THERAPEUTIC USES OF GENOME EDITING WITH CRISPR/Cas SYSTEMS

• US 20150176013A1
• Filed: 10/08/2014
• Published: 06/25/2015
• Est. Priority Date: 04/04/2013
• Status: Abandoned Application

• Current Assignee: Children's Medical Center Corporation, President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Children's Medical Center Corporation, President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Musunuru, Kiran, Cowan, Chad A., Rossi, Derrick J.
• Application Number:
  US14/509,787
• Publication Number:
  US 20150176013A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

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

Claims

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%.

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.

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.

13. A method according to claim 1, wherein the target motif is a 20-nucleotide DNA sequence.

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.

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.

19. A method according to claim 1, wherein the target motif is G(N)₁₉NGG.

21. A method according to claim 1, wherein the target motif is (N)₂₀NGG.

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.

53. A method according to claim 1, wherein the target polynucleotide sequence is CCR5.

56. A method according to claim 1, wherein the target polynucleotide sequence is CXCR4.

87. A method according to claim 1, wherein the Cas protein is encoded by a modified nucleic acid.

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.

123. A method according to claim 1, wherein the cell comprises a primary cell.

127. A method according to claim 1, wherein the target polynucleotide sequence is B2M.

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.

145. A method according to claim 134, wherein the target polynucleotide sequence comprises CXCR4.

150. A method according to claim 134, wherein the target polynucleotide sequence comprises B2M.

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.

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.

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.

7. (canceled)

10-12. -12. (canceled)

14. (canceled)

16. (canceled)

18. (canceled)

20. (canceled)

22-43. -43. (canceled)

45-52. -52. (canceled)

54-55. -55. (canceled)

57-67. -67. (canceled)

69-86. -86. (canceled)

88. (canceled)

91-122. -122. (canceled)

124-126. -126. (canceled)

128-133. -133. (canceled)

136-144. -144. (canceled)

146-149. -149. (canceled)

151-361. -361. (canceled)