Multiplexed shrnas and uses thereof

Abstract

Nucleic acid molecules such as shRNA dusters and artificial miRNA dusters are disclosed. Also disclosed are methods of use, compositions, cells, viral particles, and kits relating to the nucleic acid molecules disclosed herein.

What is claimed is: 
     
         1 . An shRNA cluster encoding a plurality of shRNA-like molecules, wherein each of the plurality of shRNA-like molecules comprises:
 a stem region comprising an artificial RNA molecule comprising a guide strand and a passenger strand, wherein the guide strand is substantially complementary to a target mRNA, wherein the artificial RNA molecule, when expressed in a cell, substantially inhibits expression of the target mRNA; and   a backbone region comprising a 5′ flanking region, a terminal loop region, and a 3′ flanking region,   provided that the backbone region of at least one shRNA-like molecule is not repeated among the plurality of shRNA-like molecules and the backbone regions of the plurality of shRNA-like molecules are not identical to the backbone regions of pri-miRNAs encoded by a naturally-occurring miRNA cluster.   
     
     
         2 . The shRNA cluster of  claim 1 , wherein the plurality of shRNA-like molecules comprise 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more shRNA-like molecules. 
     
     
         3 . The shRNA cluster of  claim 1 , wherein the plurality of shRNA-like molecules comprise 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more shRNA-like molecules. 
     
     
         4 . The shRNA cluster of  claim 1 , wherein the shRNA cluster encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more miRNA-based shRNA molecules (shRNA-miR molecules) and/or the shRNA cluster encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more shRNA molecules that are not derived from naturally-occurring pri-miRNAs. 
     
     
         5 . The shRNA cluster of  claim 1 , wherein the target mRNA is encoded by a human gene chosen from CCR5 or CXCR4 or a viral gene chosen from Gag, Env, Tat, Pol2, Pol1, or Vif of HIV-1. 
     
     
         6 . An artificial miRNA cluster encoding a plurality of modified pri-miRNA-like molecules, the artificial miRNA cluster comprising:
 a first nucleotide sequence encoding a first modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a first miRNA; and   a second nucleotide sequence encoding a second modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a second miRNA,   provided that the first and second miRNAs are not in the same naturally-occurring miRNA cluster.   
     
     
         7 . The artificial miRNA cluster of  claim 6 , wherein each of the plurality of modified pri-miRNA-like molecules comprises:
 a stem region comprising an artificial RNA molecule comprising a guide strand and a passenger strand, wherein the guide strand is substantially complementary to a target mRNA;   a terminal loop region that is at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, a terminal loop region of a naturally-occurring pri-miRNA;   a 5′ flanking region that is at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, a 5′ flanking region of a naturally-occurring pri-miRNA; and   a 3′ flanking region that is at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, a 3′ flanking region of a naturally-occurring pri-miRNA.   
     
     
         8 . The artificial miRNA cluster of  claim 6 , wherein the artificial miRNA cluster further comprises a third nucleotide sequence encoding a third modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a third miRNA, wherein the third miRNA is not in the same naturally-occurring miRNA cluster as the first, second, or both miRNAs. 
     
     
         9 . The artificial miRNA cluster of  claim 8 , wherein the artificial miRNA cluster further comprises a fourth nucleotide sequence encoding a fourth modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a fourth miRNA, wherein the fourth miRNA is not in the same naturally-occurring miRNA cluster as the first, second, or third miRNA. 
     
     
         10 . The artificial miRNA cluster of  claim 9 , wherein the artificial miRNA cluster further comprises a fifth nucleotide sequence encoding a fifth modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a fifth miRNA, wherein the fifth miRNA is not in the same naturally-occurring miRNA cluster as the first, second, third, or fourth miRNA. 
     
     
         11 . The artificial miRNA cluster of  claim 10 , wherein the artificial miRNA cluster further comprises a sixth nucleotide sequence encoding a sixth modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a sixth miRNA, wherein the sixth miRNA is not in the same naturally-occurring miRNA cluster as the first, second, third, fourth, or fifth miRNA. 
     
     
         12 . The artificial miRNA cluster of  claim 11 , wherein the artificial miRNA cluster further comprises a seventh nucleotide sequence encoding a seventh modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a seventh miRNA, wherein the seventh miRNA is not in the same naturally-occurring miRNA cluster as the first, second, third, fourth, fifth, or sixth miRNA. 
     
     
         13 . The artificial miRNA cluster of  claim 7 , wherein the plurality of artificial RNA molecules, when expressed in a cell, substantially inhibit the expression of 2, 3, 4, 5, 6, 7, or all of the genes chosen from human CCR5, human CXCR4, HIV-1 Gag, HIV-1 Env, HIV-1 Tat, HIV-1 Pol2, HIV-1 Pol1, or HIV-1 Vif. 
     
     
         14 . A cell comprising the artificial miRNA cluster of  claim 6 . 
     
     
         15 . A method of treating or preventing a disorder, the method comprising administering the cell of  claim 14  to a subject, thereby treating or preventing the disorder. 
     
     
         16 . The method of  claim 15 , wherein the disorder is an HIV-1 infection or AIDS. 
     
     
         17 . A pharmaceutical composition comprising the cell of  claim 14 . 
     
     
         18 . A method of producing a cell that expresses a plurality of artificial RNA molecules, the method comprising contacting a cell with the artificial miRNA cluster of  claim 6 , and culturing the cell under conditions that allow expression of the artificial RNA molecules. 
     
     
         19 . A viral particle comprising the artificial miRNA cluster of  claim 6 . 
     
     
         20 . A method of designing a nucleic acid molecule, the method comprising altering a backbone region of a naturally-occurring pri-miRNA, wherein the backbone region comprises a 5′ flanking region, a terminal loop region, and a 3′ flanking region, wherein the method comprises one, two, or all of the following:
 1) adding one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides in one, two, or all of the 5′ flanking region, the terminal loop region, or the 3′ flanking region; 
 2) deleting one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides in one, two, or all of the 5′ flanking region, the terminal loop region, or the 3′ flanking region; or 
 3) substituting one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides in one, two, or all of the 5′ flanking region, the terminal loop region, or the 3′ flanking region.