Targeted augmentation of nuclear gene output

Abstract

Provided herein are methods and compositions for increasing production of a target protein or functional RNA by a cell.

1 .- 71 . (canceled) 
     
     
         72 . A method of treating a disease or condition in a subject in need thereof, wherein the subject has a deficient amount or activity of a first protein or a first functional RNA; wherein the deficient amount or activity of the first protein or the first functional RNA is caused by haploinsufficiency of the first protein or the first functional RNA, the method comprising:
 administering a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises (i) an antisense oligomer (ASO) or a vector encoding the ASO and (ii) a pharmaceutically acceptable excipient;   wherein the ASO binds to targeted region of a retained-intron-containing pre-mRNA (MC pre-mRNA) that comprises a retained intron, an exon flanking a 5′ splice site of the retained intron, and an exon flanking a 3′ splice site of the retained intron, and wherein the RIC pre-mRNA encodes a target protein or a target functional RNA; wherein the first protein is different from the target protein and the first functional RNA is different from the target functional RNA;   wherein the ASO increases splicing efficiency of the RIC pre-mRNA by modulating splicing of the retained intron from the RIC pre-mRNA in a cell of the subject as compared to a cell of a subject with the disease or condition that has not been administered the pharmaceutical composition.   
     
     
         73 . The method of  claim 72 , wherein the target protein or the target functional RNA is a compensating protein or a compensating functional RNA that functionally augments or replaces the first protein, or the first functional RNA. 
     
     
         74 . The method of  claim 72 , wherein expression of the target protein or the target functional RNA is increased in a cell of the subject as compared to a cell of a subject with the disease or condition that has not been administered the pharmaceutical composition. 
     
     
         75 . The method of  claim 72 , wherein administration of the pharmaceutical composition promotes constitutive splicing of the retained intron from the RIC pre-mRNA. 
     
     
         76 . The method of  claim 72 , wherein the haploinsufficiency is caused by a mutation. 
     
     
         77 . The method of  claim 72 , wherein cells of the subject produce the target protein or the target functional RNA in a form that is fully functional compared to a corresponding wild-type protein or wild-type functional RNA. 
     
     
         78 . The method of  claim 72 , wherein a total amount of an mRNA encoding the target protein or the target functional RNA produced in the cell of the subject is increased by at least about 1.1-fold compared to a total amount of an mRNA encoding the target protein or the target functional RNA produced in a cell of a subject with the disease or condition that has not been administered the pharmaceutical composition. 
     
     
         79 . The method of  claim 72 , wherein the targeted region of the RIC pre-mRNA is within a region +6 relative to the 5′ splice site of the retained intron to −16 relative to the 3′ splice site of the retained intron. 
     
     
         80 . The method of  claim 72 , wherein the ASO comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. 
     
     
         81 . The method of  claim 72 , wherein the ASO comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2′-O-methyl moiety, a 2′-Fluoro moiety, or a 2′-O-methoxyethyl moiety. 
     
     
         82 . The method of  claim 72 , wherein the ASO comprises a modified sugar moiety. 
     
     
         83 . The method of  claim 72 , wherein the ASO consists of from 8 to 50 nucleobases. 
     
     
         84 . The method of  claim 83 , wherein the disease or condition is selected from the group consisting of thrombotic thrombocytopenic purpura, tuberous sclerosis complex, polycystic kidney disease, familial dysautonomia, retinitis pigmentosa type 10, retinitis pigmentosa type 11, cystic fibrosis, retinoblastoma, beta thalassemia, and sickle cell disease. 
     
     
         85 . The method of  claim 72 , wherein the subject is a human. 
     
     
         86 . The method of  claim 72 , wherein the subject is a non-human animal. 
     
     
         87 . The method of  claim 72 , wherein the pharmaceutical composition is administered to the subject by intravitreal injection, intrathecal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, subretinal injection, intracerebroventricular injection, intramuscular injection, topical application, or implantation. 
     
     
         88 . The method of  claim 72 , wherein the ASO is encoded by a viral vector. 
     
     
         89 . The method of  claim 88 , where the viral vector is a adenovirus vector. 
     
     
         90 . The method of  claim 72 , wherein the ASO does not increase the amount of the target protein or the target functional RNA by modulating aberrant splicing resulting from mutation of a gene encoding the target protein or the target functional RNA. 
     
     
         91 . A method for identifying from among a set of antisense oligomers (ASOs) an ASO that increases an amount of mRNA encoding a target protein or a target functional RNA by inducing constitutive splicing of a retained intron from a retained-intron-containing pre-mRNA (RIC pre-mRNA) that comprises at least one retained intron and that encodes the target protein or the target functional RNA, wherein the ASOs in the set are tiled every 1 to 5 nucleotides, and wherein each ASO in the set of ASOs hybridizes to a target region of the RIC pre-mRNA that is about 100 nucleotides upstream of the 5′ splice site of the at least one retained intron to about 100 nucleotides downstream of the 5′ splice site of the at least one retained intron; or
 about 100 nucleotides upstream of the 3′ splice site of the at least one retained intron to about 100 nucleotides downstream of the 3′ splice site of the at least one retained intron; 
 the method comprising: (a) delivering a first ASO in the set of ASOs to a cell comprising the RIC pre-mRNA; (b) measuring an amount of the RIC pre-mRNA and measuring an amount of mRNA encoding the target protein or the target functional RNA in the cell to which the first ASO was delivered; (c) measuring an amount of the RIC pre-mRNA and measuring an amount of mRNA encoding a target protein or a target functional RNA in a control cell; and (d) comparing the amounts of the RIC pre-mRNA and the mRNA measured in (b) and (c); wherein the first ASO is identified as an ASO that increases an amount of mRNA encoding the target protein or the target functional RNA by inducing constitutive splicing of the at least one retained intron from the RIC pre-mRNA based on an observed decrease in the amount of the RIC pre-mRNA and an observed increase in the amount of mRNA encoding the target protein or the target functional RNA in the cell to which the first ASO was delivered compared to a control cell; and repeating steps (a) through (d) with additional ASOs in the set of ASOs as needed to identify an ASO that increases an amount of mRNA from a gene in a cell by inducing constitutive splicing of a retained intron from the RIC pre-mRNA. 
 
     
     
         92 . A pharmaceutical composition to comprising (i) an antisense oligomer (ASO) or a vector encoding the ASO and (ii) a pharmaceutically acceptable excipient;
 wherein the ASO comprises a sequence that hybridizes to a targeted region of a retained-intron-containing pre-mRNA (RIC pre-mRNA), wherein the RIC pre-mRNA comprises a retained intron, an exon flanking a 5′ splice site of the retained intron, and an exon flanking a 3′ splice site of the retained intron, and wherein the RIC pre-mRNA encodes a target protein or a target functional RNA; wherein the ASO promotes splicing of the RIC pre-mRNA by modulating splicing of the retained intron from the RIC pre-mRNA.