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 sickle cell disease (SCD)-associated polynucleotide sequence in a cell ex vivo comprising contacting the SCD-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 SCD-associated polynucleotide sequence comprising nucleotides located between position 5246806 and position 5248263 of human chromosome 11, wherein the target SCD-associated polynucleotide sequence is cleaved. 
     
     
         2 . A method for altering a target sickle cell disease (SCD)-associated polynucleotide sequence in a cell ex vivo comprising (a) contacting the SCD-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 SCD-associated polynucleotide sequence, wherein the target SCD-associated polynucleotide sequence is cleaved, and (b) contacting the cleaved target SCD-associated polynucleotide sequences with an exogenously introduced DNA repair template to initiate homology-directed repair of the target SCD-associated polynucleotide. 
     
     
         3 . A method for altering a target HBB gene sequence in a cell ex vivo comprising (a) contacting the HBB 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 HBB gene sequence, wherein the target HBB gene sequence is cleaved, and (b) contacting the cleaved target HBB gene sequences with an exogenously introduced DNA repair template to initiate homology-directed repair of the target HBB 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. 
     
     
         11 . A method according to  claim 2  or  3 , wherein the target motif is located between position 5246806 and position 5248263 of human chromosome 11.