THERAPEUTIC USES OF GENOME EDITING WITH CRISPR/Cas SYSTEMS

Abstract

Disclosed herein are methods, compositions, and kits for high efficiency, site-specific genomic editing of cells for treating or preventing genetic blood disorders.

What is claimed is: 
     
         1 . A method for altering a target severe combined immune deficiency disease (SCID)-associated polynucleotide sequence in a cell ex vivo comprising contacting the SCID-associated polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target SCID-associated polynucleotide sequence, wherein the target SCID-associated polynucleotide sequence is cleaved. 
     
     
         2 . A method for altering a target sever combined immune deficiency disease (SCID)-associated polynucleotide sequence in a cell ex vivo comprising (a) contacting the SCID-associated polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target SCID-associated polynucleotide sequence, wherein the target SCID-associated polynucleotide sequence is cleaved, and (b) contacting the cleaved target SCID-associated polynucleotide sequences with an exogenously introduced DNA repair template to initiate homology-directed repair of the target SCID-associated polynucleotide. 
     
     
         3 . A method for altering a target IL2RG gene sequence in a cell ex vivo comprising (a) contacting the IL2RG gene sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target IL2RG gene sequence, wherein the target IL2RG gene sequence is cleaved, and (b) contacting the cleaved target IL2RG gene sequences with an exogenously introduced DNA repair template to initiate homology-directed repair of the target IL2RG gene sequence. 
     
     
         4 . A method according to any one of  claims 1 - 3 , wherein the Cas protein is  Streptococcus pyogenes  Cas9 protein or a functional portion thereof selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. 
     
     
         5 . A method according to  claim 4 , wherein the Cas protein is  Streptococcus pyogenes  Cas9 protein. 
     
     
         6 . A method according to any one of  claims 1 - 3 , wherein the Cas protein is complexed with the one to two ribonucleic acids. 
     
     
         7 . A method according to any one of  claims 1 - 3 , wherein the target motif is G(N) 19 NGG or (N) 20 NGG. 
     
     
         8 . A method according to any one of  claims 1 - 3 , wherein the cell is selected from the group consisting of a peripheral blood cell, a stem cell, a pluripotent cell, a hematopoietic stem cell, a CD34+ cell, a CD34+ mobilized peripheral blood cell, a CD34+ cord blood cell, a CD34+ bone marrow cell, a CD34 + CD38-Lineage-CD90 + CD45RA −  cell, a primary human cell, a non-transformed human cell, and combinations thereof. 
     
     
         9 . A method according to any one of  claims 1 - 3 , wherein the Cas protein is encoded by a nucleic acid sequence comprising a modified nucleic acid selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5 ′-triphosphate, 4-thiouridine-5′-triphosphate, 5,6-dihydrouridine-5′-triphosphate, and 5-azauridine-5′-triphosphate. 
     
     
         10 . A method according to any one of  claims 1 - 3 , wherein at least one of the ribonucleic acids is a modified ribonucleic acid comprising one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 5,6-dihydrouridine-5′-triphosphate, and 5-azauridine-5′-triphosphate.