Compositions and methods comprising engineered short nuclear rna (snrna)

Abstract

SnRNA systems comprising engineered stem loops are disclosed herein.

What is claimed is: 
     
         1 . An RNA-targeting nucleic acid molecule comprising an engineered snRNA esnRNA, wherein the esnRNA system comprises an engineered stem loop (eSL) comprising one or more nucleic acid sequences selected from SEQ ID NO: 1-SEQ ID NO: 11, SEQ ID NO: 146-SEQ ID NO: 148, SEQ ID NO: 163, or SEQ ID NO: 186-205. 
     
     
         2 . The RNA-targeting nucleic acid molecule of  claim 1 , wherein the esnRNA comprises a targeting sequence (TS) that targets a target RNA of interest. 
     
     
         3 . The RNA-targeting nucleic acid molecule of  claim 2 , wherein the target RNA is pre-mRNA or mRNA sequence. 
     
     
         4 . The RNA-targeting nucleic acid molecule of  claim 2 , wherein the target RNA of interest is a microsatellite repeat RNA. 
     
     
         5 . The RNA-targeting nucleic acid molecule of  claim 4 , wherein the microsatellite repeat RNA is selected from the group consisting of CUG, CAG, and GGGGCC+CCCCGG. 
     
     
         6 . The RNA-targeting nucleic acid molecule of  claim 2 , wherein the target RNA is a sequence encoding DMD. 
     
     
         7 . The RNA-targeting nucleic acid molecule of  claim 6 , wherein the targeting sequence is selected from SEQ ID NO: 206 or SEQ ID NO: 207. 
     
     
         8 . The RNA-targeting nucleic acid molecule of  claim 6 , wherein the targeting sequence comprises one or more nucleic acid sequences set forth in SEQ ID NO: 208-SEQ ID NO: 227. 
     
     
         9 . The RNA-targeting nucleic acid molecule of  claim 1 , wherein the esnRNA comprises two targeting sequences that target two RNAs of interest. 
     
     
         10 . The RNA-targeting nucleic acid molecule of  claim 9 , wherein the two TSs are a fusion sequence. 
     
     
         11 . The RNA-targeting nucleic acid molecule of  claim 1 , wherein the esnRNA comprises an Sm binding domain (SmBD) selected from the group consisting of a U1, U2, U4, and U5 SmBD. 
     
     
         12 . The RNA-targeting nucleic acid molecule of  claim 11 , wherein the SmBD comprises a nucleic acid sequence set forth in any one of SEQ ID NO: 31-SEQ ID NO: 38, or SEQ ID NO: 164. 
     
     
         13 . The RNA-targeting nucleic acid molecule of  claim 1 , wherein the esnRNA comprises a 5′ interaction stabilizer domain (5′ISD) comprising a nucleotide sequence selected any one of SEQ ID NO: 12-SEQ ID NO: 23. 
     
     
         14 . The RNA targeting nucleic acid of  claim 1 , wherein the esnRNA comprises a nucleic acid sequence set forth in any one of SEQ ID NO: 65-SEQ ID NO: 119 or SEQ ID NO: 179-SEQ ID NO: 185. 
     
     
         15 . A vector comprising one or more esnRNA of  claim 1 . 
     
     
         16 . The vector of  claim 15 , wherein the viral vector is an AAV vector. 
     
     
         17 . The AAV vector of  claim 16 , wherein the snRNA is operably linked to a promoter. 
     
     
         18 . The AAV vector of  claim 16 , wherein the snRNA is operably linked to a U7 promoter or a U1 promoter. 
     
     
         19 . The AAV vector of  claim 6 , wherein the snRNA is operably linked to a downstream terminator (DT). 
     
     
         20 . The AAV vector of  claim 16 , wherein the snRNA is operably linked to a U7 downstream terminator or a U1 downstream terminator. 
     
     
         21 . The AAV vector of  claim 16 , wherein the vector comprises at least one, at least two, at least three, at least four, or at least five esnRNA. 
     
     
         22 . The AAV vector of  claim 21 , wherein the least one, at least two, at least three, at least four, or at least five esnRNA each target the same target RNA sequences. 
     
     
         23 . The AAV vector of  claim 21 , wherein the least one, at least two, at least three, at least four, or at least five esnRNA target two or more target RNA sequences 
     
     
         24 . The AAV vector of  claim 21 , wherein each esnRNA is separated by a buffer sequence. 
     
     
         25 . The AAV vector of  claim 24 , wherein the buffer sequence comprises a nucleic acid sequence set forth in any one SEQ ID NO: 24-SEQ ID NO: 30. 
     
     
         26 . The AAV vector of  claim 20 , wherein the vector comprises a nucleic acid sequence set forth in any one of SEQ ID NO: 123-SEQ ID NO: 143, SEQ ID NO: 168-SEQ ID NO: 178, or SEQ ID NO: 231-SEQ ID NO: 233. 
     
     
         27 . A method of targeting one or more target RNAs of interest and blocking, knocking down, editing, exon-skipping or splicing the one or more target RNAs, comprising contacting the esnRNA of  claim 1  with a cell comprising the one or more target RNAs. 
     
     
         28 . A DMD exon 51 RNA-targeting nucleic acid molecule comprising a spacer sequence set forth in any one of SEQ ID NO: 206-SEQ ID NO: 230. 
     
     
         29 . A method of treating a disease or disorder in a subject comprising administering an RNA-targeting nucleic acid molecule of  claim 1  or an AAV vector of  claim 15 . 
     
     
         30 . The method of  claim 29 , wherein the disease or disorder is associated with a toxic repeat RNA sequence. 
     
     
         31 . The method of  claim 29 , wherein the toxic repeat RNA sequence is a CAG, CUG, GGCCCC, CCGGG, or GGCCC+CCGGGG RNA repeat. 
     
     
         32 . The method of  claim 29 , wherein the disease or disorder is myotonic dystrophy (DM1) or Huntington's disease (HD). 
     
     
         33 . The method of  claim 29 , wherein the disease or disorder is Duchenne Muscular Dystrophy. 
     
     
         34 . The method of  claim 33 , wherein the RNA-targeting nucleic acid molecule or AAV vector targets an RNA sequence encoding dystrophin (DMD). 
     
     
         35 . The method of  claim 34 , wherein the RNA sequence encoding DMD comprises an intronic or exonic sequence. 
     
     
         36 . The method of  claim 35 , wherein the exonic sequence comprises exon 51, or a flanking region thereof, of DMD. 
     
     
         37 . The method of  claim 29 , wherein the administration is administration is intravenous, intramuscular, subpial, intrathecal, intraparenchymal, intrathecal, intrastriatal, subcutaneous, intradermal, intraperitoneal, intratumoral, intravenous, intraocular, and/or parenteral administration.