CRISPR/CAS 9-MEDIATED INTEGRATION OF POLYNUCLEOTIDES BY SEQUENTIAL HOMOLOGOUS RECOMBINATION OF AAV DONOR VECTORS

• US 20200131539A1
• Filed: 04/23/2018
• Published: 04/30/2020
• Est. Priority Date: 04/21/2017
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Bak, Rasmus O., Porteus, Matthew, Vaidyanathan, Sriram
• Granted Patent: US 11,773,409 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 35/76 Viruses; Subviral particles; Bacteriophages
• CPC Class Codes: A61K 39/235 Adenoviridae
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: C07K 14/075 Adenoviridae
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/8616 Special methods for targeting systems
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/313 Phosphorodithioates
• CPC Class Codes: C12N 2710/10043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14141 Use of virus, viral particle or viral elements as a vector
• CPC Class Codes: C12N 2800/40 Systems of functionally co-operating vectors
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present invention relates to a system and method for efficiently modifying the genome of cells to treat diseases via sequential homologous recombination using CRISPR/Cas-mediated genome editing with donor DNA delivered by two or more adeno-associated virus (AAV) vectors.

Claims

1. A system for CRISPR/Cas9-mediated integration of a target polynucleotide into a target genetic locus in a cell comprising:

(a) a first targeting AAV vector comprising a single guide RNA (sgRNA) target site with a protospacer-adjacent motif (PAM), a first donor template, a 5′

homology arm that is homologous to a first portion of the targetlocus, and a 3′

homology arm that is homologous to a second portion of the target locus that is not overlapping or substantially not overlapping with the first portion of the target locus,

wherein the sgRNA target site is recognized by a target locus-specific sgRNA, wherein the first donor template comprises a first nucleotide sequence of the target polynucleotide;

(b) a second targeting AAV vector comprising a second donor template, a 5′

homology arm that is homologous to a first portion of the first donor template, a 3′

homology arm that is homologous to a second portion of the first targeting AAV vector,

wherein the first portion of the first donor template and the second portion of the first targeting AAV vector are not overlapping, the second donor template comprises a second nucleotide sequence of the target polynucleotide, and the nucleotide sequence of the target polynucleotide is split between the first donor template and the second donor template;

(c) the target locus-specific sgRNA; and

(d) a CRISPR-associated protein 9 (Cas9) polypeptide or a polynucleotide encoding the Cas9 polypeptide.

2. The system of claim 1, wherein the target locus-specific sgRNA and Cas9 polypeptide are complexed together to form a Cas9 ribonucleoprotein.

3. The system of claim 1 or 2, wherein the target locus-specific sgRNA comprises a synthetic sgRNA of SEQ ID NO:

1 or SEQ ID NO;

14.

4. The system of any one of claims 1 to 3, wherein the target locus-specific sgRNA comprises one or more modified nucleotides.

5. The system of claim 4, wherein the modified nucleotides comprise a modification in a ribose group, a phosphate group, a nucleobase, or a combination thereof.

6. The system of claim 5, wherein the modification in a ribose group comprises a modification at the 2′

position of the ribose group.

7. The system of claim 6, wherein the modification at the 2′

position of the ribose group is selected from the group consisting of 2′

-O-methyl, 2′

-fluoro, 2′

-deoxy, s′

-O-(2-methoxyethyl), and a combination thereof.

8. The system of claim 5, wherein the phosphate group comprises a phosphorothioate or 3′

-thioPACE modification.

9. The system of any one of claims 4 to 8, wherein the modified nucleotides are selected from the group consisting of a 2′

-O-methyl (M) nucleotide, a 2′

-O-methyl 3 ′

-phosphorothioate (MS) nucleotide, a 2′

-O-methyl 3′

-thioPACE (MSP) nucleotide, and a combination thereof.

10. The system of any one of claims 1 to 9, wherein the first targeting AAV vector has an AAV capsid polypeptide selected from the group consisting of an AAV-DJ, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVbb2, AAVcy5, AAVrh10, AAVrh20, AAVrh39, AAVrh43, AAVrh64R1, AAVhu37, engineered AAV, and chimeric AAV capsid polypeptide.

11. The system of any one of claims 1 to 10, wherein the second targeting AAV vector has an AAV capsid polypeptide selected from the group consisting of an AAV-DJ, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVbb2, AAVcy5, AAVrh10, AAVrh20, AAVrh39, AAVrh43, AAVrh64R1, AAVhu37, engineered AAV, and chimeric AAV capsid polypeptide.

12. The system of any one of claims 1 to 11, wherein the first targeting AAV vector and the second targeting AAV vector have the same AAV capsid polypeptide.

13. The system of any one of claims 1 to 12, wherein the cell is isolated from a subject.

14. The system of claim 13, wherein the subject has a genetic disease.

15. A method of introducing a target polynucleotide into a target genetic locus in a cell comprising introducing into the cell:

(a) a first targeting AAV vector comprising a single guide RNA (sgRNA) target site with a protospacer-adjacent motif(PAM), a first donor template, a 5′

homology arm that is homologous to a first portion of the target locus, and a 3′

homology arm that is homologous to a second portion of the target locus that is not overlapping or substantially not overlapping with the first portion of the target locus,

wherein the sgRNA target site is recognized by a target locus-specific sgRNA, wherein the first donor template comprises a first nucleotide sequence of the target polynucleotide(s);

(b) a second targeting AAV vector comprising a second donor template, a 5′

homology arm that is homologous to a first portion of the first donor template, a 3′

homology arm that is homologous to a second portion of the first targeting AAV vector,

wherein the first portion of the first donor template and the second portion of the first targeting AAV vector are not overlapping, the second donor template comprises a second nucleotide sequence of the target polynucleotide(s), and the nucleotide sequence of the target polynucleotide(s) is split between the first donor template and the second donor template;

(c) the target locus-specific sgRNA; and

(d) a CRISPR-associated protein 9 (Cas9) polypeptide or a polynucleotide encoding the Cas9 polypeptide.

16. The method of claim 15, wherein the target locus-specific sgRNA and Cas9 polypeptide are complexed together to form a Cas9 ribonucleoprotein before introduction into the cell.

17. The method of claim 15 or 16, wherein the target locus-specific sgRNA comprises a synthetic sgRNA of SEQ ID NO:

1 or SEQ ID NO;

14.

18. The method of any one of claims 15 to 17, wherein the target locus-specific sgRNA comprises one or more modified nucleotides.

19. The system of claim 18, wherein the modified nucleotides comprise a modification in a ribose group, a phosphate group, a nucleobase, or a combination thereof.

20. The method of claim 19, wherein the modification in a ribose group comprises a modification at the 2′

position of the ribose group.

21. The method of claim 20, wherein the modification at the 2′

position of the ribose group is selected from the group consisting of 2′

-O-methyl, 2′

-fluoro, 2′

-deoxy, s′

-O-(2-methoxyethyl), and a combination thereof.

22. The method of claim 19, wherein the phosphate group comprises a phosphorothioate or 3′

-thioPACE modification.

23. The method of any one of claims 18 to 22, wherein the modified nucleotides are selected from the group consisting of a 2′

-O-methyl (M) nucleotide, a 2′

-O-methyl 3 ′

-phosphorothioate (MS) nucleotide, a 2′

-O-methyl 3′

-thioPACE (MSP) nucleotide, and a combination thereof.

24. The method of any one of claims 15 to 23, wherein the first targeting AAV vector has an AAV capsid polypeptide selected from the group consisting of a AAV-DJ, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVbb2, AAVcy5, AAVrh10, AAVrh20, AAVrh39, AAVrh43, AAVrh64R1, AAVhu37, engineered AAV, and chimeric AAV capsid polypeptide.

25. The method of any one of claims 15 to 24, wherein the second targeting AAV vector has an AAV capsid polypeptide selected from the group consisting of an AAV-DJ, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVbb2, AAVcy5, AAVrh10, AAVrh20, AAVrh39, AAVrh43, AAVrh64R1, AAVhu37, engineered AAV, and chimeric AAV capsid polypeptide.

26. The method of any one of claims 15 to 25, wherein the first targeting AAV vector and the second targeting AAV vector have the same AAV capsid polypeptide.

27. The method of any one of claims 15 to 26, further comprising selecting the cell containing the target polynucleotide.

28. The method of any one of claims 15 to 25, wherein the cell is isolated from a subject prior to performing said method.

29. The method of claim 28, further comprising administering the cell containing the target polynucleotide into the subject.

30. The method of any one of claims 15 to 29, wherein the cell is selected from the group consisting of an immune cell, a muscle cell, a liver cell, a skin cell, a retinal cell, an airway cell, a lung cell, and a stem cell.

31. The method of any one of claims 28 to 30, wherein the subject has a genetic disease.

32. A method of treating a genetic disease in a subject, the method comprising administering to the subject:

(a) a first targeting AAV vector comprising a single guide RNA (sgRNA) target site with a protospacer-adjacent motif (PAM), a first donor template, a 5′

homology arm that is homologous to a first portion of a target genetic locus, and a 3′

homology arm that is homologous to a second portion of the target locus that is not overlapping or substantially not overlapping with the first portion of the target locus,

wherein the sgRNA target site is recognized by a target locus-specific sgRNA, and the first donor template comprises a first nucleotide sequence of a target polynucleotide(s);

(b) a second targeting AAV vector comprising a second donor template, a 5′

homology arm that is homologous to a first portion of the first donor template, a 3′

homology arm that is homologous to a second portion of the first targeting AAV vector,

wherein the first portion of the first donor template and the second portion of the first targeting AAV vector are not overlapping, the second donor template comprises a second nucleotide sequence of the target polynucleotide, and the nucleotide sequence of the target polynucleotide(s) is split between the first donor template and the second donor template;

(c) the target locus-specific sgRNA; and

(d) a CRISPR-associated protein 9 (Cas9) polypeptide or a polynucleotide encoding the Cas9 polypeptide.

33. The method of claim 32, wherein the target locus-specific sgRNA and Cas9 polypeptide are complexed together to form a Cas9 ribonucleoprotein before administering to the subject.

34. The method of claim 32 or 33, wherein the target locus-specific sgRNA comprises a synthetic sgRNA of SEQ ID NO:

1 or SEQ ID NO;

14.

35. The method of any one of claims 32 to 34, wherein the target locus-specific sgRNA comprises one or more modified nucleotides.

36. The method of claim 34, wherein the modified nucleotides comprise a modification in a ribose group, a phosphate group, a nucleobase, or a combination thereof.

37. The method of claim 35, wherein the modification in a ribose group comprises a modification at the 2′

position of the ribose group.

38. The method of claim 36, wherein the modification at the 2′

position of the ribose group is selected from the group consisting of 2′

-O-methyl, 2′

-fluoro, 2′

-deoxy, s′

-O-(2-methoxyethyl), and a combination thereof.

39. The method of claim 35, wherein the phosphate group comprises a phosphorothioate or 3′

-thioPACE modification.

40. The method of any one of claims 34 to 38, wherein the modified nucleotides are selected from the group consisting of a 2′

-O-methyl (M) nucleotide, a 2′

-O-methyl 3 ′

-phosphorothioate (MS) nucleotide, a 2′

-O-methyl 3′

-thioPACE (MSP) nucleotide, and a combination thereof.

41. The method of any one of claims 32 to 39, wherein the first targeting AAV vector has an AAV capsid polypeptide selected from the group consisting of an AAV-DJ, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVbb2, AAVcy5, AAVrh10, AAVrh20, AAVrh39, AAVrh43, AAVrh64R1, AAVhu37, engineered AAV, and chimeric AAV capsid polypeptide.

42. The method of any one of claims 32 to 40, wherein the second targeting AAV vector has an AAV capsid polypeptide selected from the group consisting of an AAV-DJ, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVbb2, AAVcy5, AAVrh10, AAVrh20, AAVrh39, AAVrh43, AAVrh64R1, AAVhu37, engineered AAV, and chimeric AAV capsid polypeptide.

43. The method of any one of claims 32 to 41, wherein the first targeting AAV vector and the second targeting AAV vector have the same AAV capsid polypeptide.