is reduced.

96. 3. The method of claim 1 or 2, wherein the therapeutic agent reduces the level of processed mRNA encoding the target protein in the cell.

97. The level of processed mRNA encoding the target protein in the cells contacted with the therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 97. The method of claim 96, wherein the antibody titer is reduced by 2 to about 5 fold, by about 2 to about 6 fold, by about 2 to about 7 fold, by about 2 to about 8 fold, by about 2 to about 9 fold, by about 3 to about 6 fold, by about 3 to about 7 fold, by about 3 to about 8 fold, by about 3 to about 9 fold, by about 4 to about 7 fold, by about 4 to about 8 fold, by about 4 to about 9 fold, by at least about 1.1 fold, by at least about 1.5 fold, by at least about 2 fold, by at least about 2.5 fold, by at least about 3 fold, by at least about 3.5 fold, by at least about 4 fold, by at least about 5 fold, or by at least about 10 fold.

98. The method of claim 1 or 2, wherein the therapeutic agent reduces the expression of the target protein in the cell.

99. the level of the target protein in the cells contacted with the therapeutic agent is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, 99. The method of claim 98, wherein the antibody titer is reduced by about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold.

100. 100. The method of any one of claims 6 or 8-99, wherein the therapeutic agent comprises a backbone modification comprising a phosphorothioate or phosphorodiamidate linkage.

101. 101. The method of any one of claims 6 or 8-100, wherein the therapeutic agent comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

102. 102. The method of any one of claims 6 or 8-101, wherein the therapeutic agent comprises at least one modified sugar moiety.

103. 103. The method of any one of claims 6 or 8-102, wherein each sugar moiety is a modified sugar moiety.

104. The therapeutic agent is selected from the group consisting of 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases 10 to 20 nucleic acid bases, 10 to 15 nucleic acid bases, 11 to 50 nucleic acid bases, 11 to 40 nucleic acid bases, 11 to 35 nucleic acid bases, 11 to 30 nucleic acid bases, 11 to 25 nucleic acid bases, 11 to 20 nucleic acid bases, 11 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, 12 to 30 nucleic acid bases, 12 to 25 nucleic acid bases, 12 to 20 nucleic acid bases, or 12 to 15 nucleic acid bases.

104. The method according to any one of claims 1 to 103.

105. 105. The method of any one of claims 1 to 104, further comprising assessing the mRNA level or expression level of the target protein.

106. The method of any one of claims 2 to 105, wherein the subject is a human.

107. The method of any one of claims 2 to 105, wherein the subject is a non-human animal.

108. 108. The method of any one of claims 2 to 107, wherein the subject is a fetus, embryo, or child.

109. 109. The method of any one of claims 1 to 108, wherein the one or more cells are ex vivo or in a tissue or organ ex vivo.

110. 109. The method of any one of claims 2-108, wherein the therapeutic agent is administered to the subject by intraventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, implantation, or intravenous injection.

111. 111. The method of any one of claims 1 to 110, wherein splicing of the alternative intron from the AIC pre-mRNA encoding the target protein or the target RNA produces a processed mRNA with a premature termination codon (PTC).

112. 112. The method of any one of claims 1 to 111, wherein splicing of the alternative intron from the AIC pre-mRNA encoding the target protein or target RNA produces a processed mRNA with a premature termination codon (PTC) that does not encode a functional target protein or that does not encode a functional RNA.

113. 113. The method of any one of claims 1 to 112, wherein splicing of the alternative intron from the AIC pre-mRNA encoding the target protein or target RNA produces a processed mRNA with a premature termination codon (PTC) that encodes a non-functional target protein or target RNA.

114. 114. The method of any one of claims 1 to 113, wherein splicing of the alternative intron from the AIC pre-mRNA encoding the target protein or target RNA produces a processed mRNA that has a lower translation or expression efficiency for producing the target protein compared to a corresponding processed mRNA that contains the alternative intron but is otherwise identical.

115. 115. The method of any one of claims 1 to 114, wherein splicing of the alternative intron from the AIC pre-mRNA encoding the target protein or the target RNA produces a processed mRNA that undergoes nonsense-mediated decay (NMD).

116. 116. The method of any one of claims 1-115, wherein splicing of the alternative intron from the AIC pre-mRNA encoding the target protein or the target RNA produces a processed mRNA that undergoes nonsense-mediated decay (NMD) in greater amounts than a corresponding processed mRNA that is otherwise identical but contains the alternative intron.

117. A therapeutic agent for use in the method of any one of claims 1 to 116.

118. 118. A pharmaceutical composition comprising the therapeutic agent of claim 117 and a pharmaceutically acceptable excipient.

119. 119. A method of treating a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 118 by intraventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, implantation, or intravenous injection.

120. 1. A composition comprising a therapeutic agent for use in a method of modulating expression of a target protein or target RNA by a cell to treat a disease or condition in a subject in need thereof, the disease or condition being associated with an aberrant protein or an aberrant RNA, wherein the aberrant protein or the aberrant RNA is abnormal in amount or activity in the subject, and the therapeutic agent modulates splicing of an alternative intron-containing pre-mRNA (AIC pre-mRNA) encoding the target protein or the target RNA; The target protein is: (a) the abnormal protein; (b) a protein that functionally activates or deactivates a cell signaling mechanism, thereby altering cellular activity associated with said disease or condition; (c) a protein that functionally enhances or replaces the abnormal protein in the subject; or (d) a protein that functionally reduces or inhibits the abnormal protein in the subject; The target RNA is: (a) the abnormal RNA; (b) an RNA that functionally activates or deactivates a cell signaling mechanism, thereby altering cellular activity associated with said disease or condition; (c) an RNA that functionally enhances or replaces the abnormal RNA in the subject; or (d) an RNA that functionally reduces or inhibits said abnormal RNA in said subject; The composition, wherein the AIC pre-mRNA comprises an alternative intron, a first portion of the exon adjacent to the 5' splice site of the alternative intron, and a second portion of the exon adjacent to the 3' splice site of the alternative intron, thereby regulating splicing of the alternative intron from the AIC pre-mRNA encoding the target protein or target RNA, thereby regulating the production or activity of the target protein or target RNA in the subject.

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