THERAPEUTIC USES OF GENOME EDITING WITH CRISPR/Cas SYSTEMS

• US 20150071889A1
• Filed: 09/12/2014
• Published: 03/12/2015
• Est. Priority Date: 04/04/2013
• Status: Active Grant

• 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, Rossi, Derrick J., Cowan, Chad A.
• Granted Patent: US 9,822,370 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• 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 mammalian 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 in cells that contain Cas protein is from about 50% to about 80%.

2. The method according to claim 1, wherein the Cas protein is Cas9 protein from any bacterial species or a functional portion thereof.

3. The method according to claim 2, 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.

4. The method according to claim 1, wherein the Cas protein is Streptococcus pyogenes Cas9 protein or a functional portion thereof.

5. The method according to claim 4, 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.

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

7. The method according to claim 6, wherein the modified nucleic acid comprises a ribonucleic acid containing at least one modified nucleotide 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.

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

9. The 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.

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

11. The method according to claim 1, wherein the target polynucleotide sequence is CCR5.

12. The method according to claim 1, wherein the target polynucleotide sequence is CXCR4.

13. 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 mammalian 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 in cells that contain 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.

14. The method according to claim 13, wherein the Cas protein is Cas9 protein from any bacterial species or a functional portion thereof.

15. The method according to claim 14, 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.

16. The method according to claim 13, wherein the Cas protein is Streptococcus pyogenes Cas9 protein or a functional portion thereof.

17. The method according to claim 16, 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.

18. The method according to claim 13, wherein the Cas protein is encoded by a modified nucleic acid.

19. The method according to claim 18, wherein the modified nucleic acid comprises a ribonucleic acid containing at least one modified nucleotide 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.

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

21. The method according to claim 13, wherein the target motif is a 20-nucleotide DNA sequence beginning with G and immediately precedes an NGG motif recognized by the Cas protein.

22. The method according to claim 13, wherein the target motif is G(N)₁₉NGG.

23. The method according to claim 13, wherein the target polynucleotide sequence is CCR5.

24. The method according to claim 13, wherein the target polynucleotide sequence is CXCR4.