Modulation of gene expression and screening for deregulated protein expression

Abstract

Disclosed herein include compositions and methods of modulating protein expression that utilizes an activator or a repressor of a non-sense mediated RNA decay switch exon (NSE). In some embodiments, also included herein are compositions and methods of modulating protein expression that uses an agent that targets a transposed element.

What is claimed is: 
     
         1 . A method of modulating protein expression, the method comprising:
 binding an agent to a target motif within a pre-processed mRNA transcript in a target cell,   wherein the pre-processed mRNA transcript is processed into a processed mRNA transcript in the target cell,   wherein the pre-processed mRNA transcript is a wild-type pre-processed mRNA transcript encoding a functional protein,   wherein binding of the agent to the target motif represses activation of a cryptic splice site of a pseudoexon within the pre-processed mRNA and represses inclusion of the pseudoexon in the processed mRNA transcript, and   wherein inclusion of the pseudoexon in the processed mRNA transcript downregulates expression of the functional protein in the target cell.   
     
     
         2 . The method of  claim 1 , wherein inclusion of the pseudoexon in the processed mRNA transcript introduces a premature termination codon (PTC) in a translational reading frame of the processed mRNA transcript. 
     
     
         3 . The method of  claim 1 , wherein the target motif is within the pseudoexon. 
     
     
         4 . The method of  claim 1 , wherein the target motif comprises the cryptic splice site. 
     
     
         5 . The method of  claim 1 , wherein the target motif is within an intronic region between two canonical exons of the pre-processed mRNA transcript. 
     
     
         6 . The method of  claim 1 , wherein the target motif is from 7 to 100 nucleotides in length. 
     
     
         7 . The method of  claim 1 , wherein the target motif is from 10 to 50 nucleotides in length. 
     
     
         8 . The method of  claim 1 , wherein the target cell is a cell of a human subject. 
     
     
         9 . The method of  claim 1 , wherein the pre-processed mRNA transcript is an endogenous pre-processed mRNA transcript. 
     
     
         10 . The method of  claim 1 , wherein the functional protein translated from the processed mRNA transcript is a wild-type protein. 
     
     
         11 . The method of  claim 1 , wherein the functional protein translated from the processed mRNA transcript is a full-length protein. 
     
     
         12 . The method of  claim 1 , wherein a protein translated from an equivalent processed mRNA transcript that is processed from the pre-processed mRNA but comprises the pseudoexon is not a full-length protein. 
     
     
         13 . The method of  claim 1 , wherein the agent comprises a polynucleic acid polymer that hybridizes to the target motif within the pre-processed mRNA transcript. 
     
     
         14 . The method of  claim 13 , wherein the polynucleic acid polymer is modified at a nucleoside moiety, at a phosphate moiety, at a 5′ terminus, at a 3′ terminus, or a combination thereof. 
     
     
         15 . The method of  claim 13 , wherein the polynucleic acid polymer comprises an artificial nucleotide selected from the group consisting of 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), 2′-O-N-methylacetamido (2-O-NMA), a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1′,5′-anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2′-fluoro N3-P5′-phosphoramidite. 
     
     
         16 . The method of  claim 13 , wherein the method comprises expressing the polynucleic acid polymer from a vector encoding the polynucleic acid polymer. 
     
     
         17 . The method of  claim 16 , wherein the vector is a viral vector. 
     
     
         18 . The method of  claim 17 , wherein the viral vector is adeno-associated viral vector. 
     
     
         19 . The method of  claim 1 , wherein the polynucleic acid polymer is associated with a delivery vehicle suitable for delivering the polynucleic acid polymer to cells. 
     
     
         20 . The method of  claim 19 , wherein the delivery vehicle comprises a cell-penetrating peptide. 
     
     
         21 . The method of  claim 19 , wherein the delivery vehicle comprises a viral vector. 
     
     
         22 . A method of treating or preventing a disease or condition in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition that comprises (i) an agent, or a vector encoding the agent, and (ii) a pharmaceutically acceptable excipient and/or a delivery vehicle,
 wherein the agent interacts with a target motif within a pre-processed mRNA transcript in a target cell of the subject,   wherein the pre-processed mRNA transcript is processed into a processed mRNA transcript in the target cell,   wherein the pre-processed mRNA transcript is a wild-type pre-processed mRNA transcript encoding a functional protein,   wherein hybridization of the polynucleic acid polymer to the target motif represses activation of a cryptic splice site of a pseudoexon within the pre-processed mRNA and represses inclusion of the pseudoexon in the processed mRNA transcript, and   wherein inclusion of the pseudoexon in the processed mRNA transcript downregulates expression of the functional protein in the target cell.   
     
     
         23 . The method of  claim 22 , wherein the disease or condition is cancer or a genetic disorder or condition selected from an autosomal dominant disorder, an autosomal recessive disorder, an X-linked dominant disorder, an X-linked recessive disorder, a Y-linked disorder, a mitochondrial disease, a multifactorial disorder and a polygenic disorder. 
     
     
         24 . The method of  claim 22 , wherein the disease or condition is an autosomal dominant disorder. 
     
     
         25 . The method of  claim 22 , wherein the target motif is within the pseudoexon. 
     
     
         26 . The method of  claim 22 , wherein the target motif is within an intronic region between two canonical exons of the pre-processed mRNA transcript. 
     
     
         27 . The method of  claim 22 , wherein the target motif is from 7 to 100 nucleotides in length. 
     
     
         28 . The method of  claim 22 , wherein the target motif is from 10 to 50 nucleotides in length. 
     
     
         29 . The method of  claim 22 , wherein the target motif comprises the cryptic splice site. 
     
     
         30 . The method of  claim 22 , wherein inclusion of the pseudoexon in the processed mRNA transcript introduces a premature termination codon (PTC) in a translational reading frame of the processed mRNA transcript. 
     
     
         31 . The method of  claim 22 , wherein the agent comprises a polynucleic acid polymer that hybridizes to the target motif within the pre-processed mRNA transcript. 
     
     
         32 . The method of  claim 31 , wherein the polynucleic acid polymer is modified at a nucleoside moiety, at a phosphate moiety, at a 5′ terminus, at a 3′ terminus, or a combination thereof. 
     
     
         33 . The method of  claim 31 , wherein the polynucleic acid polymer comprises an artificial nucleotide selected from the group consisting of 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), 2′-O-N-methylacetamido (2-O-NMA), a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1′,5′-anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2′-fluoro N3-P5′-phosphoramidite. 
     
     
         34 . The method of  claim 31 , wherein the functional protein translated from the processed mRNA transcript is a full-length protein. 
     
     
         35 . The method of  claim 31 , wherein a protein translated from an equivalent processed mRNA transcript that is processed from the pre-processed mRNA transcript but comprises the pseudoexon is not a full-length protein. 
     
     
         36 . The method of  claim 31 , wherein the pharmaceutical composition comprises the vector encoding the polynucleic acid polymer, and wherein the vector is a viral vector. 
     
     
         37 . The method of  claim 31 , wherein the pharmaceutical composition comprises the polynucleic acid polymer, wherein the polynucleic acid polymer is associated with the delivery vehicle suitable for delivering the polynucleic acid polymer to cells of the subject, and wherein the delivery vehicle comprises a cell-penetrating peptide or a viral vector. 
     
     
         38 . The method of  claim 22 , wherein the pharmaceutical composition further comprises a genomic editing molecule or a polynucleotide encoding the genomic editing molecule. 
     
     
         39 . A pharmaceutical composition comprising:
 an agent, or a vector encoding the agent, wherein the agent interacts with a target motif within a wild-type pre-processed mRNA transcript to repress activation of a cryptic splice site of a pseudoexon within the pre-processed mRNA; and   a pharmaceutically acceptable excipient and/or a delivery vehicle.