US2015176013A1PendingUtilityA1

THERAPEUTIC USES OF GENOME EDITING WITH CRISPR/Cas SYSTEMS

60
Assignee: HARVARD COLLEGEPriority: Apr 4, 2013Filed: Oct 8, 2014Published: Jun 25, 2015
Est. expiryApr 4, 2033(~6.7 yrs left)· nominal 20-yr term from priority
Inventors:Kiran MusunuruChad A. CowanDerrick J. Rossi
A61P 35/00A61P 31/00A61P 43/00A61P 31/18A61K 48/00C12N 15/907C12N 15/63C12N 5/0606
60
PatentIndex Score
0
Cited by
0
References
0
Claims

Abstract

Disclosed herein are methods, compositions, and kits for high efficiency, site-specific genomic editing of cells.

Claims

exact text as granted — not AI-modified
1 . A method for altering a target polynucleotide sequence in a cell comprising contacting the 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 polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%. 
     
     
         2 . A method for treating or preventing a disorder associated with expression of a polynucleotide sequence in a subject, the method comprising (a) altering a target polynucleotide sequence in a cell ex vivo by contacting the 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 polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequence. 
     
     
         3 . A method for simultaneously altering multiple target polynucleotide sequences in a cell comprising contacting the polynucleotide sequences with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%. 
     
     
         4 . A method for treating or preventing a disorder associated with expression of polynucleotide sequences in a subject, the method comprising (a) altering target polynucleotide sequences in a cell ex vivo by contacting the polynucleotide sequences with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequences. 
     
     
         5 . A method according to  claim 1 , wherein the Cas protein is  Streptococcus pyogenes  Cas9 protein or a functional portion thereof. 
     
     
         6 . The method according to  claim 5 , wherein the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. 
     
     
         7 . (canceled) 
     
     
         8 . A method according to  claim 1 , wherein the Cas protein is Cas9 protein from any bacterial species or functional portion thereof. 
     
     
         9 . The method according to  claim 8 , wherein the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. 
     
     
         10 - 12 . (canceled) 
     
     
         13 . A method according to  claim 1 , wherein the target motif is a 20-nucleotide DNA sequence. 
     
     
         14 . (canceled) 
     
     
         15 . A method according to  claim 1 , wherein the target motif is a 20-nucleotide DNA sequence beginning with G and immediately precedes an NGG motif recognized by the Cas protein. 
     
     
         16 . (canceled) 
     
     
         17 . A method according to  claim 1 , wherein the target motif is a 20-nucleotide DNA sequence and immediately precedes an NGG motif recognized by the Cas protein. 
     
     
         18 . (canceled) 
     
     
         19 . A method according to  claim 1 , wherein the target motif is G(N) 19 NGG. 
     
     
         20 . (canceled) 
     
     
         21 . A method according to  claim 1 , wherein the target motif is (N) 20 NGG. 
     
     
         22 - 43 . (canceled) 
     
     
         44 . A method according to  claim 1 , 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, and a hepatocyte. 
     
     
         45 - 52 . (canceled) 
     
     
         53 . A method according to  claim 1 , wherein the target polynucleotide sequence is CCR5. 
     
     
         54 - 55 . (canceled) 
     
     
         56 . A method according to  claim 1 , wherein the target polynucleotide sequence is CXCR4. 
     
     
         57 - 67 . (canceled) 
     
     
         68 . A method according to  claim 2 , wherein the disorder is selected from the group consisting of a genetic disorder, a monogenic disorder, human HIV infection, and AIDS. 
     
     
         69 - 86 . (canceled) 
     
     
         87 . A method according to  claim 1 , wherein the Cas protein is encoded by a modified nucleic acid. 
     
     
         88 . (canceled) 
     
     
         89 . A method according to  claim 1 , 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. 
     
     
         90 . A method according to  claim 1 , wherein any of the Cas protein or the ribonucleic acids are expressed from a plasmid. 
     
     
         91 - 122 . (canceled) 
     
     
         123 . A method according to  claim 1 , wherein the cell comprises a primary cell. 
     
     
         124 - 126 . (canceled) 
     
     
         127 . A method according to  claim 1 , wherein the target polynucleotide sequence is B2M. 
     
     
         128 - 133 . (canceled) 
     
     
         134 . A method according to  claim 1 , wherein the one to two ribonucleic acids comprise two guide ribonucleic acid sequences. 
     
     
         135 . A method according to  claim 134 , wherein the target polynucleotide sequence comprises CCR5. 
     
     
         136 - 144 . (canceled) 
     
     
         145 . A method according to  claim 134 , wherein the target polynucleotide sequence comprises CXCR4. 
     
     
         146 - 149 . (canceled) 
     
     
         150 . A method according to  claim 134 , wherein the target polynucleotide sequence comprises B2M. 
     
     
         151 - 361 . (canceled)

Join the waitlist — get patent alerts

Track US2015176013A1 — get alerts on status changes and closely related new filings.

We store only your email — no account needed. See our privacy policy.