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.

1 .- 12 . (canceled) 
     
     
         13 . A method for altering a target beta thalassemia-associated polynucleotide sequence in a cell comprising contacting the beta thalassemia-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 beta thalassemia-associated polynucleotide sequence, wherein the target beta thalassemia-associated polynucleotide sequence is cleaved, and wherein the efficiency of alteration is from about 50% to about 80%. 
     
     
         14 .- 18 . (canceled) 
     
     
         19 . The method according to  claim 13 , 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. 
     
     
         20 .- 21 . (canceled) 
     
     
         22 . The method according to  claim 13 , wherein the Cas protein is Cas9 protein from any bacterial species or 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. 
     
     
         23 .- 24 . (canceled) 
     
     
         25 . The method according to  claim 13 , wherein the Cas protein is complexed with the one to two ribonucleic acids. 
     
     
         26 .- 32 . (canceled) 
     
     
         33 . The method according to  claim 13 , wherein the target motif is G(N)19NGG. 
     
     
         34 .- 41 . (canceled) 
     
     
         42 . The method according to  claim 13 , wherein the alteration results in reduced expression of the target polynucleotide sequence, a knock out of the target polynucleotide sequence, or correction of the target polynucleotide sequence from an undesired sequence to a desired sequence. 
     
     
         43 .- 57 . (canceled) 
     
     
         58 . The method according to  claim 13 , 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. 
     
     
         59 .- 107 . (canceled) 
     
     
         108 . The method according to  claim 13 , wherein the target polynucleotide sequence is HBB. 
     
     
         109 . The method according to  claim 108 , wherein at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of SEQ ID NOS. 814-908, or at least a 12 nucleotide fragment thereof. 
     
     
         110 . The method according to  claim 108 , wherein at least one of the one to two ribonucleic acids comprises a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOS. 814-908, or at least a 12 nucleotide fragment thereof. 
     
     
         111 .- 173 . (canceled) 
     
     
         174 . The method according to  claim 13 , wherein the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. 
     
     
         175 .- 193 . (canceled) 
     
     
         194 . The method according to  claim 13 , 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. 
     
     
         195 .- 272 . (canceled) 
     
     
         273 . The method according to  claim 13 , wherein the cell was selected for Cas protein expression. 
     
     
         274 . The method according to  claim 13 , wherein the target motif of the target beta thalassemia-associated polynucleotide sequence comprises nucleotides located between position 5246806 and position 5248263 of human chromosome 11.