Methods of modulating expression of target nucleic acid sequences in a cell

Patent metadata

Field	Value
Publication number	US-11674138-B2
Application number	US-201816486586-A
Country	US
Kind code	B2
Filing date	Mar 13, 2018
Priority date	Mar 13, 2017
Publication date	Jun 13, 2023
Grant date	Jun 13, 2023

Abstract

The present disclosure provides methods and compositions of modulating expression of a target nucleic acid sequence in a cell. The method comprises introducing into the cell a nucleic acid sequence encoding a Cas9 fusion protein and a guide RNA, wherein the Cas9 fusion protein and the guide RNA are expressed and co-localize at a target site and modulate the expression of the target nucleic acid sequence.

First claim

What is claimed is:

1. A method of activating expression of a target nucleic acid sequence in a eukaryotic cell comprising:
introducing into the cell a nucleic acid sequence encoding a Cas9 fusion protein and a guide RNA, wherein the Cas9 fusion protein comprises a nuclease-null Cas9 (dCas9) fused with an activation domain of VP64, an activation domain of p65, and an activation domain of RTA, wherein the activation domain of p65 comprises a protein sequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, wherein the Cas9 fusion protein and the guide RNA are expressed and co-localize at a target site and activate the expression of the target nucleic acid sequence.

2. The method of claim 1 wherein the activation domains of VP64, p65 and RTA comprises a tripartite VP64-p65-RTA (VPR) fusion wherein each of the activation domains of VP64, p65 and RTA is truncated.

3. The method of claim 1 wherein a sequence encoding the Cas9 fusion protein is flanked by a promoter sequence at its 5′ end and a terminator sequence at its 3′ end wherein the promoter sequence is truncated.

4. The method of claim 3 wherein the terminator sequence is truncated.

5. The method of claim 4 wherein the terminator sequence comprises a short 17nt sNRP-1 (SEQ ID NO: 35), a 34nt dual sNRP-1 (SEQ ID NO: 36), a 50nt synthetic (SEQ ID NO: 37), or a 250 nt bGHR terminator.

6. The method of claim 1 wherein the activation domain of VP64 is a modified activation domain comprising protein sequence of SEQ ID NO: 17.

7. The method of claim 6 wherein the protein sequence is encoded by nucleic acid sequence of SEQ ID NO: 1.

8. The method of claim 1 wherein the activation domain of p65 is encoded by nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, respectively.

9. The method of claim 1 wherein the activation domain of RTA is a modified activation domain comprising RTA deletion protein.

10. The method of claim 9 wherein the RTA deletion protein comprises protein sequence of SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

11. The method of claim 10 wherein the protein sequence is encoded by nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, respectively.

12. The method of claim 1 wherein the activation domain of VP64 is a modified activation domain comprising protein sequence of SEQ ID NO: 17 and the activation domain of RTA is a modified activation domain comprising SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

13. A method of activating expression of a target nucleic acid sequence in a eukaryotic cell comprising:
introducing into the cell a nucleic acid sequence encoding a Cas9 fusion protein and a guide RNA, wherein the Cas9 fusion protein comprises a nuclease-null Cas9 (dCas9) fused with an activation domain of VP64, an activation domain of p65, and an activation domain of RTA, wherein the activation domain of RTA comprises a protein sequence of SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32,
wherein the Cas9 fusion protein and the guide RNA are expressed and co-localize at a target site and activate the expression of the target nucleic acid sequence.

14. The method of claim 13 wherein the protein sequence is encoded by nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, respectively.

15. The method of claim 13 wherein the activation domains of VP64, p65 and RTA comprises a tripartite VP64-p65-RTA (VPR) fusion wherein each of the activation domains of VP64, p65 and RTA is truncated.

16. The method of claim 13 wherein a sequence encoding the Cas9 fusion protein is flanked by a promoter sequence at its 5′ end and a terminator sequence at its 3′ end wherein the promoter sequence is truncated.

17. The method of claim 16 wherein the terminator sequence is truncated.

18. The method of claim 16 wherein the terminator sequence comprises a short 17nt sNRP-1 (SEQ ID NO: 35), a 34nt dual sNRP-1 (SEQ ID NO: 36), a 50nt synthetic (SEQ ID NO: 37), or a 250 nt bGHR terminator.

19. The method of claim 13 wherein the activation domain of VP64 is a modified activation domain comprising protein sequence of SEQ ID NO: 17.

20. The method of claim 19 wherein the protein sequence is encoded by nucleic acid sequence of SEQ ID NO: 1.

21. The method of claim 13 wherein the activation domain of p65 is a modified activation domain comprising p65 deletion protein.

22. The method of claim 21 wherein the p65 deletion protein comprises protein sequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

23. The method of claim 13 wherein the activation domain of p65 is encoded by nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, respectively.

24. The method of claim 13 wherein the activation domain of VP64 is a modified activation domain comprising protein sequence of SEQ ID NO: 17 and wherein the activation domain of p65 is a modified activation domain comprising protein sequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

25. The method of claim 1 wherein the nucleic acid sequence encoding a Cas9 fusion protein and a guide RNA is within an AAV which is introduce into the eukaryotic cell.

26. The method of claim 15 wherein the nucleic acid sequence encoding a Cas9 fusion protein and a guide RNA is within an AAV which is introduce into the eukaryotic cell.

27. The method of claim 1 wherein the Cas9 is spCas9, St1Cas9 or SaCas9.

28. The method of claim 13 wherein the Cas9 is spCas9, St1Cas9 or SaCas9.

Assignees

• Harvard College

Inventors

• Vora Suhani D
• Chavez Alejandro
• Cheng Jing
• Pruitt Benjamin W

Classifications

• C12N2750/14143

  viral genome or elements thereof as genetic vector · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C07K14/315

  from Streptococcus (G), e.g. Enterococci · CPC title

• C07K14/31

  from Staphylococcus (G) · CPC title