Antisense oligomers for the treatment of nonsense-mediated RNA decay-based pathologies and diseases

Abstract

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

Description

[Technical field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 212,981, filed June 21, 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002]

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

[0003] PHIP-related disorder, also known as Chung Jansen syndrome (CHUJANS), is characterized by global developmental delay evident from infancy, intellectual developmental disability or learning difficulties, behavioral abnormalities, dysmorphic features, and obesity. The severity of its phenotype and additional features varies. CHUJANS is a rare condition caused by alterations in the pleckstrin homology domain interacting protein (PHIP) gene. This gene plays an important role in several processes related to the development of the brain and nervous system. The PHIP gene is also involved in regulating insulin in nervous tissue. To date, PHIP-related disorder is not known to cause diabetes, but people with this disorder are at increased risk of becoming overweight, a risk factor for diabetes. The most common signs and symptoms include mild to severe learning problems, behavioral problems, and a tendency to become overweight. PHIP-related disorder is an autosomal dominant condition. Many people with PHIP-related disorder have mild to severe intellectual disabilities. People with PHIP-related disorders who do not have intellectual disability often have speech problems, global developmental delays in early childhood, and learning problems. Currently, there are no approved disease-modifying treatments for patients with PHIP-related disorders, and a need exists for such treatments. Summary of the Invention

[0004]

[0004] Provided herein is a method for regulating expression of a target protein in a cell having a pre-mRNA transcribed from a target gene and including a nonsense-mediated RNA decay-inducing exon (NMD exon), comprising contacting the cell with an agent or a vector encoding the agent, whereby the agent regulates splicing of the NMD exon from the pre-mRNA, thereby regulating the level of processed mRNA processed from the pre-mRNA and regulating expression of the target protein in the cell, wherein the target gene is the PHIP gene.

[0005]

[0005] In some aspects, the agent (a) binds to a targeted portion of a pre-mRNA, (b) modulates the binding of a factor involved in splicing of an NMD exon, or a combination of (a) and (b).

[0006]

[0006] In some aspects, the agent interferes with the binding of a factor involved in splicing of the NMD exon to the region of the targeting moiety.

[0007]

[0007] In some embodiments, the targeting portion of the pre-mRNA is proximal to an NMD exon.

[0008]

[0008] In some embodiments, the targeting portion of the pre-mRNA is located up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of the 5' end of the NMD exon.

[0009]

[0009] In some embodiments, the targeting portion of the pre-mRNA is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, or about 1 nucleotide upstream of the 5' end of the NMD exon.

[0010]

[0010] In some embodiments, the targeting portion of the pre-mRNA is up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of the 3' end of the NMD exon.

[0011]

[0011] In some embodiments, the targeting portion of the pre-mRNA is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, or about 1 nucleotide downstream of the 3' end of the NMD exon.

[0012]

[0012] In some aspects, the targeting portion of the pre-mRNA is up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of the genomic site GRCh38 / hg38:chr6 79004373.

[0013]

[0013] In some embodiments, the targeting portion of the pre-mRNA is located about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of genomic site GRCh38 / hg38:chr6 79004373.

[0014]

[0014] In some aspects, the targeted portion of the pre-mRNA is up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of the genomic site GRCh38 / hg38:chr6 79004436.

[0015]

[0015] In some embodiments, the targeting portion of the pre-mRNA is located about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of genomic site GRCh38 / hg38:chr6 79004436.

[0016]

[0016] In some embodiments, the targeting portion of the pre-mRNA is located in an intron region between two canonical exon regions of the pre-mRNA, the intron region containing the NMD exon.

[0017]

[0017] In some embodiments, the targeted portion of the pre-mRNA at least partially overlaps with the NMD exon.

[0018]

[0018] In some embodiments, the targeting portion of the pre-mRNA at least partially overlaps with an intron upstream or downstream of the NMD exon.

[0019]

[0019] In some embodiments, the targeting portion of the pre-mRNA comprises a 5' NMD exon-intron junction or a 3' NMD exon-intron junction.

[0020]

[0020] In some embodiments, the targeting portion of the pre-mRNA is within an NMD exon.

[0021]

[0021] In some aspects, the targeting portion of the pre-mRNA includes about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides of an NMD exon.

[0022]

[0022] In some aspects, the NMD exon (a) is located within an intronic sequence having at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO:2, and / or (b) contains a sequence having at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO:3.

[0023]

[0023] In some embodiments, the NMD exon comprises the sequence of SEQ ID NO:3.

[0024]

[0024] In some embodiments, the targeted portion of the pre-mRNA is within the nonsense-mediated RNA decay-inducing exon GRCh38 / hg38:chr6 79004373-79004436.

[0025]

[0025] In some aspects, the targeted portion of the pre-mRNA is upstream or downstream of the nonsense-mediated RNA decay-inducing exon GRCh38 / hg38:chr6 79004373-79004436.

[0026]

[0026] In some embodiments, the targeting portion of the pre-mRNA comprises the exon-intron junction of the nonsense-mediated RNA decay-induced exon GRCh38 / hg38:chr6 79004373-79004436.

[0027] In some aspects, the target protein expressed from the processed mRNA is a full-length or wild-type PHIP protein.

[0028] In some aspects, the target protein expressed from the processed mRNA is a functional PHIP protein.

[0029] In some embodiments, the target protein expressed from the processed mRNA is at least partially functional compared to the wild-type PHIP protein.

[0030] In some aspects, the target protein expressed from the processed mRNA is at least partially functional compared to the full-length wild-type PHIP protein.

[0031]

[0031] In some embodiments, the target protein expressed from the processed mRNA is a PHIP protein lacking the amino acid sequence encoded by the nonsense-mediated RNA decay-inducing exon GRCh38 / hg38:chr6 79004373-79004436.

[0032]

[0032] In some aspects, the methods promote the exclusion of an NMD exon from a pre-mRNA.

[0033]

[0033] In some embodiments, the exclusion of NMD exons from pre-mRNA in cells contacted with the agent is about 1.1 to about 10 fold, about 1.5 to about 10 fold, about 2 to about 10 fold, about 3 to about 10 fold, about 4 to about 10 fold, about 1.1 to about 5 fold, about 1.1 to about 6 fold, about 1.1 to about 7 fold, about 1.1 to about 8 fold, about 1.1 to about 9 fold, about 2 to about 5 fold, about 2 to about 6 fold, fold, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0034]

[0034] In some aspects, the methods result in increased levels of processed mRNA in the cell.

[0035]

[0035] In some embodiments, the level of processed mRNA in a cell contacted with an 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, The increase is 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.

[0036]

[0036] In some aspects, the methods result in increased expression of a target protein in a cell.

[0037]

[0037] In some embodiments, the level of the target protein expressed from the processed mRNA in the cells contacted with the drug is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, The increase is 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.

[0038]

[0038] In some aspects, the agent comprises an antisense oligomer having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 4-180.

[0039]

[0039] In some aspects, the agent further comprises a gene editing molecule.

[0040]

[0040] In some embodiments, the gene editing molecule includes CRISPR-Cas9.

[0041] In some aspects, the agent is an antisense oligomer (ASO), and the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

[0042]

[0042] In some aspects, the agent is an antisense oligomer (ASO), and the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-NMA moiety, or a 2'-O-methoxyethyl moiety.

[0043]

[0043] In some aspects, the agent is an antisense oligomer (ASO), and the antisense oligomer comprises at least one modified sugar moiety.

[0044]

[0044] In some aspects, each sugar moiety is a modified sugar moiety.

[0045] In some embodiments, the agent is an antisense oligomer (ASO), and the antisense oligomer is selected from the group consisting of 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 nucleobases, 8-15 nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 nucleobases, 10-50 nucleobases, 10-40 nucleobases, The nucleic acid comprises 10-35 nucleic acid bases, 10-30 nucleic acid bases, 10-25 nucleic acid bases, 10-20 nucleic acid bases, 10-15 nucleic acid bases, 11-50 nucleic acid bases, 11-40 nucleic acid bases, 11-35 nucleic acid bases, 11-30 nucleic acid bases, 11-25 nucleic acid bases, 11-20 nucleic acid bases, 11-15 nucleic acid bases, 12-50 nucleic acid bases, 12-40 nucleic acid bases, 12-35 nucleic acid bases, 12-30 nucleic acid bases, 12-25 nucleic acid bases, 12-20 nucleic acid bases, or 12-15 nucleic acid bases.

[0046]

[0046] In some aspects, the method includes contacting the cell with a vector encoding the agent, wherein the vector is a viral vector.

[0047]

[0047] In some aspects, the viral vector comprises an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, a herpes simplex viral (HSV) viral vector, or a retroviral vector.

[0048]

[0048] In some aspects, the viral vector comprises an adeno-associated viral (AAV) vector.

[0049]

[0049] In some aspects, the agent comprises a modified snRNA.

[0050]

[0050] In some aspects, the modified human snRNA is a modified U1 snRNA.

[0051]

[0051] In some aspects, the modified human snRNA is a modified U7 snRNA.

[0052]

[0052] In some embodiments, a portion of the single-stranded nucleotide sequence of the modified human snRNA comprises a sequence that binds to a targeting portion of a pre-mRNA.

[0053]

[0053] In some aspects, the method includes contacting a cell with a vector encoding the modified snRNA.

[0054]

[0054] In some aspects, the method further comprises assessing the mRNA level or expression level of the target protein.

[0055] In some aspects, the agent is a therapeutic agent.

[0056]

[0056] Also provided herein is a pharmaceutical composition comprising a therapeutic agent described herein or a vector encoding a therapeutic agent described herein and a pharma- ceutically acceptable excipient.

[0057]

[0057] Also provided herein is a pharmaceutical composition comprising a therapeutic agent or a vector encoding a therapeutic agent and a pharma- ceutically acceptable excipient, wherein the therapeutic agent comprises an antisense oligomer having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 4-180.

[0058]

[0058] In some aspects, the therapeutic agent comprises an antisense oligomer having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs:55-180.

[0059]

[0059] In some aspects, the pharmaceutical composition is formulated for intraventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, implantation, or intravenous injection.

[0060]

[0060] In some embodiments, the pharmaceutical composition is formulated for intrathecal or intracerebrospinal injection.

[0061] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent.

[0062] In some embodiments, the second therapeutic agent comprises a small molecule.

[0063] In some embodiments, the second therapeutic agent comprises an antisense oligomer.

[0064] In some aspects, the second therapeutic agent corrects intron retention.

[0065]

[0065] Also provided herein is a composition comprising an antisense oligomer having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 4-180, wherein the antisense oligomer comprises a backbone modification, a sugar moiety modification, or a combination thereof.

[0066]

[0066] Also provided in the present specification is a composition comprising a viral vector encoding a polynucleotide comprising an antisense oligomer, wherein the antisense oligomer consists of an sequence selected from the group consisting of SEQ ID NOs: 4-180.

[0067]

[0067] In some aspects, the polynucleotide further comprises a modified snRNA.

[0068]

[0068] In some embodiments, the modified human snRNA is a modified U1 snRNA.

[0069]

[0069] In some aspects, the modified human snRNA is a modified U7 snRNA.

[0070]

[0070] In some aspects, the antisense oligomer has at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 55-180.

[0071]

[0071] Also provided herein is a method for treating a disease or condition in a subject or reducing the likelihood of developing a disease or condition by regulating expression of a target protein in cells of a subject in need thereof, the method comprising contacting cells of the subject with a pharmaceutical composition described herein.

[0072] In some aspects, the disease or condition is associated with a loss-of-function mutation in the PHIP gene.

[0073]

[0073] In some aspects, the disease or condition is associated with haploinsufficiency of the PHIP gene, and the subject has a first allele that encodes a functional PHIP protein and a second allele in which PHIP protein is not produced or is produced at reduced levels, or a second allele that encodes a non-functional or partially functional PHIP protein.

[0074]

[0074] In some aspects, the disease or condition comprises an intellectual disability disease or condition.

[0075]

[0075] In some aspects, the disease or condition includes Chung-Jansen syndrome (CHUJANS), autosomal dominant disorder, intellectual disability, speech delay, anxiety, autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), aggression, facial dysmorphism, cafe au lait spots, overweight syndrome caused by PHIP haploinsufficiency, developmental delay, obesity, or dysmorphism.

[0076]

[0076] In some aspects, the disease or condition includes Chung-Jansen syndrome.

[0077]

[0077] In some aspects, the disease or condition comprises intellectual disability.

[0078]

[0078] In some aspects, the disease or condition is associated with an autosomal recessive mutation in the PHIP gene and the subject has (i) a first allele such that PHIP protein is not produced or is produced at reduced levels compared to the wild-type allele, or (ii) the produced PHIP protein is non-functional or partially functional compared to the wild-type allele, and (iii) a second allele such that PHIP protein is produced at reduced levels compared to the wild-type allele and the produced PHIP protein is at least partially functional compared to the wild-type allele, or (iv) the produced PHIP protein is partially functional compared to the wild-type allele.

[0079]

[0079] In some embodiments, the subject is a human.

[0080]

[0080] In some embodiments, the subject is a non-human animal.

[0081]

[0081] In some aspects, the subject is a fetus, embryo, or child.

[0082]

[0082] In some aspects, the cells are ex vivo.

[0083]

[0083] In some aspects, the pharmaceutical composition is administered 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.

[0084]

[0084] In some embodiments, the pharmaceutical composition is administered by intrathecal or intracerebrospinal injection.

[0085]

[0085] In some aspects, the method treats a disease or condition.

[0086]

[0086] Also provided herein is a composition comprising an agent or a vector encoding the agent that regulates splicing of a nonsense-mediated RNA decay-inducing exon (NMD exon) from a pre-mRNA that is transcribed from a target gene and contains the NMD exon, thereby regulating the level of processed mRNA processed from the pre-mRNA, and regulating expression of a target protein in a cell having the pre-mRNA, wherein the target gene is the PHIP gene.

[0087]

[0087] Also provided herein is a composition comprising an agent or a vector encoding the agent that treats a disease or condition in a subject by regulating splicing of a nonsense-mediated RNA decay-inducing exon (NMD exon) from a pre-mRNA that is transcribed from a target gene and contains the NMD exon, thereby regulating the level of processed mRNA processed from the pre-mRNA and regulating expression of a target protein in cells of a subject in need of treatment for the disease or condition, wherein the target gene is the PHIP gene.

[0088]

[0088] Also provided herein are pharmaceutical compositions comprising a composition described herein and a pharma- ceutically acceptable excipient and / or delivery vehicle.

[0089] In some aspects, the target protein is pleckstrin homology domain interacting protein (PHIP). Reference

[0090] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief description of the drawings]

[0090] [Figure 1]

[0092] 1A-1B are schematic diagrams of target mRNAs containing nonsense-mediated mRNA decay-inducing exons (NMD exon mRNAs) and therapeutic-mediated elimination of nonsense-mediated mRNA decay-inducing exons, which increase expression of full-length target proteins or functional RNAs. FIG. 1A shows a cell separated into nuclear and cytoplasmic compartments. In the nucleus, the pre-mRNA transcript of the target gene undergoes splicing to generate mRNA, which is transported to the cytoplasm and translated into the target protein. For this target gene, a certain fraction of the mRNA contains the nonsense-mediated mRNA decay-inducing exon that is degraded in the cytoplasm (NMD exon-containing mature mRNA), and thus does not result in target protein production. FIG. 1B shows an example of the same cell separated into nuclear and cytoplasmic compartments. Treatment with a therapeutic agent, e.g., an antisense oligomer (ASO), promotes the elimination of the nonsense-mediated mRNA decay-inducing exon, resulting in an increase in productive mRNA, which is then translated into higher levels of the target protein. [Diagram 2]

[0093] FIG. 2 shows an exemplary schematic of a novel nonsense-mediated decay (NMD) exon inclusion event (exon X) identified in the PHIP gene that results in a nonproductive mRNA transcript that is degraded by NMD. [Diagram 3]

[0094] Figure 3 shows a UCSC genome browser snapshot of a region in the PHIP gene containing an NMD-induced exon inclusion event (GRCh38 / hg38:chr6 79004373-79004436) indicated by the shaded area and black bar at the top (exons are rectangles, introns are lines with arrowheads). RNA sequencing was derived from human neural progenitor cells (ReN) and human astrocytes treated with cycloheximide (CHX) or DMSO control. [Figure 4A]

[0095] Figure 4A shows confirmation of NMD-induced exons via puromycin or cycloheximide treatment in HEK293, SK-N-AS, and ReN VM cell lines. RT-PCR analysis using total RNA from water-treated (H), DMSO-treated (D), puromycin-treated (P), or cycloheximide-treated (C) cells confirmed the presence of a band corresponding to the NMD-induced exon 15x (GRCh38 / hg38:chr6 79004373-79004436) of the PHIP gene. [Figure 4B]

[0096] Figure 4B shows confirmation of NMD-induced exons via puromycin or cycloheximide treatment in U87 cell lines and astrocytes. RT-PCR analysis using total RNA from water-treated (H), DMSO-treated (D), puromycin-treated (P), or cycloheximide-treated (C) cells confirmed the presence of a band corresponding to the NMD-induced exon 15x (GRCh38 / hg38:chr6 79004373-79004436) of the PHIP gene. [Figure 4C]

[0097] Figure 4C shows confirmation of NMD-induced exons via puromycin or cycloheximide treatment in mouse embryonic fibroblasts. RT-PCR analysis using total RNA from water-treated (H), DMSO-treated (D), puromycin-treated (P), or cycloheximide-treated (C) cells confirmed the presence of a band corresponding to the NMD-induced exon 15x (GRCh38 / hg38:chr6 79004373-79004436) of the PHIP gene. [Diagram 5]

[0098] FIG. 5 shows the conservation of the NMD exonic event (15X) in non-human primates. [Figure 6]

[0099] Figure 6 shows an exemplary ASO walk around the PHIP exon 15x (GRCh38 / hg38:chr6 79004373-79004436) region. A graphical representation of ASO walks performed around the PHIP exon 15x (GRCh38 / hg38:chr6 79004373-79004436) region targeting sequences upstream of the 3' splice site, across the 3' splice site, exon 15x, across the 5' splice site, and downstream of the 5' splice site. ASOs were designed to cover these regions by moving 5 nucleotides at a time, or 3 nucleotides between the start or end of the exon and adjacent ASOs across the splice site region. [Figure 7]

[0100] Figure 7 shows PHIP exon 15x (GRCh38 / hg38:chr6 79004373 79004436) region ASO walking assessed by Taqman RT-qPCR (for productive mRNA fold change) and RT-PCR (for non-productive mRNA fold change) in ReN VM cells nucleofected with 3 μM of the indicated ASO in the presence of cycloheximide. Gel images and graphs of fold change of PHIP productive mRNA product compared to the control gene are plotted. [Figure 8]

[0101] Figure 8 shows PHIP exon 15x (GRCh38 / hg38:chr6 79004373 79004436) region ASO walking assessed by Taqman RT-qPCR in ReN cells nucleofected with 3 μM of the applicable ASO in the absence of cycloheximide. Gel images and graphs of % PHIP non-productive mRNA product, fold change of canonical PHIP mRNA, and fold change of total PHIP mRNA compared to the control gene are plotted. [Figure 9]

[0102] Figure 9 shows PHIP exon 15x (GRCh38 / hg38:chr6 79004373 79004436) region ASO walking assessed by Taqman RT-qPCR in HEK293 cells transfected with 120 nM of the applicable ASO in the presence of cycloheximide. Gel images and graphs of fold change of PHIP productive mRNA product compared to the control gene are plotted. [Figure 10A]

[0103] Figures 10A-10D show PHIP exon 15x (GRCh38 / hg38:chr6 79004373 79004436) region ASO walking assessed by Taqman RT-qPCR in HEK293 cells transfected with 120 nM of the applicable ASO in the absence of cycloheximide. Gel images (Figures 10A-10C) and graphs of % PHIP non-productive mRNA product, fold change of canonical PHIP mRNA, and fold change of total PHIP mRNA relative to the control gene (Figure 10D) are plotted. [Figure 10B] Figures 10A-10D show PHIP exon 15x (GRCh38 / hg38:chr6 79004373 79004436) region ASO walking assessed by Taqman RT-qPCR in HEK293 cells transfected with 120 nM of the applicable ASO in the absence of cycloheximide. Gel images (Figures 10A-10C) and graphs of % PHIP non-productive mRNA product, fold change of canonical PHIP mRNA, and fold change of total PHIP mRNA relative to the control gene (Figure 10D) are plotted. [Figure 10C] Figures 10A-10D show PHIP exon 15x (GRCh38 / hg38:chr6 79004373 79004436) region ASO walking assessed by Taqman RT-qPCR in HEK293 cells transfected with 120 nM of the applicable ASO in the absence of cycloheximide. Gel images (Figures 10A-10C) and graphs of % PHIP non-productive mRNA product, fold change of canonical PHIP mRNA, and fold change of total PHIP mRNA relative to the control gene (Figure 10D) are plotted. [Figure 10D] Figures 10A-10D show PHIP exon 15x (GRCh38 / hg38:chr6 79004373 79004436) region ASO walking assessed by Taqman RT-qPCR in HEK293 cells transfected with 120 nM of the applicable ASO in the absence of cycloheximide. Gel images (Figures 10A-10C) and graphs of % PHIP non-productive mRNA product, fold change of canonical PHIP mRNA, and fold change of total PHIP mRNA relative to the control gene (Figure 10D) are plotted. [Figure 11]

[0104] Figure 11 shows an exemplary ASO microwalk around the PHIP exon 15x (GRCh38 / hg38:chr6 79004373 to 79004436) region. [Figure 12]

[0105] Figure 12 shows PHIP exon 15x (GRCh38 / hg38:chr6 79004373-79004436) region ASO microwalking assessed by Taqman RT-qPCR in ReN cells nucleofected with 3 μM of the applicable ASO in the presence of cycloheximide. Gel images and graphs of fold change of PHIP productive mRNA product compared to the control gene are plotted. [Figure 13]

[0106] Figure 13 shows PHIP exon 15x (GRCh38 / hg38:chr6 79004373-79004436) region ASO microwalking assessed by Taqman RT-qPCR in HEK293 cells transfected with 120 nM of the applicable ASO in the presence of cycloheximide. Gel images and graphs of fold change of PHIP productive mRNA product compared to the control gene are plotted. [Figure 14]

[0107] Figure 14A shows gel images demonstrating modulation of exon 15x splicing from the PHIP pre-mRNA by different concentrations of an exemplary ASO, and Figure 14B is a bar graph summarizing quantification of the fold change in canonical PHIP mRNA abundance and the change in non-productive PHIP mRNA abundance in response to treatment with different concentrations of an exemplary ASO. [Figure 15]

[0108] Figures 15A and 15B demonstrate that exemplary ASOs (IVS14X:-3, IVS14X-Ex15XX:7, Ex15X:19, and Ex15X:23) caused an increase in PHIP protein levels. Figure 15A shows Western blotting images of PHIP protein under different treatment conditions, and Figure 15B is a bar graph summarizing the quantification of changes in PHIP protein levels in response to different treatments. [Figure 16A]

[0109] Figures 16A-16D demonstrate that treatment with an exemplary ASO via nucleofection resulted in a decrease in the amount of non-productive PHIP transcripts with exon 15x (Figure 16B) and an increase in the amount of PHIP transcripts without exon 15x as shown by Taqman qPCR using probes spanning exons 15 and 16 (canonical; Figure 16C) and exons 35 and 36 (GE; Figure 16D). Figure 16A shows gel images of RT-PCR amplification products under different treatment conditions. [Figure 16B] Figures 16A-16D demonstrate that treatment with an exemplary ASO via nucleofection resulted in a decrease in the amount of non-productive PHIP transcripts with exon 15x (Figure 16B) and an increase in the amount of PHIP transcripts without exon 15x as shown by Taqman qPCR using probes spanning exons 15 and 16 (canonical; Figure 16C) and exons 35 and 36 (GE; Figure 16D). Figure 16A shows gel images of RT-PCR amplification products under different treatment conditions. [Figure 16C]Figures 16A-16D demonstrate that treatment with an exemplary ASO via nucleofection resulted in a decrease in the amount of non-productive PHIP transcripts with exon 15x (Figure 16B) and an increase in the amount of PHIP transcripts without exon 15x as shown by Taqman qPCR using probes spanning exons 15 and 16 (canonical; Figure 16C) and exons 35 and 36 (GE; Figure 16D). Figure 16A shows gel images of RT-PCR amplification products under different treatment conditions. [Figure 16D] Figures 16A-16D demonstrate that treatment with an exemplary ASO via nucleofection resulted in a decrease in the amount of non-productive PHIP transcripts with exon 15x (Figure 16B) and an increase in the amount of PHIP transcripts without exon 15x as shown by Taqman qPCR using probes spanning exons 15 and 16 (canonical; Figure 16C) and exons 35 and 36 (GE; Figure 16D). Figure 16A shows gel images of RT-PCR amplification products under different treatment conditions. [Figure 17A]

[0110] Figures 17A-17D demonstrate that treatment with an exemplary ASO via free incorporation resulted in a decrease in the amount of non-productive PHIP transcripts with exon 15x (Figure 17B) and an increase in the amount of PHIP transcripts without exon 15x as shown by Taqman qPCR using probes spanning exons 15 and 16 (canonical; Figure 17C) and exons 35 and 36 (GE; Figure 17D). Figure 17A shows gel images of RT-PCR amplification products under different treatment conditions. [Figure 17B]Figures 17A-17D demonstrate that treatment with an exemplary ASO via free incorporation resulted in a decrease in the amount of non-productive PHIP transcripts with exon 15x (Figure 17B) and an increase in the amount of PHIP transcripts without exon 15x as shown by Taqman qPCR using probes spanning exons 15 and 16 (canonical; Figure 17C) and exons 35 and 36 (GE; Figure 17D). Figure 17A shows gel images of RT-PCR amplification products under different treatment conditions. [Figure 17C] Figures 17A-17D demonstrate that treatment with an exemplary ASO via free incorporation resulted in a decrease in the amount of non-productive PHIP transcripts with exon 15x (Figure 17B) and an increase in the amount of PHIP transcripts without exon 15x as shown by Taqman qPCR using probes spanning exons 15 and 16 (canonical; Figure 17C) and exons 35 and 36 (GE; Figure 17D). Figure 17A shows gel images of RT-PCR amplification products under different treatment conditions. [Figure 17D] Figures 17A-17D demonstrate that treatment with an exemplary ASO via free incorporation resulted in a decrease in the amount of non-productive PHIP transcripts with exon 15x (Figure 17B) and an increase in the amount of PHIP transcripts without exon 15x as shown by Taqman qPCR using probes spanning exons 15 and 16 (canonical; Figure 17C) and exons 35 and 36 (GE; Figure 17D). Figure 17A shows gel images of RT-PCR amplification products under different treatment conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0091]

[0111] Alternative splicing events in the PHIP (pleckstrin homology interacting protein) gene can result in non-productive mRNA transcripts that can result in aberrant or reduced protein expression, and therapeutic agents that can target alternative splicing events in the PHIP gene can regulate the expression levels of functional proteins and / or inhibit aberrant protein expression in Chung Jansen syndrome (CHUJANS) patients. Such therapeutic agents can be used to treat pathologies caused by PHIP protein deficiency.

[0092]

[0112] One alternative splicing event that may result in a non-productive mRNA transcript is the inclusion of an additional exon in the mRNA transcript, which may induce nonsense-mediated mRNA decay. The present disclosure provides compositions and methods for modulating alternative splicing of PHIP to increase the production of mature mRNA encoding a protein, and thus the translated functional PHIP protein. These compositions and methods include antisense oligomers (ASOs) that can cause exon skipping, e.g., pseudoexon skipping, and promote constitutive splicing of the PHIP pre-mRNA. In various embodiments, functional PHIP protein can be increased using the methods of the present disclosure to treat pathologies caused by PHIP protein deficiency. mRNA splicing

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

[0093]

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

[0094]

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

[0095]

[0116] Sequences marking exon-intron boundaries are degenerate signals of various strengths that can occur frequently within human genes. In multi-exon genes, different pairs of splice sites can be linked together in many different combinations to generate a wide variety of transcripts from a single gene. This is commonly referred to as alternative pre-mRNA splicing. Although most mRNA isoforms produced by alternative splicing can be exported from the nucleus and translated into functional polypeptides, different mRNA isoforms derived from a single gene can vary greatly in their translation efficiency. Those mRNA isoforms that have a premature stop codon (PTC) at least 50 bp upstream of the exon junction complex are likely to be targeted for degradation by the nonsense-mediated mRNA decay (NMD) pathway. Mutations in traditional (BPS / PPT / 3'ss / 5'ss) and auxiliary splicing motifs can lead to aberrant splicing, e.g., exon skipping, or cryptic (or spurious) exon inclusion or splice site activation, and can be a significant contributor to human morbidity and mortality. Both aberrant and alternative splicing patterns can be influenced by naturally occurring DNA variants in exons and introns.

[0096]

[0117] Considering that exon-intron boundaries can occur at any of three codon positions, it is clear that only a subset of alternative splicing events can maintain the canonical open reading frame. For example, only exons divisible by 3 can be skipped or included in the mRNA without any change in the reading frame. Splicing events that are not in phase with compatibility can induce frameshifts. Unless reversed by downstream events, frameshifts will certainly result in one or more PTCs, possibly resulting in their subsequent degradation by NMD. NMD is a translation-coupled mechanism that excludes mRNAs that contain PTCs. NMD can function as a surveillance pathway that exists in all eukaryotes. NMD can reduce failures in gene expression by eliminating mRNA transcripts that contain premature stop codons. Translation of these aberrant mRNAs can potentially result in deleterious gain-of-function or dominant-negative activity of the resulting protein. NMD targets not only transcripts with PTCs but also a broad range of mRNA isoforms expressed from many endogenous genes, suggesting that NMD is a master regulator driving both fine and coarse regulation of steady-state RNA levels in cells.

[0097]

[0118] An NMD-inducing exon ("NIE" or "NMD exon") is an exon, or a pseudoexon, that is a region within an intron, that can activate the NMD pathway when included in a mature RNA transcript. In a constitutive splicing event, the intron containing the NMD exon is typically excised, but during an alternative or aberrant splicing event, the intron or a portion thereof (e.g., the NMD exon) may be retained. A mature mRNA transcript containing such an NMD exon may be non-productive due to a frameshift that induces the NMD pathway. Inclusion of the NMD exon in a mature RNA transcript may downregulate gene expression. An mRNA transcript containing an NMD exon may be referred to in the present disclosure as an "NIE-containing mRNA" or an "NMD exon mRNA."

[0098]

[0119] Cryptic (or false splice sites) have the same splicing recognition sequence as true splice sites, but are not used in the splicing reaction. They are 10 times more abundant than true splice sites in the human genome and are usually suppressed by molecular mechanisms that are not yet fully understood. Cryptic 5' splice sites have the consensus NNN / GUNNNN or NNN / GCNNNN, where N is any nucleotide and / N is the exon-intron boundary. Cryptic 3' splice sites have the consensus NAG / N. Their activation is positively influenced by the surrounding nucleotides that make them more similar to the optimal consensus of the original splice sites, i.e., MAG / GURAGU and YAG / G, respectively, where M is C or A, R is G or A, and Y is C or U.

[0099]

[0120] Splice sites and their regulatory sequences can be readily identified by the skilled artisan using suitable publicly available algorithms, e.g. as 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.

[0100]

[0121] A cryptic splice site or splicing control sequence may compete with a splice site of an NMD exon for an RNA binding protein such as U2AF, in some embodiments, an agent may bind to a cryptic splice site or splicing control sequence and prevent binding of the RNA binding protein, thereby favoring binding of the RNA binding protein to the NMD exon splice site.

[0101]

[0122] In some embodiments, the cryptic splice site may not include the 5' or 3' splice site of the NMD exon. In some embodiments, the cryptic splice site may be at least 10 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, or at least 200 nucleotides upstream of the NMD exon 5' splice site. In some embodiments, the cryptic splice site may be at least 10 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, or at least 200 nucleotides downstream of the NMD exon 3' splice site. Target transcript

[0123] In some embodiments, the disclosed methods and compositions take advantage of the presence of an NMD exon in the pre-mRNA transcribed from the PHIP gene. Splicing of the identified PHIP NMD exon pre-mRNA species to produce a functional mature PHIP mRNA can be induced using an agent such as an ASO that stimulates exon skipping of the NMD exon. Induction of exon skipping can result in inhibition of the NMD pathway. The resulting mature PHIP mRNA is normally translated without activating the NMD pathway, thereby increasing the amount of PHIP protein in the patient's cells and reducing symptoms of conditions or diseases associated with PHIP deficiency, such as Chung-Jansen syndrome (CHUJANS), autosomal dominant disorders, intellectual disability, speech delay, anxiety, autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), aggressive behavior, facial dysmorphism, cafe au lait spots, overweight syndrome, developmental delay, obesity, or dysmorphism caused by PHIP haploinsufficiency.

[0102]

[0124] In some embodiments, the disease or condition that can be treated or ameliorated using the methods or compositions disclosed herein is not directly related to the target protein (gene) targeted by the therapeutic agent. In some embodiments, the therapeutic agents provided herein may target a protein (gene) that is not directly related to the disease or condition, but modulation of the expression of the target protein (gene) can treat or ameliorate the disease or condition.

[0103]

[0125] In various embodiments, the disclosure provides therapeutic agents that can target PHIP precursor mRNA transcripts to modulate splicing or protein expression levels. The therapeutic agent can be a small molecule, polynucleotide, or polypeptide. In some embodiments, the therapeutic agent is an ASO. Various regions or sequences of the PHIP precursor mRNA can be targeted by therapeutic agents, such as ASOs. In some embodiments, the ASO targets a PHIP precursor mRNA transcript that contains an NMD exon. In some embodiments, the ASO targets a sequence within the NMD exon of the PHIP precursor mRNA transcript. In some embodiments, the ASO targets a sequence upstream (or 5') from the 5' end (3'ss) of the NMD exon of the PHIP precursor mRNA transcript. In some embodiments, the ASO targets a sequence downstream (or 3') from the 3' end (5'ss) of the NMD exon of the PHIP precursor mRNA transcript. In some embodiments, the ASO targets a sequence within an intron adjacent to the 5' end of the NMD exon of the PHIP precursor mRNA transcript. In some embodiments, the ASO targets a sequence within an intron adjacent to the 3' end of the NMD exon of the PHIP precursor mRNA transcript. In some embodiments, the ASO targets a sequence that includes an NMD exon-intron boundary of the PHIP precursor mRNA transcript. The NMD exon-intron boundary may refer to the junction of an intron sequence with an NMD exon region. The intron sequence may be adjacent to the 5' end of the NMD exon or the 3' end of the NMD exon. In some embodiments, the ASO targets a sequence within an exon of the PHIP precursor mRNA transcript. In some embodiments, the ASO targets a sequence within an intron of the PHIP precursor mRNA transcript. In some embodiments, the ASO targets a sequence that includes both a portion of an intron and a portion of an exon of the PHIP precursor mRNA transcript.

[0104]

[0126] In some embodiments, the ASO targets a sequence about 4 to about 300 nucleotides upstream (or on the 5' side) from the 5' end of the NMD exon. In some embodiments, the ASO targets a sequence about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides upstream (or on the 5' side) from the 5' end of the NMD exon region. In some embodiments, the ASO may target a sequence more than 300 nucleotides upstream from the 5' end of the NMD exon. In some embodiments, the ASO targets a sequence about 4 to about 300 nucleotides downstream (or on the 3' side) from the 3' end of the NMD exon. In some embodiments, the ASO targets a sequence about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides downstream from the 3' end of the NMD exon. In some embodiments, the ASO targets a sequence more than 300 nucleotides downstream from the 3' end of the NMD exon.

[0105]

[0127] In some embodiments, the PHIP NMD exon-containing pre-mRNA transcript is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the PHIP NMD exon pre-mRNA transcript comprises a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:3.

[0106]

[0128] In some embodiments, the PHIP NMD exon-containing precursor-mRNA transcript (or NMD exon mRNA) comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the PHIP NMD exon-containing precursor-mRNA transcript (or NMD exon mRNA) is encoded by a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the targeted portion of the NMD exon mRNA comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO: 3.

[0107]

[0129] In some embodiments, the ASO targets exon 15x of a PHIP NMD exon-containing pre-mRNA that contains NIE exon 15 (exon 15x) of a PHIP NMD exon-containing pre-mRNA that contains NIE exon 15. In some embodiments, the ASO targets exon (GRCh38 / hg38:chr6 79004373-79004436) of PHIP.

[0108]

[0130] In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream (or 5') of the 5' end of exon 15x of PHIP, which is exon 15x of PHIP. In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream (or 5') of GRCh38 / hg38:chr6 79004373 of PHIP.

[0109]

[0131] In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') from the 5' end of exon 15x of PHIP. In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') from GRCh38 / hg38:chr6 79004373 of PHIP.

[0110]

[0132] In some embodiments, the ASO targets a sequence about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') from the 3' end of exon 15x of PHIP. In some embodiments, the ASO targets a sequence about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') from GRCh38 / hg38:chr6 79004436 of PHIP.

[0111]

[0133] In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') from the 3' end of exon 15x of PHIP. In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') from GRCh38 / hg38:chr6 79004436 of PHIP.

[0112]

[0134] In some embodiments, the ASO has a sequence complementary to a targeted portion of the NMD exon mRNA set forth in SEQ ID NO:3.

[0113]

[0135] In some embodiments, the ASO targets a sequence upstream from the 5' end of an NMD exon. For example, an ASO targeting a sequence upstream from the 5' end of an NMD exon (e.g., an exon of PHIP (GRCh38 / hg38:chr6 79004373-79004436)) can include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO:3.

[0114]

[0136] In some embodiments, the ASO targets a sequence containing an exon-intron boundary (or junction). For example, an ASO targeting a sequence containing an exon-intron boundary can include a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to at least 8 consecutive nucleic acids of SEQ ID NO:3. In some embodiments, the ASO targets a sequence downstream from the 3' end of an NMD exon. For example, an ASO targeting a sequence downstream from the 3' end of an NMD exon (e.g., exon 15x of PHIP) can include a sequence that has at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO:3 or at least 8 consecutive nucleic acids of SEQ ID NO:3. For example, an ASO targeting a sequence downstream from the 3' end of an NMD exon (e.g., exon of PHIP (GRCh38 / hg38:chr6 79004373-79004436)) can include SEQ ID NO: 3, or a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to at least 8 contiguous nucleic acids of SEQ ID NO: 3. In some embodiments, the ASO targets a sequence within an NMD exon.

[0115]

[0137] In some embodiments, the ASO targets exon 15x of a PHIP NMD exon-containing pre-mRNA that contains exon 15, or NIE exon 15x of a PHIP NMD exon-containing pre-mRNA that contains NIE exon 16. In some embodiments, the ASO targets a sequence downstream (or 3') from the 5' end of exon 15x of a PHIP pre-mRNA. In some embodiments, the ASO targets a sequence upstream (or 5') from the 3' end of exon 15x of a PHIP pre-mRNA.

[0116]

[0138] In some embodiments, the targeted portion of the PHIP NMD exon-containing pre-mRNA is in intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39. In some embodiments, hybridization of the ASO to the targeting portion of the NMD exon pre-mRNA results in exon skipping of at least one NMD exon within intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39, followed by increased PHIP protein production. In some embodiments, the targeting portion of the PHIP NMD exon-containing pre-mRNA is in intron 15 of PHIP. In some embodiments, the targeting portion of the PHIP NMD exon-containing pre-mRNA is intron (GRCh38 / hg38:chr6 79003858-79015082) of PHIP.

[0117]

[0139] In some embodiments, the methods and compositions of the disclosure are used to increase expression of PHIP by inducing exon skipping of a pseudoexon in a PHIP NMD exon-containing pre-mRNA. In some embodiments, the pseudoexon is a sequence within any of introns 1-39. In some embodiments, the pseudoexon is a sequence within any of introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39. In some embodiments, the pseudoexon can be a PHIP intron or a portion thereof. In some embodiments, the pseudoexon is within intron 15 of PHIP. In some embodiments, the pseudoexon is within an intron of PHIP (GRCh38 / hg38:chr6 79003858 79015082).

[0118]

[0140] In some embodiments, the PHIP precursor mRNA transcript is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the PHIP precursor mRNA transcript comprises a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:3.

[0119]

[0141] In some embodiments, the PHIP precursor mRNA transcript (or NMD exon mRNA) comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the PHIP precursor mRNA transcript (or NMD exon mRNA) is encoded by a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the targeting portion of the PHIP precursor mRNA comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO: 3.

[0120]

[0142] In some embodiments, the ASO targets exon 15 of the PHIP pre-mRNA. In some embodiments, the ASO targets exon (GRCh38 / hg38:chr6 79015081 79015216) of the PHIP pre-mRNA. In some embodiments, the ASO targets exon 16 of the PHIP pre-mRNA and the ASO targets exon (GRCh38 / hg38:chr6 79003729 79003858) of the PHIP pre-mRNA.

[0121]

[0143] In some embodiments, the ASO has a sequence complementary to a targeted portion of the NMD exon mRNA set forth in SEQ ID NO:3. Protein expression

[0144] In some embodiments, the methods described herein are used to increase the production of functional PHIP protein or RNA. As used herein, the term "functional" refers to the amount of activity or function of a PHIP protein or RNA necessary to eliminate any one or more symptoms of the condition or disease being treated, e.g., Chung-Jansen syndrome. In some embodiments, the methods are used to increase the production of partially functional PHIP protein or RNA. As used herein, the term "partially functional" refers to any amount of activity or function of a PHIP protein or RNA that is less than the amount of activity or function necessary to eliminate or prevent any one or more symptoms of the disease or condition. In some embodiments, a partially functional protein or RNA may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% less activity than a fully functional protein or RNA.

[0122]

[0145] In some embodiments, the method is a method of increasing expression of PHIP protein by cells of a subject having a PHIP pre-mRNA, the subject having a disease or condition caused by a defective amount of activity of PHIP protein, e.g., Chung-Jansen syndrome, the defective amount of PHIP protein being caused by haploinsufficiency of the PHIP protein. In such embodiments, the subject has a first allele that encodes a functional PHIP protein and a second allele in which no PHIP protein is produced. In another such embodiment, the subject has a first allele that encodes a functional PHIP protein and a second allele that encodes a non-functional PHIP protein. In another such embodiment, the subject has a first allele that encodes a functional PHIP protein and a second allele that encodes a partially functional PHIP protein. In any of these embodiments, the antisense oligomer binds to the targeted portion of the PHIP pre-mRNA transcribed from the second allele, thereby inducing exon skipping of the pseudoexon from the pre-mRNA, causing an increase in the level of mature mRNA encoding the functional PHIP protein and an increase in expression of the PHIP protein in the cells of the subject.

[0123]

[0146] In some embodiments, the method is for increasing expression of PHIP protein by cells of a subject having a PHIP precursor mRNA, the subject having a disease or condition caused by a deficient amount of activity of PHIP protein, the deficient amount of PHIP protein being caused by autosomal recessive inheritance.

[0124]

[0147] In some embodiments, the method is for increasing expression of PHIP protein by cells of a subject having a PHIP precursor mRNA, the subject having a disease or condition caused by a deficient amount of activity of PHIP protein, e.g., Chung-Jansen syndrome, the deficient amount of PHIP protein being caused by autosomal dominant inheritance.

[0125]

[0148] In related embodiments, the method is a method of increasing expression of a protein or functional RNA using an ASO. In some embodiments, the ASO can be used to increase expression of PHIP protein in cells of a subject having a PHIP precursor mRNA, the subject having a deficiency in the amount or function of PHIP protein, e.g., Chung-Jansen syndrome.

[0126]

[0149] In some embodiments, a pre-mRNA transcript that encodes a protein that causes a disease or pathology is targeted by an agent, e.g., an oligonucleotide, as described herein. In some cases, it is an NMD exon-containing pre-mRNA transcript that is targeted by an agent, e.g., an oligonucleotide, as described herein. In some embodiments, an NMD exon-containing pre-mRNA transcript that encodes a protein that does not cause a disease is targeted by an ASO. For example, a disease that is the result of a mutation or deficiency of a first protein in a particular pathway may be ameliorated by targeting a pre-mRNA that encodes a second protein, thereby increasing the production of the second protein. In some embodiments, the function of the second protein can compensate for the mutation or deficiency of the first protein, which causes the disease or pathology.

[0127]

[0150] In some embodiments, the subject has (a) a first mutant allele, whereby (i) PHIP protein is produced at a reduced level compared to production from a wild-type allele, (ii) PHIP protein is produced in a form that has reduced functionality compared to an equivalent wild-type protein, or (iii) no PHIP protein or functional RNA is produced, and (b) a second mutant allele, whereby (i) PHIP protein is produced at a reduced level compared to production from a wild-type allele, (ii) PHIP protein is produced in a form that has reduced functionality compared to an equivalent wild-type protein, or (iii) no PHIP protein is produced, and an NMD exon-containing pre-mRNA is transcribed from the first allele and / or the second allele. In these embodiments, the ASO binds to a targeted portion of the NMD exon-containing pre-mRNA transcribed from the first allele or the second allele, thereby inducing exon skipping of the pseudoexon from the NMD exon-containing pre-mRNA, resulting in an increase in the level of the mRNA encoding the PHIP protein and an increase in expression of the target protein or functional RNA in the cells of the subject. In these embodiments, the target protein or functional RNA having an increased expression level resulting from exon skipping of the pseudoexon from the NMD exon-containing pre-mRNA can be in a form that has reduced function (partially functional) compared to the equivalent wild-type protein, or that has full function (fully functional) compared to the equivalent wild-type protein.

[0128]

[0151] In some embodiments, the level of mRNA encoding a PHIP protein is increased by 1.1 to 10 fold when compared to the amount of mRNA encoding a PHIP protein produced in a control cell, e.g., a cell that is not treated with an antisense oligomer, or a cell that is treated with an antisense oligomer that does not bind to a targeted portion of the PHIP precursor mRNA.

[0129]

[0152] In some embodiments, subjects treated using the methods of the present disclosure express a partially functional PHIP protein from one allele, where the partially functional PHIP protein may be caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion. In some embodiments, subjects treated using the methods of the present disclosure express a non-functional PHIP protein from one allele, where the non-functional PHIP protein may be caused by a frameshift mutation, a nonsense mutation, a missense mutation, a partial gene deletion in one allele. In some embodiments, subjects treated using the methods of the present disclosure have a PHIP full gene deletion in one allele. Exon inclusion

[0153] As used herein, an "NMD exon-containing pre-mRNA" is a pre-mRNA transcript that contains at least one pseudoexon. Alternative or aberrant splicing can result in the inclusion of at least one pseudoexon in a mature mRNA transcript. The terms "mature mRNA" and "fully spliced ​​mRNA" are used interchangeably herein to refer to a fully processed mRNA. The inclusion of at least one pseudoexon can result in a non-productive mRNA, leading to NMD of the mature mRNA. NMD exon-containing mature mRNAs can sometimes lead to aberrant protein expression.

[0130]

[0154] In some embodiments, the included pseudo-exon is the pseudo-exon that is most abundant in a population of NMD exon-containing pre-mRNAs transcribed from a gene encoding a target protein in a cell. In some embodiments, the included pseudo-exon is the pseudo-exon that is most abundant in a population of NMD exon-containing pre-mRNAs transcribed from a gene encoding a target protein in a cell, the population of NMD exon-containing pre-mRNAs comprising more than one included pseudo-exon. In some embodiments, an antisense oligomer targeted to the pseudo-exon that is most abundant in a population of NMD exon-containing pre-mRNAs encoding a target protein induces exon skipping of one or more pseudo-exons in the population, including the pseudo-exon that the antisense oligomer targets or binds. In some embodiments, the targeted region is in a pseudo-exon that is the pseudo-exon that is most abundant in the NMD exon-containing pre-mRNAs encoding a PHIP protein.

[0131]

[0155] The degree of exon inclusion can be expressed as the exon inclusion ratio, e.g., the percentage of transcripts that include a given pseudoexon. Briefly, the % exon inclusion can be calculated as the percentage of the amount of RNA transcripts with exon inclusion relative to the sum of the average amount of RNA transcripts with exon inclusion and the average amount of RNA transcripts with exon exclusion.

[0132]

[0156] In some embodiments, an included pseudoexon is an exon identified as an included pseudoexon based on a determination of an inclusion rate of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%. In an embodiment, the included pseudoexons are from about 5% to about 100%, from about 5% to about 95%, from about 5% to about 90%, from about 5% to about 85%, from about 5% to about 80%, from about 5% to about 75%, from about 5% to about 70%, from about 5% to about 65%, from about 5% to about 60%, from about 5% to about 55%, from about 5% to about 50%, from about 5% to about 45%, from about 5% to about 40%, from about 5% to about 35%, from about 5% to about 30%, from about 5% to about 25%, from about 5% to about 20%, from about 5% to about 15%, from about 10% to about 100%, from about 10% to about 95%, from about 10% to about 90%, About 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 100%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 70%, About 15% to about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 100%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45% , about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 25% to about 100%, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, or about 25% to about 35% inclusion rate.ENCODE data (e.g., as described in Tilgner et al., 2012, "Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs", Genome Research 22(9):1616-25) can be used to assist in identifying exon inclusion.

[0133]

[0157] In some embodiments, contacting a cell with an ASO that is complementary to a targeted portion of a PHIP precursor mRNA transcript results in an increase in the amount of PHIP protein produced of at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%, compared to the amount of protein produced by the cell in the absence / treatment of the ASO. In some embodiments, the total amount of PHIP protein produced by cells contacted with an antisense oligomer is about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 5 ... An increase of 0% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%. In some embodiments, the total amount of PHIP protein produced by cells contacted with an antisense oligomer is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 3 to about 10 times, about 4 to about 10 times, about 5 to about 10 times, about 5 to about 10 times, about 6 to about 10 times, about 7 to about 10 times, about 8 to about 10 times, about 10 ... fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold. The control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the pre-mRNA.

[0134]

[0158] In some embodiments, contacting a cell with an ASO that is complementary to a targeted portion of a PHIP precursor mRNA transcript results in an increase in the amount of mRNA encoding PHIP, including the mature mRNA encoding the target protein, hi some embodiments, the amount of mRNA encoding a PHIP protein, or the mature mRNA encoding a PHIP protein, is increased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the amount of protein produced by the cell in the absence / treatment of the ASO. In some embodiments, the total amount of mRNA encoding a PHIP protein, or mature mRNA encoding a PHIP protein produced in a cell contacted with an antisense oligomer, is about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 300%, about 30% to about 40% or about 50% of the total amount of mature RNA produced in an untreated cell, e.g., an untreated cell or a cell treated with a control compound. an increase of about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%.In some embodiments, the total amount of mRNA encoding a PHIP protein, or mature mRNA encoding a PHIP protein produced in a cell contacted with an antisense oligomer, is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 ...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 1.5 to about 10 times, about 2 to about 10 times, about 2 to about 10 times, about 2 to about 10 times, about 2 to about 10 times, about 2 to about 10 times, about 2 to .1 to about 7 fold, about 1.1 to about 8 fold, about 1.1 to about 9 fold, about 2 to about 5 fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold. The control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the PHIP NMD exon-containing pre-mRNA.

[0135]

[0159] The NMD exon can be any length. In some embodiments, the NMD exon includes the complete sequence of the intron, in which case it can be referred to as intron retention. In some embodiments, the NMD exon can be a portion of an intron. In some embodiments, the NMD exon can be a 5' end portion of an intron that includes a 5'ss sequence. In some embodiments, the NMD exon can be a 3' end portion of an intron that includes a 3'ss sequence. In some embodiments, the NMD exon can be a portion within an intron without the inclusion of a 5'ss sequence. In some embodiments, the NMD exon can be a portion within an intron without the inclusion of a 3'ss sequence. In some embodiments, the NMD exon can be a portion within an intron without the inclusion of either a 5'ss or a 3'ss sequence. In some embodiments, the NMD exon can be from 5 nucleotides to 10 nucleotides in length, from 10 nucleotides to 15 nucleotides in length, from 15 nucleotides to 20 nucleotides in length, from 20 nucleotides to 25 nucleotides in length, from 25 nucleotides to 30 nucleotides in length, from 30 nucleotides to 35 nucleotides in length, from 35 nucleotides to 40 nucleotides in length, from 40 nucleotides to 45 nucleotides in length, from 45 nucleotides to 50 nucleotides in length, from 50 nucleotides to 55 nucleotides in length, from 55 nucleotides to 60 nucleotides in length, from 60 nucleotides to 65 nucleotides in length, from 65 nucleotides to 70 nucleotides in length, from 70 nucleotides to 75 nucleotides in length, from 75 nucleotides to 80 nucleotides in length, from 80 nucleotides to 85 nucleotides in length, from 85 nucleotides to 90 nucleotides in length, from 90 nucleotides to 95 nucleotides in length, or from 95 nucleotides to 100 nucleotides in length. In some embodiments, the NMD exon can be at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides in length, at least 90 nucleotides, or at least 100 nucleotides in length.In some embodiments, the NMD exon can be 100-200 nucleotides, 200-300 nucleotides, 300-400 nucleotides, 400-500 nucleotides, 500-600 nucleotides, 600-700 nucleotides, 700-800 nucleotides, 800-900 nucleotides, 900-1,000 nucleotides in length, In some embodiments, the NMD exon can be greater than 1,000 nucleotides in length.

[0136]

[0160] Inclusion of the pseudoexon can result in frameshifting and introduction of a premature stop codon (PIC) into the mature mRNA transcript, making the transcript a target for NMD. A mature mRNA transcript containing an NMD exon can be a non-productive mRNA transcript that does not result in protein expression. The PIC can be present at any position downstream of the NMD exon. In some embodiments, the PIC can be present in any exon downstream of the NMD exon. In some embodiments, the PIC can be present within the NMD exon. For example, inclusion of exon 15x of PHIP into an mRNA transcript encoded by the PHIP gene can direct the PIC to the mRNA transcript. For example, inclusion of exon (GRCh38 / hg38:chr6 79004373-79004436) of PHIP into an mRNA transcript encoded by PHIP. Therapeutic Agents

[0161] In various embodiments of the present disclosure, compositions and methods are provided that include a therapeutic agent that modulates the protein expression level of PHIP. In some embodiments, compositions and methods are provided herein that modulate alternative splicing of PHIP pre-mRNA. In some embodiments, compositions and methods are provided herein that induce exon skipping in the splicing of PHIP pre-mRNA, e.g., induce skipping of a pseudoexon during splicing of PHIP pre-mRNA. In other embodiments, a therapeutic agent may be used to induce exon inclusion to reduce protein expression levels.

[0137]

[0162] The therapeutic agent disclosed herein can be an NIE inhibitor. The therapeutic agent may comprise a polynucleic acid polymer.

[0138]

[0163] According to one aspect of the disclosure, provided herein is a method for treating or preventing a condition or disease associated with a deficiency in functional PHIP protein, comprising administering to a subject an NIE inhibitor to increase the level of functional PHIP protein, wherein the agent binds to a region of a pre-mRNA transcript to reduce inclusion of an NMD exon in the mature transcript. For example, provided herein is a method for treating or preventing a condition associated with a deficiency in functional PHIP protein, comprising administering to a subject an NIE inhibitor to increase the level of functional PHIP protein, wherein the agent binds to a region of an intron containing an NMD exon (e.g., exon 15x of PHIP) of a pre-mRNA transcript, or to an NMD exon activating regulatory sequence in the same intron. For example, provided herein is a method of treating or preventing a condition associated with a deficiency of functional PHIP protein, comprising administering to a subject an NIE inhibitor to increase the level of functional PHIP protein, wherein the agent binds to a region of an intron containing an NMD exon of a pre-mRNA transcript (e.g., exon (GRCh38 / hg38:chr6 79004373-79004436) of PHIP), or to an NMD exon activating regulatory sequence in the same intron.

[0139]

[0164] When referring to reducing NMD exon inclusion in mature mRNA, the reduction may be complete, e.g., 100%, or partial. The reduction may be clinically significant. The reduction / correction may be compared to the level of NMD exon inclusion in subjects without treatment, or compared to the amount of NMD exon inclusion in a population of similar subjects. The reduction / correction may be at least 10% less NMD exon inclusion compared to the average subject, or subject before treatment. The reduction may be at least 20% less NMD exon inclusion compared to the average subject, or subject before treatment. The reduction may be at least 40% less NMD exon inclusion compared to the average subject, or subject before treatment. The reduction may be at least 50% less NMD exon inclusion compared to the average subject, or subject before treatment. The reduction may be at least 60% less NMD exon inclusion compared to the average subject, or subject before treatment. The reduction may be at least 80% less NMD exon inclusion compared to the average subject, or subject before treatment. The reduction can be at least 90% less NMD exon inclusion compared to the average subject, or compared to a subject prior to treatment.

[0140]

[0165] When referring to increasing active PHIP protein levels, the increase may be clinically significant. The increase may be compared to the level of active PHIP protein in a subject without treatment, or compared to the amount of active PHIP protein in a population of similar subjects. The increase may be at least 10% more active PHIP protein compared to the average subject, or subject before treatment. The increase may be at least 20% more active PHIP protein compared to the average subject, or subject before treatment. The increase may be at least 40% more active PHIP protein compared to the average subject, or subject before treatment. The increase may be at least 50% more active PHIP protein compared to the average subject, or subject before treatment. The increase may be at least 80% more active PHIP protein compared to the average subject, or subject before treatment. The increase may be at least 100% more active PHIP protein compared to the average subject, or subject before treatment. The increase may be at least 200% more active PHIP protein compared to the average subject, or subject before treatment. The increase can be at least 500% more active PHIP protein compared to the average subject, or compared to the subject prior to treatment.

[0141]

[0166] In embodiments where the NIE inhibitor comprises a polynucleic acid polymer, the polynucleic acid polymer can be about 50 nucleotides in length. The polynucleic acid polymer can be about 45 nucleotides in length. The polynucleic acid polymer can be about 40 nucleotides in length. The polynucleic acid polymer can be about 35 nucleotides in length. The polynucleic acid polymer can be about 30 nucleotides in length. The polynucleic acid polymer can be about 24 nucleotides in length. The polynucleic acid polymer can be about 25 nucleotides in length. The polynucleic acid polymer can be about 20 nucleotides in length. The polynucleic acid polymer can be about 19 nucleotides in length. The polynucleic acid polymer can be about 18 nucleotides in length. The polynucleic acid polymer can be about 17 nucleotides in length. The polynucleic acid polymer can be about 16 nucleotides in length. The polynucleic acid polymer can be about 15 nucleotides in length. The polynucleic acid polymer can be about 14 nucleotides in length. The polynucleic acid polymer can be about 13 nucleotides in length. The polynucleic acid polymer can be about 12 nucleotides in length. The polynucleic acid polymer can be about 11 nucleotides in length. The polynucleic acid polymer can be about 10 nucleotides in length. The polynucleic acid polymer can be about 10 to about 50 nucleotides in length. The polynucleic acid polymer can be about 10 to about 45 nucleotides in length. The polynucleic acid polymer can be about 10 to about 40 nucleotides in length. The polynucleic acid polymer can be about 10 to about 35 nucleotides in length. The polynucleic acid polymer can be about 10 to about 30 nucleotides in length. The polynucleic acid polymer can be about 10 to about 25 nucleotides in length. The polynucleic acid polymer can be about 10 to about 20 nucleotides in length. The polynucleic acid polymer can be about 15 to about 25 nucleotides in length. The polynucleic acid polymer can be about 15 to about 30 nucleotides in length. The polynucleic acid polymer can be about 12 to about 30 nucleotides in length.

[0142]

[0167] The sequence of the polynucleic acid polymer can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% complementary to the target sequence of the mRNA transcript, such as a partially processed mRNA transcript. The sequence of the polynucleic acid polymer can be 100% complementary to the target sequence of the pre-mRNA transcript.

[0143]

[0168] The sequence of the polynucleic acid polymer may have no more than four mismatches to the target sequence of the precursor mRNA transcript. The sequence of the polynucleic acid polymer may have no more than three mismatches to the target sequence of the precursor mRNA transcript. The sequence of the polynucleic acid polymer may have no more than two mismatches to the target sequence of the precursor mRNA transcript. The sequence of the polynucleic acid polymer may have no more than one mismatch to the target sequence of the precursor mRNA transcript. The sequence of the polynucleic acid polymer may have no mismatches to the target sequence of the precursor mRNA transcript.

[0144]

[0169] The polynucleic acid polymer can specifically hybridize to the target sequence of the pre-mRNA transcript. For example, the polynucleic acid polymer can have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence complementarity to the target sequence of the pre-mRNA transcript. Hybridization can be performed under highly stringent hybridization conditions.

[0145]

[0170] The polynucleic acid polymer comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2-5. The polynucleic acid polymer may comprise a sequence having 100% sequence identity to SEQ ID NO:3.

[0146]

[0171] When referring to polynucleic acid polymer sequences, one skilled in the art will understand that one or more substitutions may be tolerated, optionally two substitutions may be tolerated in the sequence, such that the polynucleic acid polymer sequence maintains the ability to hybridize to a target sequence, or to be recognized as a target sequence if the substitution is in the target sequence. The sequence identity criteria may be determined by BLAST sequence alignment using standard / default parameters. For example, a sequence can have 99% identity and still function according to the present disclosure. In other embodiments, a sequence can have 98% identity and still function according to the present disclosure. In another embodiment, a sequence can have 95% identity and still function according to the present disclosure. In another embodiment, a sequence can have 90% identity and still function according to the present disclosure. Antisense oligomers

[0172] Provided herein are compositions comprising antisense oligomers that induce exon skipping by binding to a targeted portion of a PHIP pre-mRNA, e.g., a PHIP NMD exon-containing pre-mRNA. As used herein, the terms "ASO" and "antisense oligomer" are used interchangeably and refer to an oligomer, such as a polynucleotide, that includes nucleobases that hybridize to a target nucleic acid (e.g., a PHIP pre-mRNA, e.g., a PHIP NMD exon-containing pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (GU). ASOs can have exact sequence complementarity or very high complementarity (e.g., sufficient complementarity to bind to the target sequence and enhance splicing at the splice site) to the target nucleic acid. ASOs are designed to bind (hybridize) to a target nucleic acid (e.g., a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, when ASO hybridizes to sites other than the intended (targeted) nucleic acid sequence, it hybridizes to a limited number of sequences that are not the target nucleic acid (a small number of sites other than the target nucleic acid). The design of ASO can take into account the presence of the nucleic acid sequence of the targeting portion of the pre-mRNA transcript or a sufficiently similar nucleic acid sequence in the genome or elsewhere in the pre-mRNA or transcriptome of the cell, so that the possibility of ASO binding to other sites and causing "off-target" effects is limited. Any antisense oligomer known in the art (e.g., in PCT / US2014 / 054151, published as WO2015 / 035091, entitled "Reducing Nonsense-Mediated mRNA Decay", incorporated herein by reference) can be used to carry out the methods described herein.

[0147]

[0173] In some embodiments, the ASO "specifically hybridizes" or is "specific" to a target nucleic acid or a targeted portion of a PHIP pre-mRNA, e.g., an NMD exon-containing pre-mRNA. Typically, such hybridization occurs at a T substantially greater than 37° C., preferably at least 50° C., typically between 60° C. and approximately 90° C. m Such hybridization preferably corresponds to stringent hybridization conditions. At a given ionic strength and pH, T m is the temperature at which 50% of the target sequence hybridizes to a complementary oligonucleotide.

[0148]

[0174] Oligomers, e.g., oligonucleotides, are "complementary" to each other when hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration. A double-stranded polynucleotide can be "complementary" to another polynucleotide when hybridization can occur between one of the strands of a first polynucleotide and a second polynucleotide. Complementarity (the degree to which one polynucleotide is complementary to another) can be quantified in terms of the proportion (e.g., percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other according to generally accepted base pairing rules. The sequence of an antisense oligomer (ASO) need not be 100% complementary to the sequence of the target nucleic acid to which it hybridizes. In certain embodiments, the ASO can include at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity to a target region within the target nucleic acid sequence to which it is targeted. For example, an ASO in which 18 of 20 nucleobases of an oligomeric compound are complementary to a target region and therefore can specifically hybridize represents 90% complementarity. In this example, the remaining non-complementary nucleobases can be clustered together or interspersed with complementary nucleobases, and do not need to be contiguous to each other or to complementary nucleobases. The complementarity percentage of an ASO with a region of a target nucleic acid can be routinely determined using BLAST (basic local alignment search tool) and PowerBLAST programs (Altschul et al., J.Mol.Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) known in the art.

[0149]

[0175] ASO does not need to hybridize to all nucleobases in the target sequence, and the nucleobases that ASO hybridizes can be continuous or non-contiguous. ASO may hybridize over one or more segments of a pre-mRNA transcript, such that intervening or adjacent segments are not involved in the hybridization event (e.g., loop or hairpin structures may be formed). In certain embodiments, ASO hybridizes to non-contiguous nucleobases in the target pre-mRNA transcript. For example, ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobases that ASO does not hybridize to.

[0150]

[0176] The ASO described herein comprises a nucleobase that is complementary to a nucleobase present in a target portion of a PHIP pre-mRNA, e.g., a NMD exon-containing pre-mRNA. The term ASO includes oligonucleotides and any other oligomeric molecules that contain a nucleobase that can hybridize to a complementary nucleobase of a target mRNA, but do not contain a sugar moiety, e.g., peptide nucleic acid (PNA). The ASO may comprise naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and / or modified backbones. In some embodiments, all nucleotides of the ASO are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described herein will be apparent to one of skill in the art and can be found, for example, in U.S. Pat. No. 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, pp. 347-355, which are incorporated herein by reference in their entireties.

[0151]

[0177] The one or more nucleobases of the ASO may be any naturally occurring unmodified nucleobase, such as adenine, guanine, cytosine, thymine, and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase to be capable of hydrogen bonding with a nucleobase present in the target pre-mRNA. Examples of modified nucleobases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethoylcytosine.

[0152]

[0178] The ASOs described herein also include backbone structures that connect the components of the oligomer. The terms "backbone structure" and "oligomer linkage" can be used interchangeably and refer to the linkage between the monomers of the ASO. In naturally occurring oligonucleotides, the backbone includes 3'-5' phosphodiester linkages that connect the sugar moieties of the oligomer. The backbone structures or oligomer linkages of the ASOs described herein include, but are not limited to, phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroaniladates, and phosphoroamidates. See, e.g., LaPlanche et al., Nucleic Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984); Stein et al., Nucleic Acids Res. 16:3209 (1988); Zon et al., Anti-Cancer Drug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, ed., Oxford University Press, Oxford England (1991)); Stec et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990). In some embodiments, the backbone structure of the ASO does not contain phosphorus, but rather contains peptide bonds, such as peptide nucleic acids (PNAs) or linking groups including carbamates, amides, and linear and cyclic hydrocarbon groups. In some embodiments, the backbone modification is a phosphorothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.

[0153]

[0179] In some embodiments, the stereochemistry at each phosphorus internucleotide linkage of the ASO backbone is random. In some embodiments, the stereochemistry at each phosphorus internucleotide linkage of the ASO backbone is controlled and not random. For example, U.S. Patent Application Publication No. 2014 / 0194610, "Methods for the Synthesis of Functionalized Nucleic Acids," incorporated herein by reference, describes methods for independently selecting the handedness of chirality at each phosphorus atom in a nucleic acid oligomer. In some embodiments, the ASOs used in the methods of the present disclosure include ASOs with non-random phosphorus internucleotide linkages, including but not limited to any ASOs listed in Tables 5 and 6 herein. In some embodiments, the compositions used in the methods of the present disclosure include pure diastereomeric ASOs. In some embodiments, a composition used in a method of the disclosure comprises an ASO having a diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

[0154]

[0180] In some embodiments, the ASO has a non-random mixture of Rp and Sp configurations in its phosphointernucleotide linkages. For example, it has been suggested that a mixture of Rp and Sp is required in antisense oligonucleotides to achieve a balance between good activity and nuclease stability (Wan et al., 2014, "Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages," Nucleic Acids Research 42(22):13456-13468, incorporated herein by reference). In some embodiments, the ASOs used in the methods of the disclosure, including but not limited to any ASO set forth herein in SEQ ID NO: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 and the remaining Sp, or about 100% Rp.In some embodiments, the ASO used in the methods of the disclosure, including but not limited to any ASO described herein, comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 consecutive nucleic acids of any one of SEQ ID NO:3, and has an Rp of about 10% to about 100%, about 15% to about 100%, about 20% to about 100%, about 25% to about 100%, about 30% to about 100%, about 35% to about 100%, about 40% to about 45%, about 50% to about 55%, about 60% to about 65%, about 70% to about 75%, about 80% to about 85%, about 90% to about 95%, about 100% to about 100%, about 100% to about 100%, about 15% to about 100%, about 20% to about 100%, about 25% to about 100%, about 30% to about 100%, about 35% to about 100%, about 40% to about 100%, about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 10 ... 0% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about 60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, or about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp and the remainder Sp.

[0155]

[0181] In some embodiments, the ASO used in the methods of the disclosure, including but not limited to any ASO described herein, comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO:3, and can be from about 5 to 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 50% Sp, at least about 60% Sp, at least about 70% Sp, at least about 80% Sp, at least about 90% Sp, at least about 100% Sp, at least about 15% Sp, at least about 20% Sp, at least about 35% Sp, at least about 40% Sp, at least about 50% Sp, at least about 60% Sp, at least about 70% Sp, at least about 80% Sp, at least about 90% Sp, at least about 10 ... Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp with the remainder Rp, or about 100% Sp. In embodiments, ASOs used in the methods of the present disclosure, including but not limited to any ASO described herein, include a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region containing at least 8 consecutive nucleic acids of SEQ ID NO:3, and may include at least 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, and the like. , about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55% Sp and the remainder Rp.

[0156]

[0182] Any of the ASOs described herein may contain modified sugar moieties or sugar analogs that contain a ribose or deoxyribose sugar moiety, or a morpholine ring, as found in naturally occurring nucleotides. Non-limiting examples of modified sugar moieties include 2' substituents, such as 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F; 2'-NMA moieties; N3'→P5' phosphoramidate, 2' dimethylaminooxyethoxy, 2' dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. As used herein, "2'-NMA" refers to a -O-CH group in place of the 2'-OH group of the ribosyl sugar moiety. 2 -C(=O)-NH-CH 3 A "2'-NMA sugar moiety" or "2'-NMA moiety" can refer to a 2'-O-CH group in place of the 2'-OH group of a ribosyl sugar moiety. 2 -C(=O)-NH-CH 3The 2'-NMA sugar moiety is in the β-D configuration unless otherwise indicated. "NMA" can mean ON-methylacetamide. In some embodiments, the sugar moiety modification is selected from 2'-O-Me, 2'F, 2'-NMA, and 2'MOE. In some embodiments, the sugar moiety modification is an additional crosslink bond, for example in locked nucleic acids (LNAs). In some embodiments, the sugar analog contains a morpholine ring, for example, a 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, for example, Jarver et al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications," Nucleic Acid Therapeutics 24(1):37-47, which is incorporated herein by reference for this purpose.

[0157]

[0183] In some embodiments, each monomer of the ASO is modified in the same way, for example, each link in the backbone of the ASO contains a phosphorothioate linkage, or each ribose sugar moiety contains a 2'-O-methyl modification. Such modifications present in each of the monomer components of the ASO are referred to as "uniform modifications". In some cases, a combination of different modifications may be desired, for example, an ASO may contain a combination of phosphorodiamidate linkages and sugar moieties containing morpholine rings (morpholinos). A combination of different modifications to an ASO is referred to as "mixed modifications" or "mixed chemistry".

[0158]

[0184] In some embodiments, the ASO comprises one or more backbone modifications. In some embodiments, the ASO comprises one or more sugar moiety modifications. In some embodiments, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs described herein, or any component of the ASO (e.g., nucleobase, sugar moiety, backbone) may be modified to achieve a desired property or activity of the ASO or to reduce an undesirable property or activity of the ASO. For example, the ASO, or one or more components of any ASO, may be modified to enhance binding affinity for the target sequence of a pre-mRNA transcript, to reduce binding to any non-target sequences, to reduce degradation by cellular nucleases (i.e., RNase H), to improve uptake of the ASO into cells and / or into the nucleus of a cell, to alter the pharmacokinetics or pharmacodynamics of the ASO, and / or to modulate the half-life of the ASO.

[0159]

[0185] In some embodiments, the ASO is composed of 2'-O-(2-methoxyethyl) (MOE) phosphorothioate modified nucleotides. ASOs composed of such nucleotides are particularly well suited for the methods disclosed herein, as oligomers with such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable for oral delivery, for example, in some embodiments described herein. See, e.g., Geary et al., J Pharmacol Exp Ther. 2001;296(3):890-7; Geary et al., J Pharmacol Exp Ther. 2001;296(3):898-904.

[0160]

[0186] Methods for synthesizing ASOs are known to those of skill in the art. Alternatively, or additionally, ASOs may be obtained from commercial suppliers.

[0161]

[0187] Unless otherwise specified, the left-hand end of a single-stranded nucleic acid (e.g., a pre-mRNA transcript, an oligonucleotide, an ASO, etc.) sequence is the 5' end, and the left-hand direction of a single-stranded or double-stranded nucleic acid sequence is referred to as the 5' direction. Similarly, the right-hand end or direction of a nucleic acid sequence (single-stranded or double-stranded) is the 3' end or direction. Generally, a region or sequence that is 5' to a reference point in a nucleic acid is referred to as "upstream" and a region or sequence that is 3' to a reference point in a nucleic acid is referred to as "downstream". Generally, the 5' direction or end of an mRNA is where the initiation or start codon is located, and the 3' end or direction is where the stop codon is located. In some aspects, nucleotides that are upstream of the reference point in a nucleic acid may be designated by a negative number, and nucleotides that are downstream of the reference point may be designated by a positive number. For example, a reference point (e.g., an exon-exon junction in an mRNA) may be designated as a "zero" site, with the nucleotide immediately adjacent to the reference point upstream being designated as a "minus one," e.g., "-1," and the nucleotide immediately adjacent to the reference point downstream being designated as a "plus one," e.g., "+1."

[0162]

[0188] In some embodiments, the ASO is complementary to (and binds to) a targeted portion of a PHIP pre-mRNA, e.g., a PHIP NMD exon-containing pre-mRNA, that is downstream (in the 3' direction) (e.g., in the direction designated by a positive number relative to the 5' splice site) of the included exon in the PHIP pre-mRNA. In some embodiments, the ASO is complementary to a targeted portion of a PHIP pre-mRNA, e.g., a PHIP NMD exon-containing pre-mRNA, that is within a region of about +1 to about +500 relative to the 5' splice site (or 3' end) of the included exon. In some embodiments, the ASO can be complementary to a targeted portion of a PHIP pre-mRNA, e.g., a PHIP NMD exon-containing pre-mRNA, that is within a region of +6 to +40,000 nucleotides relative to the 5' splice site (or 3' end) of the included exon.In some embodiments, the ASO is about +1 to about +40,000, about +1 to about +30,000, about +1 to about +20,000, about +1 to about +15,000, about +1 to about +10,000, about +1 to about +5,000, about +1 to about +4,000, about +1 to about +3,000, about +1 to about +2,000, about +1 to about +1,000, about + 1 to about +500, about +1 to about +490, about +1 to about +480, about +1 to about +470, about +1 to about +460, about +1 to about +450, about +1 to about +440, about +1 to about +430, about +1 to about +420, about +1 to about +410, about +1 to about +400, about +1 to about +390, about +1 to about +380, about +1 to about +370, about +1 to about +360, about +1 to about +350, about +1 to about +340, About +1 to about +330, about +1 to about +320, about +1 to about +310, about +1 to about +300, about +1 to about +290, about +1 to about +280, about +1 to about +270, about +1 to about +260, about +1 to about +250, about +1 to about +240, about +1 to about +230, about +1 to about +220, about +1 to about +210, about +1 to about +200, about +1 to about +190, about +1 to about +180, about +1 to about +17 is complementary to a targeting portion within a region of 0, about +1 to about +160, about +1 to about +150, about +1 to about +140, about +1 to about +130, about +1 to about +120, about +1 to about +110, about +1 to about +100, about +1 to about +90, about +1 to about +80, about +1 to about +70, about +1 to about +60, about +1 to about +50, about +1 to about +40, about +1 to about +30, or about +1 to about +20. In some embodiments, the ASO is complementary to a targeting portion that is within a region of about +1 to about +100, about +100 to about +200, about +200 to about +300, about +300 to about +400, or about +400 to about +500 relative to the 5' splice site (or 3' end) of the encompassing exon.

[0163]

[0189] In some embodiments, the ASO is complementary to (or binds to) a targeted portion of a PHIP precursor mRNA, e.g., a PHIP NMD exon-containing pre-mRNA, that is upstream (in the 5' direction) (e.g., in the direction designated by a negative number relative to the 5' splice site) of the 5' splice site (or 3' end) of the included exon in the PHIP precursor mRNA, e.g., the PHIP NMD exon-containing pre-mRNA. In some embodiments, the ASO is complementary to a targeted portion of a PHIP precursor mRNA, e.g., a PHIP NMD exon-containing pre-mRNA, that is within a region of about -4 to about -270 relative to the 5' splice site (or 3' end) of the included exon. In some embodiments, the ASO can be complementary to a targeted portion of a PHIP precursor mRNA, e.g., a PHIP NMD exon-containing pre-mRNA, that is within a region of -1 to -40,000 nucleotides relative to the 5' splice site (or 3' end) of the included exon.In some embodiments, the ASO is about -1 to about -40,000, about -1 to about -30,000, about -1 to about -20,000, about -1 to about -15,000, about -1 to about -10,000, about -1 to about -5,000, about -1 to about -4,000, about -1 to about -3,000, about -1 to about -2,000, about -1 to about -1,000, about - 1 to about -500, about -1 to about -490, about -1 to about -480, about -1 to about -470, about -1 to about -460, about -1 to about -450, about -1 to about -440, about -1 to about -430, about -1 to about -420, about -1 to about -410, about -1 to about -400, about -1 to about -390, about -1 to about -380, about -1 to about -370, about -1 to about -360, about -1 to about -350, about -1 to about -340, About -1 to about -330, about -1 to about -320, about -1 to about -310, about -1 to about -300, about -1 to about -290, about -1 to about -280, about -1 to about -270, about -1 to about -260, about -1 to about -250, about -1 to about -240, about -1 to about -230, about -1 to about -220, about -1 to about -210, about -1 to about -200, about -1 to about -190, about -1 to about -180, about -1 to about -17 0, about -1 to about -160, about -1 to about -150, about -1 to about -140, about -1 to about -130, about -1 to about -120, about -1 to about -110, about -1 to about -100, about -1 to about -90, about -1 to about -80, about -1 to about -70, about -1 to about -60, about -1 to about -50, about -1 to about -40, about -1 to about -30, or about -1 to about -20.

[0164]

[0190] In some embodiments, the ASO is complementary to a targeted portion of a PHIP pre-mRNA, e.g., a PHIP NMD exon-containing pre-mRNA, that is upstream (in the 5' direction) (e.g., in the direction designated by a negative number) of the 3' splice site (or 5' end) of the included exon in the PHIP pre-mRNA. In some embodiments, the ASO is complementary to a targeted portion of a PHIP pre-mRNA, e.g., a PHIP NMD exon-containing pre-mRNA, that is within a region of about -1 to about -500 relative to the 3' splice site (or 5' end) of the included exon. In some embodiments, the ASO is complementary to a targeted portion of a PHIP pre-mRNA, e.g., a PHIP NMD exon-containing pre-mRNA, that is within a region of -1 to -40,000 relative to the 3' splice site of the included exon.In some embodiments, the ASO is about -1 to about -40,000, about -1 to about -30,000, about -1 to about -20,000, about -1 to about -15,000, about -1 to about -10,000, about -1 to about -5,000, about -1 to about -4,000, about -1 to about -3,000, about -1 to about -2,000, about -1 to about -1,000, about -1 to about -50 0, about -1 to about -490, about -1 to about -480, about -1 to about -470, about -1 to about -460, about -1 to about -450, about -1 to about -440, about -1 to about -430, about -1 to about -420, about -1 to about -410, about -1 to about -400, about -1 to about -390, about -1 to about -380, about -1 to about -370, about -1 to about -360, about -1 to about -350, about -1 to about -340, about -1 to about about -330, about -1 to about -320, about -1 to about -310, about -1 to about -300, about -1 to about -290, about -1 to about -280, about -1 to about -270, about -1 to about -260, about -1 to about -250, about -1 to about -240, about -1 to about -230, about -1 to about -220, about -1 to about -210, about -1 to about -200, about -1 to about -190, about -1 to about -180, about -1 to about -170, The ASO is complementary to a targeting moiety within a region of about -1 to about -160, about -1 to about -150, about -1 to about -140, about -1 to about -130, about -1 to about -120, about -1 to about -110, about -1 to about -100, about -1 to about -90, about -1 to about -80, about -1 to about -70, about -1 to about -60, about -1 to about -50, about -1 to about -40, about -1 to about -30, or about -1 to about -20. In some embodiments, the ASO is complementary to a targeting moiety within a region of about -1 to about -100, about -100 to about -200, about -200 to about -300, about -300 to about -400, or about -400 to about -500 relative to the 3' splice site of the encompassing exon.

[0165]

[0191] In some embodiments, the ASO is complementary to a targeted portion of a PHIP pre-mRNA, e.g., a PHIP NMD exon-containing pre-mRNA, that is downstream (in the 3' direction) (e.g., in the direction designated by a positive number) of the 3' splice site (5' end) of an included exon in a PHIP pre-mRNA, e.g., a PHIP NMD exon-containing pre-mRNA. In some embodiments, the ASO is complementary to a targeted portion of a PHIP pre-mRNA that is within a region of about +1 to about +40,000 relative to the 3' splice site of the included exon. In some embodiments, the ASO is at least about +1 to about +40,000, about +1 to about +30,000, about +1 to about +20,000, about +1 to about +15,000, about +1 to about +10,000, about +1 to about +5,000, about +1 to about +4,000, about +1 to about +3,000, about +1 to about +2,000, about +1 to about +1,000, about +1 to about +500, About +1 to about +490, about +1 to about +480, about +1 to about +470, about +1 to about +460, about +1 to about +450, about +1 to about +440, about +1 to about +430, about +1 to about +420, about +1 to about +410, about +1 to about +400, about +1 to about +390, about +1 to about +380, about +1 to about +370, about +1 to about +360, about +1 to about +350, about +1 to about +340, about +1 to about +330 , about +1 to about +320, about +1 to about +310, about +1 to about +300, about +1 to about +290, about +1 to about +280, about +1 to about +270, about +1 to about +260, about +1 to about +250, about +1 to about +240, about +1 to about +230, about +1 to about +220, about +1 to about +210, about +1 to about +200, about +1 to about +190, about +1 to about +180, about +1 to about +170, about +1 to about +16 or about +1 to about +10.

[0166]

[0192] In some embodiments, the targeted portion of a PHIP pre-mRNA, e.g., a PHIP NMD exon-containing pre-mRNA, is within the region of +100 relative to the 5' splice site (3' end) of the included exon to -100 relative to the 3' splice site (5' end) of the included exon. In some embodiments, the targeted portion of a PHIP NMD exon-containing pre-mRNA is within an NMD exon. In some embodiments, the targeted portion of a PHIP NMD exon-containing pre-mRNA includes the boundary between the pseudoexon and the intron.

[0167]

[0193] ASOs can be any length suitable for effective enhancement of specific binding and splicing. In some embodiments, ASOs are 8-50 nucleobases. For example, ASOs can be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases long. In some embodiments, ASOs are more than 50 nucleobases long. In some embodiments, the ASO is 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 nucleobases, 8-15 nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 nucleobases, 10-50 nucleobases, 10-40 nucleobases, 10-35 nucleobases, 10-30 nucleobases, 10-25 nucleobases, 10-20 nucleobases, 10-15 nucleobases, 11-50 nucleobases, 11-40 nucleobases, 11-35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11-15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12 ~30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, 12-15 nucleobases, 13-50 nucleobases, 13-40 nucleobases, 13-35 nucleobases, 13-30 nucleobases, 13-25 nucleobases, 13-20 nucleobases, 14-50 nucleobases, 14-40 nucleobases, 14-35 nucleobases, 14-30 nucleobases, 14-25 nucleobases, 14-20 nucleobases , 15-50 nucleobases, 15-40 nucleobases, 15-35 nucleobases, 15-30 nucleobases, 15-25 nucleobases, 15-20 nucleobases, 20-50 nucleobases, 20-40 nucleobases, 20-35 nucleobases, 20-30 nucleobases, 20-25 nucleobases, 25-50 nucleobases, 25-40 nucleobases, 25-35 nucleobases, or 25-30 nucleobases in length. In some embodiments, the ASO is 18 nucleotides in length. In some embodiments, the ASO is 15 nucleotides in length. In some embodiments, the ASO is 25 nucleotides in length.

[0168]

[0194] In some embodiments, two or more ASOs are used that have different chemical properties but are complementary to the same targeting portion of a pre-mRNA, e.g., an NMD exon-containing pre-mRNA. In some embodiments, two or more ASOs are used that are complementary to different targeting portions of a pre-mRNA, e.g., an NMD exon-containing pre-mRNA.

[0169]

[0195] In some embodiments, the antisense oligonucleotides of the present disclosure are chemically linked to one or more moieties or conjugates, such as targeting moieties or other conjugates that enhance the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholesteryl moieties, aliphatic chains, such as dodecanediol or undecyl residues, polyamines or polyethylene glycol chains, or adamantane acetic acid. Oligonucleotides containing lipophilic moieties and preparation methods are described in the published literature. In embodiments, the antisense oligonucleotides are conjugated to moieties, such as, but not limited to, abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), lipids, or polyhydrocarbon compounds. The conjugate can be linked to one or more of any nucleotides, including antisense oligonucleotides, at any of several positions of the sugar, base, or phosphate group, for example, using a linker, as understood in the art and described in the literature. The linker can include a bivalent or trivalent branched linker. In an embodiment, the conjugate is attached to the 3' end of the antisense oligonucleotide. Methods for preparing oligonucleotide conjugates are described, for example, in U.S. Patent No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides," incorporated herein by reference.

[0170]

[0196] In some embodiments, the nucleic acid targeted by the ASO is a PHIP pre-mRNA, e.g., an NMD exon-containing pre-mRNA, expressed in a cell, e.g., a eukaryotic cell. In some embodiments, the term "cell" may refer to a population of cells. In some embodiments, the cell is in a subject. In some embodiments, the cell is isolated from a subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is a pathology or disease-related cell or cell line. In some embodiments, the cell is in vitro (e.g., in cell culture). Pharmaceutical Compositions

[0197] The pharmaceutical compositions or preparations comprising the described compositions and agents for use in any of the described methods, such as antisense oligonucleotides, can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the open literature.In embodiments, the pharmaceutical compositions or preparations for treating subjects comprise an effective amount of any antisense oligomer as described herein, or its pharmaceutically acceptable salt, solvate, hydrate, or ester.The pharmaceutical preparations comprising antisense oligomers can further comprise pharmaceutically acceptable excipients, diluents, or carriers.

[0171]

[0198] Pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and represent a reasonable benefit / risk ratio. (See, e.g., S. M. Berge et al., J. Pharmaceutical Sciences, 66:1-19 (1977), incorporated herein by reference for this purpose. Salts may be added in the form of a salt during the final isolation and purification of the compounds. They can be prepared in situ or separately by reacting the free base form with a suitable organic acid. Examples of pharma- ceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other documented methodologies such as ion exchange. Other pharma-ceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptone, etc. Examples of the salts include glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc.Further pharma-ceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed where appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates.

[0172]

[0199] In some embodiments, the composition is formulated into any of many possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. In embodiments, the composition is formulated as a suspension in an aqueous, non-aqueous, or mixed medium. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. Suspensions may also contain stabilizers. In embodiments, pharmaceutical formulations or compositions of the present disclosure include, but are not limited to, solutions, emulsions, microemulsions, foams, or liposome-containing formulations (e.g., cationic or non-cationic liposomes).

[0173]

[0200] The pharmaceutical compositions or formulations described herein may optionally include one or more penetration enhancers, carriers, excipients, or other active or inactive ingredients known to those skilled in the art or described in the published literature. In embodiments, liposomes also include sterically stabilized liposomes, e.g., liposomes that include one or more specialized lipids. These specialized lipids result in liposomes with enhanced circulation life. In embodiments, the sterically stabilized liposomes include one or more glycolipids or are derivatized with one or more hydrophilic polymers, e.g., polyethylene glycol (PEG) moieties. In some embodiments, a surfactant is included in the pharmaceutical formulation or composition. The use of surfactants in drug products, formulations, and emulsions is well known in the art. In embodiments, the present disclosure uses a penetration enhancer to provide efficient delivery of antisense oligonucleotides, e.g., to aid diffusion across cell membranes and / or to enhance the permeability of lipophilic drugs. In some embodiments, the penetration enhancer is a surfactant, a fatty acid, a bile salt, a chelating agent, or a non-chelating non-surfactant.

[0174]

[0201] In some embodiments, the pharmaceutical formulation comprises more than one antisense oligonucleotide. In embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent. Combination therapy

[0202] In some embodiments, the ASO disclosed in the present disclosure can be used in combination with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents can include small molecules. For example, the one or more additional therapeutic agents can include small molecules described in WO2016128343A1, WO2017053982A1, WO2016196386A1, WO201428459A1, WO201524876A2, WO2013119916A2, and WO2014209841A2, which are incorporated herein in their entirety. In some embodiments, the one or more additional therapeutic agents include ASO that can be used to correct intron retention. Treatment of the subject

[0203] Any of the compositions provided herein can be administered to an individual. An "individual" can be used interchangeably with a "subject" or a "patient." An individual can be a mammal, e.g., a human, or an animal such as a non-human primate, rodent, rabbit, rat, mouse, horse, donkey, goat, cat, dog, cow, pig, or sheep. In embodiments, an individual is a human. In embodiments, an individual is a fetus, embryo, or child. In other embodiments, an individual can be another eukaryotic organism, e.g., a plant. In some embodiments, a composition provided herein is administered to a cell ex vivo.

[0175]

[0204] In some embodiments, the compositions provided herein are administered to an individual as a method of treating a disease or disorder. In some embodiments, the individual has a genetic disease, such as any of the diseases described herein. In some embodiments, the individual is at risk of having a disease, such as any of the diseases described herein. In some embodiments, the individual is at increased risk of having a disease or disorder caused by an insufficient amount of protein or insufficient activity of a protein. When an individual is at "increased risk" of having a disease or disorder caused by an insufficient amount of protein or insufficient activity of a protein, the method involves a preventative or prophylactic treatment. For example, an individual may be at increased risk of having such a disease or disorder due to a family history of the disease. Typically, an individual at increased risk of having such a disease or disorder will benefit from a prophylactic treatment (e.g., by preventing or delaying the onset or progression of the disease or disorder). In an embodiment, a fetus is treated in utero, for example, by administering an ASO composition to the fetus directly or indirectly (e.g., via the mother).

[0176]

[0205] In some cases, the subject pharmaceutical compositions and methods are applicable to the treatment of conditions or diseases associated with PHIP deficiency. In some cases, the subject pharmaceutical compositions and methods are applicable to the treatment of Chung-Jansen syndrome (CHUJANS), autosomal dominant disorders, intellectual disability, speech delay, anxiety, autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), aggressive behavior, facial dysmorphism, cafe au lait spots, overweight syndrome caused by PHIP haploinsufficiency, developmental delay, obesity, or dysmorphism.

[0177]

[0206] In some cases, the therapeutic agent comprises an oligonucleotide. In some cases, the therapeutic agent comprises a vector, e.g., a viral vector, that expresses an oligonucleotide that binds to a target region of a pre-mRNA that encodes a target peptide sequence. The methods provided herein can be adapted to contact a cell with an agent, e.g., a vector that encodes an oligonucleotide, such that the agent binds to the pre-mRNA in the cell and modulates pre-mRNA processing. In some cases, the viral vector comprises an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, a herpes simplex viral (HSV) viral vector, a retroviral vector, or any applicable viral vector. In some cases, the therapeutic agent comprises a gene editing tool that is configured to modify a gene that encodes a target peptide sequence such that a region of the gene that encodes an inefficient translation region is deleted. In some cases, the gene editing tool comprises a vector, e.g., a viral vector, for gene editing based on CRISPR-Cas9, TALEN, zinc finger, or other applicable technology.

[0178]

[0207] The suitable route of administration of the ASO of the present disclosure may vary depending on the cell type to which delivery of the ASO is desired. The ASO of the present disclosure may be administered to a patient parenterally, for example, by intravitreal, intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous, or intravenous injection.

[0179]

[0208] In embodiments, antisense oligonucleotides are administered by any method known in the art together with one or more agents that can facilitate the penetration of the antisense oligonucleotides of interest across the blood-brain barrier.For example, delivery of agents by administration of adenoviral vectors to motor neurons of muscle tissue is described in U.S. Patent No. 6,632,427, "Adenoviral-vector-mediated gene transfer into medullary motor neurons," which is incorporated herein by reference.Direct delivery of vectors to the brain, for example, the striatum, thalamus, hippocampus, or substantia nigra, is described in U.S. Patent No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain," which is incorporated herein by reference.

[0180]

[0209] In some embodiments, the antisense oligonucleotide is linked or conjugated to an agent that provides desirable pharmaceutical or pharmacodynamic properties. In embodiments, the antisense oligonucleotide is coupled to an agent known in the art that enhances penetration or transport across the blood-brain barrier, such as an antibody against the transferrin receptor. In embodiments, the antisense oligonucleotide is linked to a viral vector, for example, to make the antisense compound more effective or to increase transport across the blood-brain barrier. In some embodiments, osmotic blood-brain barrier disruption is achieved by the addition of sugars, such as mesoerythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myoinositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, This is supported by infusions of adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-) lyxose, or amino acids such as glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine.Methods and materials for enhancing blood-brain barrier penetration are described, for example, in U.S. Pat. No. 9,193,969, entitled "Compositions and methods for selective delivery of oligonucleotide molecules to specific neuron types," U.S. Pat. No. 4,866,042, entitled "Method for the delivery of genetic material across the blood brain barrier," U.S. Pat. No. 6,294,520, entitled "Material for passage through the blood-brain barrier," and U.S. Pat. No. 6,936,589, entitled "Parental delivery systems," each of which is incorporated herein by reference.

[0181]

[0210] In some embodiments, the therapeutic agent comprises a modified snRNA, e.g., a modified human snRNA. In some embodiments, the therapeutic agent comprises a vector, e.g., a viral vector, encoding the modified snRNA. In some embodiments, the modified snRNA is a modified U1 snRNA (see, e.g., Alanis et al., Human Molecular Genetics, 2012, Vol. 21, No. 11, pp. 2389-2398). In some embodiments, the modified snRNA is a modified U7 snRNA (see, e.g., Gadgil et al., J Gene Med. 2021;23:e3321). In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that hybridizes to a PHIP NMD exon-containing pre-mRNA. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that includes one or more sequences of the ASOs disclosed herein. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that hybridizes to the sequence of a PHIP NMD exon-containing pre-mRNA having a mutation. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that includes two or more sequences that hybridize to two or more target regions of a PHIP NMD exon-containing pre-mRNA. For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to at least eight consecutive nucleic acids of a PHIP NMD exon-containing pre-mRNA. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that hybridizes to any target region of a PHIP NMD exon-containing pre-mRNA disclosed herein. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that includes two or more sequences that hybridize to two or more target regions of a PHIP NMD exon-containing pre-mRNA.For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to one or more sequences of an intron containing an NMD exon (e.g., exon 15x of PHIP (e.g., exon (GRCh38 / hg38:chr6 79004373-79004436)) of a pre-mRNA transcript, or to an NMD exon activating regulatory sequence in the same intron. For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to a region within an NMD exon or a region upstream or downstream of an NMD exon (e.g., exon 15x of PHIP (e.g., exon (GRCh38 / hg38:chr6 79004373-79004436))). In some embodiments, the modified snRNA has a 5' region that is modified to include a single-stranded nucleotide sequence that hybridizes to a PHIP NMD exon-containing pre-mRNA.

[0182]

[0211] For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to a region that overlaps with an intron upstream of an NMD exon and an NMD exon (e.g., exon 15x of PHIP (e.g., exon (GRCh38 / hg38:chr6 79004373-79004436))). For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to a region that overlaps with an intron downstream of an NMD exon and an NMD exon (e.g., exon 15x of PHIP (e.g., exon (GRCh38 / hg38:chr6 79004373-79004436))).

[0183]

[0212] For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that is complementary to an intron sequence downstream of an NMD exon (e.g., exon 15x of PHIP (e.g., exon (GRCh38 / hg38:chr6 79004373-79004436))). For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that is complementary to a 3' splice site of an intron sequence upstream or downstream of an NMD exon (e.g., exon 15x of PHIP (e.g., exon (GRCh38 / hg38:chr6 79004373-79004436))). For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that is complementary to the 5' splice site of an intron sequence downstream of an NMD exon (e.g., exon 15x of PHIP (e.g., exon (GRCh38 / hg38:chr6 79004373-79004436) of PHIP).

[0184]

[0213] For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that is complementary to an intron sequence upstream of an NMD exon (e.g., exon 15x of PHIP (e.g., exon (GRCh38 / hg38:chr6 79004373-79004436))). For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that is complementary to a splice site of an intron sequence upstream of an NMD exon (e.g., exon 15x of PHIP (e.g., exon (GRCh38 / hg38:chr6 79004373-79004436))). For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that is complementary to a 3' splice site of an intron sequence upstream of an NMD exon (e.g., exon 15x of PHIP (e.g., exon (GRCh38 / hg38:chr6 79004373-79004436))). For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that is complementary to a 5' splice site of an intron sequence upstream of an NMD exon (e.g., exon 15x of PHIP (e.g., exon (GRCh38 / hg38:chr6 79004373-79004436))).

[0185]

[0214] In some embodiments, subjects treated using the methods and compositions are evaluated for improvement in their condition using any method known and described in the art. Methods for identifying additional ASOs that induce exon skipping

[0215] Methods for identifying or determining ASOs that induce exon skipping of PHIP NMD exon-containing pre-mRNA are also within the scope of the present disclosure. For example, the method can include identifying or determining ASOs that induce pseudoexon skipping of PHIP NMD exon-containing pre-mRNA. ASOs that specifically hybridize to different nucleotides in the target region of the pre-mRNA may be screened to identify or determine ASOs that enhance the rate and / or extent of splicing of the target intron. In some embodiments, the ASOs may block or interfere with the binding site of a splicing repressor / silencer. Any method known in the art may be used to identify (determine) ASOs that result in a desired effect (e.g., pseudoexon skipping, protein or functional RNA production) when hybridized to the target region of an exon. These methods can also be used to identify ASOs that induce exon skipping of included exons by binding to a targeted region in an intron adjacent to an included exon or a non-included exon. One example of a method that can be used is provided below.

[0186]

[0216] A round of screening, referred to as ASO "walking", may be performed using ASOs designed to hybridize to a target region of the pre-mRNA. For example, the ASOs used for ASO walking may be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 3' splice site of the included exon (e.g., a portion of the sequence of the exon located upstream of the target / included exon) to approximately 100 nucleotides downstream of the 3' splice site of the target / included exon, and / or from approximately 100 nucleotides upstream of the 5' splice site of the included exon to approximately 100 nucleotides downstream of the 5' splice site of the target / included exon (e.g., a portion of the sequence of the exon located downstream of the target / included exon). For example, a first ASO of 15 nucleotides in length may be designed to specifically hybridize to nucleotides +6 to +20 relative to the 3' splice site of the target / included exon. The second ASO may be designed to specifically hybridize to nucleotides +11 to +25 relative to the 3' splice site of the target / inclusive exon. The ASO is designed to span the target region of the pre-mRNA. In embodiments, the ASO can be tiled more closely, for example, every 1, 2, 3, or 4 nucleotides. Additionally, the ASO can be tiled from 100 nucleotides downstream of the 5' splice site to 100 nucleotides upstream of the 3' splice site. In some embodiments, the ASO can be tiled from about 1,160 nucleotides upstream of the 3' splice site to about 500 nucleotides downstream of the 5' splice site. In some embodiments, the ASO can be tiled from about 500 nucleotides upstream of the 3' splice site to about 1,920 nucleotides downstream of the 3' splice site.

[0187]

[0217] 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 a target pre-mRNA (e.g., an NMD exon-containing pre-mRNA described herein). The exon skipping effect of each ASO may be assessed by any method known in the art, for example, by reverse transcription (RT)-PCR using primers spanning the splice junction, as described in Example 2. A reduction or absence of longer RT-PCR products in ASO-treated cells compared to control ASO-treated cells, produced using primers spanning a region containing an inclusion exon (e.g., including an exon adjacent to an NMD exon), indicates enhanced splicing of the target NMD exon. In some embodiments, exon skipping efficiency (or splicing efficiency of splicing introns containing NMD exons), the ratio of spliced ​​pre-mRNA to unspliced ​​pre-mRNA, the rate of splicing, or the extent of splicing can be improved using the ASOs described herein. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine whether each ASO has achieved the desired effect (e.g., enhanced functional protein production). Any method known in the art for evaluating and / or quantifying protein production can be used, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0188]

[0218] A second round of screening, termed ASO "microwalking", may be performed using ASOs designed to hybridize to target regions of the pre-mRNA. The ASOs used for ASO microwalking are tiled nucleotide by nucleotide to further refine the nucleotide acid sequence of the pre-mRNA that results in exon skipping (or enhanced splicing of NMD exons) when hybridized with the ASO.

[0189]

[0219] The region defined by the ASO promoting splicing of the target intron is explored in more detail by ASO "microwalking" with ASOs spaced at one nucleotide steps apart, and by longer ASOs, typically 18-25 nucleotides.

[0190]

[0220] As described above for ASO walking, ASO microwalking is performed by delivering one or more ASOs, or a control ASO (an ASO with a scrambled sequence that is not expected to hybridize to the target region), for example by transfection, to a disease-related cell line expressing the target pre-mRNA. The splicing-inducing effect of each ASO may be evaluated by any method known in the art, for example by reverse transcription (RT)-PCR using primers spanning the NMD exon, as described herein (see, for example, Example 2). Reduction or absence of longer RT-PCR products produced using primers spanning the NMD exon in ASO-treated cells compared to those in control ASO-treated cells indicates enhanced exon skipping (or splicing of the target intron containing the NMD exon). In some embodiments, exon skipping efficiency (or splicing efficiency of splicing introns containing NMD exons), the ratio of spliced ​​pre-mRNA to unspliced ​​pre-mRNA, the rate of splicing, or the extent of splicing can be improved using the ASOs described herein. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine whether each ASO has achieved the desired effect (e.g., enhanced functional protein production). Any method known in the art for evaluating and / or quantifying protein production can be used, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0191]

[0221] ASOs that result in exon skipping (or enhanced splicing of introns containing NMD exons) and increased protein production when hybridized to a region of a pre-mRNA may be tested in vivo using animal models, such as transgenic mouse models with full-length human genes knocked in, or humanized mouse models of disease. The suitable route of administration of the ASO may vary depending on the disease and / or cell type to which delivery of the ASO is desired. The ASO may be administered, for example, by intravitreal, intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous, or intravenous injection. After administration, cells, tissues, and / or organs of the model animal can be evaluated to determine the efficacy of ASO treatment, for example, by assessing splicing (e.g., efficiency, speed, extent) and protein production by methods known in the art and described herein. The animal model may also be any phenotypic or behavioral indicator of disease or disease severity.

[0192]

[0222] Methods for identifying or validating NMD-induced exons in the presence of an NMD inhibitor, such as cycloheximide, are also within the scope of the present disclosure. An exemplary method is provided in Example 3. Specific embodiments Embodiment 1. A method of regulating expression of a target protein in a cell having a pre-mRNA transcribed from a target gene and including a nonsense-mediated RNA decay-induced exon (NMD exon), comprising contacting the cell with an agent or a vector encoding the agent, whereby the agent regulates splicing of the NMD exon from the pre-mRNA, thereby regulating the level of processed mRNA processed from the pre-mRNA and regulating expression of the target protein in the cell, wherein the target gene is a PHIP gene.

[0193] Embodiment 2. The agent is a. Binds to a targeting portion of a pre-mRNA; b. modulates the binding of factors involved in splicing of NMD exons; or c. A combination of (a) and (b); 2. The method of embodiment 1.

[0194] Embodiment 3. The method of embodiment 2, wherein the agent interferes with the binding of a factor involved in splicing of the NMD exon to the region of the targeting moiety.

[0195] Embodiment 4. The method of embodiment 2, wherein the targeting portion of the pre-mRNA is proximal to an NMD exon.

[0196] Embodiment 5. The method of embodiment 2, wherein the targeting portion of the pre-mRNA is up to about 1500, about 1000, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 80, about 70, about 60, or about 50 nucleotides upstream of the 5' end of the NMD exon.

[0197] Embodiment 6. The method of embodiment 2, wherein the targeting portion of the pre-mRNA is at least about 1500, about 1000, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 10, about 5, about 4, about 2, about 1 nucleotide upstream of the 5' end of the NMD exon.

[0198] Embodiment 7. The method of embodiment 2, wherein the targeting portion of the pre-mRNA is up to about 1500, about 1000, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 80, about 70, about 60, or about 50 nucleotides downstream of the 3' end of the NMD exon.

[0199] Embodiment 8. The method of embodiment 2, wherein the targeting portion of the pre-mRNA is at least about 1500, about 1000, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 10, about 5, about 4, about 2, about 1 nucleotide downstream of the 3' end of the NMD exon.

[0200] Embodiment 9. The method of embodiment 2, wherein the targeting portion of the pre-mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of the genomic site GRCh38 / hg38:chr6 79004373.

[0201] Embodiment 10. The method of embodiment 2, wherein the targeting portion of the pre-mRNA is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of the genomic site GRCh38 / hg38:chr6 79004373.

[0202] Embodiment 11. The method of embodiment 2, wherein the targeting portion of the pre-mRNA is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of the genomic site GRCh38 / hg38:chr6 79004436.

[0203] Embodiment 12. The method of embodiment 2, wherein the targeting portion of the pre-mRNA is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of the genomic site GRCh38 / hg38:chr6 79004436.

[0204] Embodiment 13. The method of embodiment 2, wherein the pre-mRNA targeting portion is located in an intron region between two canonical exon regions of the pre-mRNA, the intron region containing an NMD exon.

[0205] Embodiment 14. The method of embodiment 2, wherein the targeting portion of the pre-mRNA at least partially overlaps with an NMD exon.

[0206] Embodiment 15. The method of embodiment 2, wherein the targeting portion of the pre-mRNA at least partially overlaps with an intron upstream or downstream of the NMD exon.

[0207] Embodiment 16. The method of embodiment 2, wherein the targeting portion of the pre-mRNA comprises a 5' NMD exon-intron junction or a 3' NMD exon-intron junction.

[0208]

[0031] Embodiment 17. The method of embodiment 2, wherein the targeting portion of the pre-mRNA is within an NMD exon.

[0209] Embodiment 18. The method of embodiment 2, wherein the targeting portion of the pre-mRNA comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous nucleotides of an NMD exon.

[0210] Embodiment 19. The method of any one of embodiments 1-18, wherein the NMD exon (a) is within an intron sequence having at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO:2, and / or (b) comprises a sequence having at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO:3.

[0211] Embodiment 20. The method of any one of embodiments 1 to 18, wherein the NMD exon comprises the sequence of SEQ ID NO:3.

[0212]

[0036] Embodiment 21. The method of embodiment 2, wherein the targeted portion of the pre-mRNA is within the nonsense-mediated RNA decay-directed exon GRCh38 / hg38:chr6 79004373-79004436.

[0213]

[0036] Embodiment 22. The method of embodiment 2, wherein the targeted portion of the pre-mRNA is upstream or downstream of the nonsense-mediated RNA decay-directed exon GRCh38 / hg38:chr6 79004373-79004436.

[0214]

[0036] Embodiment 23. The method of embodiment 2, wherein the targeting portion of the pre-mRNA comprises the exon-intron junction of the nonsense-mediated RNA decay-directed exon GRCh38 / hg38:chr6 79004373-79004436.

[0215] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the target protein expressed from the processed mRNA is a full-length PHIP protein or a wild-type PHIP protein.

[0216] Embodiment 25. The method of any one of embodiments 1 to 23, wherein the target protein expressed from the processed mRNA is a functional PHIP protein.

[0217] Embodiment 26. The method of any one of embodiments 1 to 23, wherein the target protein expressed from the processed mRNA is at least partially functional compared to wild-type PHIP protein.

[0218] Embodiment 27. The method of any one of embodiments 1-23, wherein the target protein expressed from the processed mRNA is at least partially functional compared to full-length wild-type PHIP protein.

[0219]

[0036] Embodiment 28. The method of any one of embodiments 1-23 or 25-27, wherein the target protein expressed from the processed mRNA is a PHIP protein lacking the amino acid sequence encoded by nonsense-mediated RNA decay-directed exons GRCh38 / hg38:chr6 79004373-79004436.

[0220] Embodiment 29. The method of any one of embodiments 1 to 28, which promotes the exclusion of an NMD exon from a pre-mRNA.

[0221] Embodiment 30. The exclusion of NMD exons from pre-mRNA in cells contacted with the agent is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times 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.

[0222] Embodiment 31. The method of any one of embodiments 1 to 30, which results in an increased level of processed mRNA in the cell.

[0223] Embodiment 32. The level of processed mRNA in cells contacted with the agent is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times , about 2 to about 9 fold, about 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.

[0224] Embodiment 33. The method of any one of embodiments 1 to 32, which results in increased expression of a target protein in a cell.

[0225] Embodiment 34. The level of a target protein expressed from processed mRNA in a cell contacted with a drug is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, The method of embodiment 33, wherein the increase is 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.

[0226] Embodiment 35. The method of any one of embodiments 1-34, wherein the agent comprises an antisense oligomer having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 4-180.

[0227] Embodiment 36. The method of any one of embodiments 1 to 34, wherein the agent further comprises a gene editing molecule.

[0228]

[0036] Embodiment 37. The method of embodiment 36, wherein the gene editing molecule comprises CRISPR-Cas9.

[0229] Embodiment 38. The method of any one of embodiments 1 to 37, wherein the agent is an antisense oligomer (ASO), and the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

[0230] Embodiment 39. The method of any one of embodiments 1 to 38, wherein the agent is an antisense oligomer (ASO), and the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-NMA moiety, or a 2'-O-methoxyethyl moiety.

[0231] Embodiment 40. The method of any one of embodiments 1-39, wherein the agent is an antisense oligomer (ASO), and the antisense oligomer comprises at least one modified sugar moiety.

[0232] Embodiment 41. The method of embodiment 40, wherein each sugar moiety is a modified sugar moiety.

[0233] Embodiment 42. The agent is an antisense oligomer (ASO), and the antisense oligomer is selected from the group consisting of 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 nucleobases, 8-15 nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 nucleobases, 10-50 nucleobases, 10-40 nucleobases, 10-35 nucleobases, 10-30 nucleobases. 42. The method of any one of the preceding claims, wherein the nucleotide sequence comprises 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.

[0234]

[0036] Embodiment 43. The method of embodiment 1, comprising contacting a cell with a vector encoding an agent, wherein the vector is a viral vector.

[0235]

[0046] Embodiment 44. The method of embodiment 43, wherein the viral vector comprises an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, a herpes simplex viral (HSV) viral vector, or a retroviral vector.

[0236]

[0046] Embodiment 45. The method of embodiment 43, wherein the viral vector comprises an adeno-associated viral (AAV) vector.

[0237]

[0036] Embodiment 46. The method of embodiment 1, wherein the agent comprises a modified snRNA.

[0238] Embodiment 47. The method of embodiment 46, wherein the modified human snRNA is a modified U1 snRNA.

[0239] Embodiment 48. The method of embodiment 46, wherein the modified human snRNA is a modified U7 snRNA.

[0240] Embodiment 49. The method of embodiment 46, wherein the portion of the single-stranded nucleotide sequence of the modified human snRNA comprises a sequence that binds to the targeting portion of the pre-mRNA.

[0241] Embodiment 50. The method of any one of embodiments 46-49, comprising contacting a cell with a vector encoding a modified snRNA.

[0242] Embodiment 51. The method of any one of embodiments 1 to 50, further comprising assessing the mRNA level or expression level of the target protein.

[0243] Embodiment 52. The method of any one of embodiments 1 to 51, wherein the agent is a therapeutic agent.

[0244] Embodiment 53. A pharmaceutical composition comprising a therapeutic agent according to embodiment 52 or a vector encoding the therapeutic agent according to embodiment 52 and a pharma- ceutically acceptable excipient.

[0245] Embodiment 54. A pharmaceutical composition comprising a therapeutic agent or a vector encoding a therapeutic agent and a pharma- ceutically acceptable excipient, wherein the therapeutic agent comprises an antisense oligomer having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 4-180.

[0246] Embodiment 55. The pharmaceutical composition of embodiment 54, wherein the therapeutic agent comprises an antisense oligomer having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 55-180.

[0247] Embodiment 56. The pharmaceutical composition of any one of embodiments 53 to 55, which is formulated for intraventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, implantation, or intravenous injection.

[0248] Embodiment 57. The pharmaceutical composition of any one of embodiments 53 to 55, which is formulated for intrathecal or intracerebrospinal injection.

[0249] Embodiment 58. The pharmaceutical composition of any one of embodiments 53-57, further comprising a second therapeutic agent.

[0250] Embodiment 59. The pharmaceutical composition of embodiment 58, wherein the second therapeutic agent comprises a small molecule.

[0251] Embodiment 60. The pharmaceutical composition of embodiment 58, wherein the second therapeutic agent comprises an antisense oligomer.

[0252] Embodiment 61. The pharmaceutical composition of embodiment 58, wherein the second therapeutic agent corrects intron retention.

[0253] Embodiment 62. A composition comprising an antisense oligomer having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 4-180, wherein the antisense oligomer comprises a backbone modification, a sugar moiety modification, or a combination thereof.

[0254] Embodiment 63. A composition comprising a viral vector encoding a polynucleotide comprising an antisense oligomer, wherein the antisense oligomer consists of a sequence selected from the group consisting of SEQ ID NOs: 4-180.

[0255] Embodiment 64. The composition of embodiment 63, wherein the polynucleotide further comprises a modified snRNA.

[0256] Embodiment 65. The composition of embodiment 64, wherein the modified human snRNA is a modified U1 snRNA.

[0257] Embodiment 66. The composition of embodiment 64, wherein the modified human snRNA is a modified U7 snRNA.

[0258] Embodiment 67. The composition of embodiment 62 or 63, wherein the antisense oligomer has at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 55-180.

[0259] Embodiment 68. A method of treating a disease or condition in a subject or reducing the likelihood of developing a disease or condition by modulating expression of a target protein in cells of a subject in need thereof, comprising contacting cells of the subject with the pharmaceutical composition of any one of embodiments 53-61.

[0260] Embodiment 69. The method of embodiment 68, wherein the disease or condition is associated with a loss-of-function mutation in the PHIP gene.

[0261] Embodiment 70. The method of embodiment 68 or 69, wherein the disease or condition is associated with haploinsufficiency of the PHIP gene and the subject has a first allele that encodes a functional PHIP protein and a second allele in which no PHIP protein is produced or is produced at reduced levels, or a second allele that encodes a non-functional or partially functional PHIP protein.

[0262] Embodiment 71. The method of any one of embodiments 68-70, wherein the disease or condition comprises an intellectual disability disease or condition.

[0263] Embodiment 72. The method of any one of embodiments 68-70, wherein the disease or condition comprises Chung-Jansen syndrome (CHUJANS), an autosomal dominant disorder, intellectual disability, speech delay, anxiety, autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), aggressive behavior, facial dysmorphism, cafe au lait spots, overweight syndrome caused by PHIP haploinsufficiency, developmental delay, obesity, or dysmorphism.

[0264] Embodiment 73. The method of any one of embodiments 68-70, wherein the disease or condition comprises Chung-Jansen syndrome.

[0265] Embodiment 74. The method of any one of embodiments 68-70, wherein the disease or condition comprises intellectual disability.

[0266] Embodiment 75. The disease or condition is associated with an autosomal recessive mutation in the PHIP gene and the subject: (i) the PHIP protein is not produced or is produced at a reduced level compared to a wild-type allele; or (ii) the PHIP protein produced is non-functional or partially functional compared to the wild-type allele; The first allele, and (iii) the PHIP protein is produced at a reduced level compared to a wild-type allele, and the PHIP protein produced is at least partially functional compared to the wild-type allele; or (iv) the PHIP protein produced is partially functional compared to the wild-type allele; The second allele, 70. The method of embodiment 68 or 69, comprising the steps of:

[0267] Embodiment 79. The method of any one of embodiments 68-76, wherein the subject is a human.

[0268] Embodiment 80. The method of any one of embodiments 68-76, wherein the subject is a non-human animal.

[0269] Embodiment 81. The method of any one of embodiments 68-76, wherein the subject is a fetus, embryo, or child.

[0270] Embodiment 82. The method of any one of embodiments 68-76, wherein the cells are ex vivo.

[0271] Embodiment 83. The method of any one of embodiments 68-76, wherein the pharmaceutical composition is administered 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.

[0272] Embodiment 84. The method of any one of embodiments 68-76, wherein the pharmaceutical composition is administered by intrathecal or intracerebrospinal injection.

[0273] Embodiment 85. The method of any one of embodiments 68-76, for treating a disease or condition.

[0274] Embodiment 86. A composition comprising an agent or a vector encoding said agent that modulates splicing of a nonsense-mediated RNA decay-directed exon (NMD exon) from a pre-mRNA transcribed from a target gene and containing the NMD exon, thereby modulating the level of processed mRNA processed from the pre-mRNA and modulating expression of a target protein in a cell harboring the pre-mRNA, wherein the target gene is a PHIP gene.

[0275] Embodiment 87. A composition comprising an agent or a vector encoding said agent for treating a disease or condition in a subject by modulating splicing of a nonsense-mediated RNA decay-directed exon (NMD exon) from a pre-mRNA transcribed from a target gene and comprising the NMD exon, thereby modulating the level of processed mRNA processed from the pre-mRNA and modulating expression of a target protein in a cell of a subject in need of such treatment, wherein the target gene is a PHIP gene.

[0276] Embodiment 88. A pharmaceutical composition comprising the composition of embodiment 84 or 85 and a pharma- ceutically acceptable excipient and / or delivery vehicle.

[0277] Embodiment 89. The method or composition or pharmaceutical composition according to any one of embodiments 1 to 88, wherein the target protein is pleckstrin homology domain interacting protein (PHIP). EXAMPLES

[0278]

[0223] The present disclosure will be more specifically illustrated by the following examples. However, it should be understood that the present disclosure is not limited in any way by these examples. Example 1: Identification of NMD-induced exon inclusion events in transcripts by RNAseq using sequencing Whole transcriptome shotgun sequencing was performed using RNA sequencing to reveal a snapshot of the transcripts produced by the genes described herein and to identify NMD exon inclusion events. To this end, polyAβ was derived from nuclear and cytoplasmic fractions of human neural progenitor cells (ReN) and human astrocytes treated with cycloheximide (CHX) or DMSO control. +RNA is isolated and a cDNA library is constructed using Illumina's TruSeq stranded mRNA library preparation kit. The library is paired-end sequenced, resulting in 100 nucleotide reads that map to the human genome (December 2017, GRCh38 / hg38 assembly). Figure 3 shows the identification of exemplary nonsense-mediated mRNA decay (NMD)-induced exons in the PHIP gene.

[0279]

[0225] Exemplary gene and intron sequences are summarized in Table 1 (SEQ ID NO: indicates the corresponding nucleotide sequence, represented by the gene number). Exemplary intron sequences are summarized in Table 2. PHIP protein sequences are provided in Table 3. Tables 4, 5 and 6 list sequences of PHIP antisense oligomers of the disclosure.

[0280] [Table 1]

[0281] [Table 2-1]

[0282] [Table 2-2]

[0283] [Table 2-3]

[0284] [Table 2-4]

[0285] [Table 2-5]

[0286] [Table 3]

[0287] Example 2: Identification of NMD exons via cycloheximide / puromycin / DMSO treatment RT-PCR analysis using total RNA from water (H), DMSO-treated (D), or puromycin (P) or cycloheximide-treated (C) human embryonic kidney cells (HEK293), human neuroblastoma cells (SK-N-AS), and human neural progenitor cells (ReN) and primers in exons 15 and 16 of the PHIP gene was used to confirm the presence of bands corresponding to NMD-induced exons. Densitometric analysis of the bands was performed to calculate the % NMD exon inclusion of the total transcript. Treatment of cells with cycloheximide or puromycin to inhibit NMD can result in an increase in products corresponding to NMD-induced exons. Figure 4A shows the confirmation of exemplary NMD exons in the PHIP gene transcript using DMSO, cycloheximide, or puromycin treatment in HEK, SK-N-AS, and ReN VM cells, respectively. U87 cells and astrocytes were similarly treated with cycloheximide (C), puromycin (P), or DMSO (D). Primers in exons 15 and 16 of the PHIP gene were used to confirm the presence of bands corresponding to NMD-inducible exons. Results for U87 cells and astrocytes are shown in Figure 4B.

[0288] Similar results were found in mouse embryonic fibroblast (MEF) cells treated with water (HO), DMSO, cycloheximide (CHX), and puromycin. Four different amplification cycles (26, 29, 32, and 35) are shown. NMD exon 15x inclusion was found to be conserved in MEF cells, as shown in FIG. 4C. Example 3: PHIP NMD events are conserved in non-human primates RNA was extracted from the indicated brain regions of cynomolgus monkeys (Macaca fascicularis). cDNA was generated and the product of interest was amplified by PCR using a forward primer in exon 15 and a reverse primer in exon 16. Two different amplification cycles (28 and 31) are shown in FIG. Example 4: NMD exon region ASO walking ASO walking was performed for the NMD exon region, targeting sequences immediately upstream of the 3' splice site, across the 3' splice site, the NMD exon, across the 5' splice site, and downstream of the 5' splice site, using 2'-MOE ASOs in a PS backbone. ASOs were designed to cover these regions by moving 5 nucleotides at a time, excluding the last 3' splice site ASO and the first exon ASO, and the last exon ASO and the first 5' splice site ASO, separated by 3 nucleotides. Figure 6 shows an ASO walk for an exemplary PHIP NMD exon region.

[0289]

[0230] The designations of ASOs can be explained as follows: IVS14X = the intronic sequence portion of the canonical intron (canonical intron 15) following the canonical exon (canonical exon 15) immediately preceding the NMD exon. Ex15X = the NMD exon (within canonical intron 15). IVS15X = the intronic sequence portion of the canonical intron (canonical intron 15) immediately following the NMD exon and before the canonical exon (canonical exon 16) immediately following the NMD exon. For ASO walking, see nomenclature above. Note that ASOs that span IVS14X and Ex15X or Ex15X and IVS15X are designated as "XX". ASOs that are entirely within Ex15X are not designated as "XX". Example 5: NMD exon region ASO walking assessed by RT-PCR ASO walking sequences were evaluated by RT-PCR and TaqMan qPCR. Treated control ASOs ("mock" or "-") or 2'-MOE ASOs targeting the PHIP NMD exon regions shown in Table 4 below were transfected into HEK293 cells using Lipofectamine RNAiMax or nucleofected into ReN cells. Products corresponding to PHIP non-productive mRNA (RT-PCR) and productive mRNA (TaqMan) were quantified, normalized to an internal control, and fold changes relative to the control were plotted. Figure 7 shows the evaluation of various exemplary ASO walking against exemplary NMD exon regions in ReN cells via RT-PCR and TaqMan qPCR. Measurement of PHIP mRNA abundance was performed by Taqman qPCR using a probe spanning exons 15 and 16, and RT-PCR using primers in exons 15 and 16, on cells following 3 hours of cycloheximide treatment and 24 hours after treatment with 3 uM of an exemplary ASO.

[0290] [Table 4-1]

[0291] [Table 4-2]

[0292]

[0232] Figure 8 shows the measurement of the amount of PHIP mRNA. Analysis was performed on ReN cells 24 hours after treatment with a few exemplary ASOs at 3 μM in the absence of cycloheximide treatment by Taqman qPCR using probes spanning exons 15 and 16 (canonical) and exons 35 and 36 (GE). Various ASOs increased the production of canonical and total (GE) PHIP mRNA and reduced the amount of non-productive mRNA produced.

[0293]

[0233] Figure 9 shows the evaluation of various exemplary ASO walking against exemplary NMD exon regions in HEK293 cells via RT-PCR and TaqMan qPCR. Measurement of PHIP mRNA levels was performed on cells 24 hours after treatment with 120 nM of exemplary ASO following 3 hours of cycloheximide treatment by Taqman qPCR using a probe spanning exon 15 and exon 16, and RT-PCR using primers in exon 15 and exon 16.

[0294]

[0234] Figures 10A-10D show the evaluation of a few exemplary ASOs via RT-PCR and TaqMan qPCR against the exemplary NMD exon region 15x. Measurement of the amount of PHIP mRNA was performed on cells 24 hours after treatment with 80 nM of the exemplary ASO in the absence of cycloheximide. As shown in the figures, various ASOs increased the production of canonical PHIP mRNA and total PHIP mRNA and reduced the amount of non-productive mRNA produced. Example 6: NMD exonic ASO microwalking assessed by RT-PCR and RT-qPCR

[0235] Figure 11 shows ASO microwalking for an exemplary PHIP NMD exon region. ASO microwalking sequences (spanning exon 15x) were evaluated by RT-PCR. Cells were transfected with treated control ASO (SMN) or 2'-MOE ASO targeting the PHIP NMD exon region, as described in Table 5 below. Products corresponding to NMD exon inclusion and full length were quantified, and % NMD exon inclusion was plotted. Figure 12 shows evaluation of various exemplary ASO walks for an exemplary NMD exon region in ReN cells. Measurement of the amount of PHIP mRNA was performed on ReN cells 24 hours after transfection of 3 uM of an exemplary ASO after 3 hours of cycloheximide treatment by Taqman qPCR using a probe spanning exon 15 and exon 16 (Figure 12).

[0295] [Table 5-1]

[0296] [Table 5-2]

[0297]

[0236] Figure 13 shows the evaluation of various exemplary ASO walks against exemplary NMD exon regions in HEK293 cells. Measurement of PHIP mRNA levels was performed on HEK293 cells 24 hours after transfection of 120 nM exemplary ASOs after 3 hours of treatment with cycloheximide by Taqman qPCR using a probe spanning exon 15 and exon 16, and RT-PCR using primers in exon 15 and exon 16 (Figure 10).

[0298]

[0237] Table 6 below provides additional exemplary PHIP NMD exon region ASO microwalking sequences (across exon 15x).

[0299] [Table 6-1]

[0300] [Table 6-2]

[0301] Example 7: Dose-response assessment

[0238] Experiments were conducted to investigate the dose-response relationship of several exemplary ASOs according to some embodiments of the present disclosure.

[0302] In one experiment, ReN VM cells were nucleofected with different concentrations of ASOs, then the cells were cultured in the presence of cycloheximide for 24 hours and then harvested for RT-PCR analysis to investigate the splicing of NMD exon (15x) in the cells. As shown in Figures 14A and 14B, the cells were treated with a control ASO ("mock"), an ASO targeting SMN mRNA ("SMN"), or an exemplary ASO, such as "IVS14X:-3" (SEQ ID NO:58), "IVS14X-Ex15XX:7" (SEQ ID NO:67), "Ex15X:19" (SEQ ID NO:95), or "Ex15X:23" (SEQ ID NO:99), at 0.33 μM, 1 μM, or 3 μM, respectively. After 24 hours, the cells were harvested for RT-PCR analysis. Primers targeting exon 15 and exon 16 were used for the RT-PCR reaction, and the amplification results were visualized as shown in FIG. 14A, where the lower band was derived from the mRNA transcript without exon 15x ("canonical PHIP mRNA") and the upper band was derived from the mRNA transcript with exon 15x ("non-productive PHIP mRNA"). Quantification of the changes in the amount of canonical PHIP mRNA and non-productive PHIP mRNA in response to different treatments is summarized in FIG. 14B (normalized by their respective amounts under "mock" conditions). For all exemplary PHIP ASOs tested, the higher the concentrations they were applied at, the more canonical PHIP transcripts and the less non-productive PHIP transcripts were observed.

[0303] In another experiment, ReN VM cells were nucleofected with 3 μM of various ASOs and cultured for 72 hours before harvesting for JESS assay to investigate PHIP protein levels in cells. Figure 15A shows Western blotting images of two replicates, and Figure 15B shows plots demonstrating that all exemplary ASOs tested (IVS14X:-3, IVS14X-Ex15XX:7, Ex15X:19, and Ex15X:23) caused an increase in PHIP protein levels compared to mock conditions, but the SMN control did not. Example 8: Modulation of splicing by exemplary ASOs

[0241] This example illustrates further experiments demonstrating modulation of splicing of NMD exon 15x from PHIP pre-mRNA by an exemplary ASO according to some embodiments of the present disclosure.

[0304] [Table 7]

[0305] In one experiment, ReN VM cells were nucleofected with 3 μM ASOs and cultured in the absence of cycloheximide for 24 hours before harvesting for RT-PCR analysis. Specifically, cells were nucleofected with one of the exemplary ASOs listed in Table 7 or a control ASO ("mock") or SMN ASO, all at 3 μM. After 24 hours, cells were harvested for RT-PCR (FIGS. 16A-16B) or RT-qPCR (FIGS. 16C-16D). As shown in FIG. 16B-16D, the exemplary ASOs resulted in a decrease in the amount of non-productive PHIP transcripts with exon 15x (FIG. 16B) and an increase in the amount of PHIP transcripts without exon 15x as shown by Taqman qPCR using probes spanning exons 15 and 16 (canonical; FIG. 16C) and exons 35 and 36 (GE; FIG. 16D).

[0306] In another experiment, ASOs were added to the culture medium of ReN VM cells at 60 μM for free uptake, and the cells were then cultured for 3 days in 3D conditions and in the absence of cycloheximide before being harvested for RT-PCR analysis. Specifically, cells were treated with one of the exemplary ASOs listed in Table 7, or a control ASO ("mock") or SMN ASO, all at 3 μM. After 24 hours, cells were harvested for RT-PCR (FIG. 17A) or RT-qPCR (FIGS. 17B-17D). As shown in Figures 17B-17D, the exemplary ASOs resulted in a decrease in the amount of non-productive PHIP transcripts with exon 15x (Figure 17B) and an increase in the amount of PHIP transcripts without exon 15x as shown by Taqman qPCR using probes spanning exons 15 and 16 (canonical; Figure 17C) and exons 35 and 36 (GE; Figure 17D).

[0307]

[0244] 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 now 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 may be used in implementing the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

Claims

**Claim 1**: A composition comprising an agent or a vector encoding the agent, wherein the agent regulates the splicing of a nonsense mutation-dependent RNA degradation mechanism-inducing exon (NMD exon) from a messenger RNA (mRNA) precursor in a cell, the mRNA precursor is transcribed from a target gene and contains the NMD exon, thereby regulating the level of the processed mRNA processed from the mRNA precursor and regulating the expression of a target protein in the cell, and the target gene is the PHIP gene. **Claim 2** The composition according to claim 1, wherein the targeting portion of the mRNA precursor is located upstream of genomic locus GRCh38 / hg38:chr6 79004373 by at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides. **Claim 3** The composition according to claim 1, wherein the targeting portion of the mRNA precursor is located upstream of genomic locus GRCh38 / hg38:chr6 79004373 by about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides. **Claim 4** The composition according to claim 1, wherein the targeting portion of the mRNA precursor is located downstream of genomic locus GRCh38 / hg38:chr6 79004436 by at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides. **Claim 5** The composition according to claim 1, wherein the targeting portion of the mRNA precursor is located downstream of genomic locus GRC h38 / hg38: chr6 79004436 by about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides.

6. The composition according to claim 1, wherein the targeting portion of the mRNA precursor is located in an intron region between two canonical exon regions of the mRNA precursor, and the intron region contains an NMD exon.

7. The targeting portion of the mRNA precursor is (a) at least partially overlapping with an NMD exon, (b) at least partially overlapping with an intron upstream or downstream of an NMD exon, (c) including a 5' NMD exon-intron junction or a 3' NMD exon-intron junction, or (d) within an NMD exon, of the composition according to claim 1.

8. The composition according to claim 1, wherein the targeting portion of the mRNA precursor comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides of an NMD exon.

9. The composition according to claim 1, wherein the targeting portion of the mRNA precursor is within the nonsense mutation-dependent RNA decay mechanism-inducing exon GRC h38 / hg38: chr6 79004373-79004436.

10. The composition according to claim 1, wherein the target protein expressed from the processed mRNA is a PHIP protein lacking the amino acid sequence encoded by the nonsense mutation-dependent RNA decay mechanism-inducing exon GRC h38 / hg38: chr6 79004373-79004436.

11. The composition according to claim 1, wherein the agent comprises an antisense oligomer having at least 80%, at least 90%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 4-161 and 163-182.

12. The composition according to claim 1, wherein the agent is an antisense oligomer (ASO), and the antisense oligomer comprises a backbone modification including phosphorothioate linkage or phosphorodiamidate linkage.

13. The composition according to claim 1, wherein the agent is an antisense oligomer (ASO), and the antisense oligomer consists of 8 to 50 nucleobases.

14. The composition according to claim 1, comprising a vector encoding the agent, wherein the vector is a viral vector.

15. The composition according to claim 14, wherein the viral vector comprises an adenoviral vector, an adeno-associated virus (AAV) vector, a lentiviral vector, a herpes simplex virus (HSV) viral vector, or a retroviral vector.

16. The composition according to claim 15, wherein the viral vector comprises an adeno-associated virus (AAV) vector.

17. The composition according to claim 1, wherein the agent comprises modified snRNA.

18. The composition according to claim 17, wherein the modified human snRNA is modified U1 snRNA.

19. The composition according to claim 17, wherein the modified human snRNA is modified U7 snRNA.

20. Use of the composition according to claim 1 for the manufacture of a medicament for treating a disease or condition or reducing the likelihood of developing a disease or condition in a subject in need thereof.

21. The use according to claim 20, wherein the disease or condition is associated with a loss-of-function mutation in the PHIP gene.

22. The use according to claim 21, wherein the disease or condition is associated with haploinsufficiency of the PHIP gene, and the subject has a first allele encoding a functional PHIP protein and a second allele that does not produce or produces PHIP protein at a reduced level, or a second allele encoding a non-functional or partially functional PHIP protein.

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