Delivery system for functional nucleases
Compositions, methods, strategies, kits, and systems for the supercharged protein-mediated delivery of functional effector proteins into cells in vivo, ex vivo, or in vitro are provided. Compositions, methods, strategies, kits, and systems for delivery of functional effector proteins using cationic lipids and cationic polymers are also provided. Functional effector proteins include, without limitation, transcriptional modulators (e.g., repressors or activators), recombinases, nucleases (e.g., RNA-programmable nucleases, such as Cas9 proteins; TALE nuclease, and zinc finger nucleases), deaminases, and other gene modifying/editing enzymes. Functional effector proteins include TALE effector proteins, e.g., TALE transcriptional activators and repressors, as well as TALE nucleases. Compositions, methods, strategies, and systems for the delivery of functional effector proteins into cells is useful for therapeutic and research purposes, including, but not limited to, the targeted manipulation of a gene associated with disease, the modulation of the expression level of a gene associated with disease, and the programming of cell fate.
1. A composition comprising a Cas9 protein and a cationic lipid, wherein the Cas9 protein is associated with a gRNA, and wherein the Cas9 protein is fused to a deaminase.
2. The composition of claim 1 , wherein the composition exhibits low toxicity when administered to a population of cells.
3. The composition of claim 2 , wherein at least 60% of the cells are viable following administration of the composition to the cells.
4. The composition of claim 1 , wherein the cationic lipid is selected from the group consisting of Lipofectamine® 2000, Lipofectamine® 3000, Lipofectamine® RNAiMAX, and Lipofectamine® LTX.
5. The composition of claim 1 , wherein the Cas9 protein comprises a wild type Cas9 protein or a Cas9 variant that comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 44, a Cas9 nickase, or a nuclease-inactivated Cas9 (dCas9) that comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 45 and comprises a D10A and/or a H840A substitution as compared to the amino acid sequence of a wild type Cas9.
6. The composition of claim 1 , wherein the deaminase comprises a cytidine deaminase.
7. A method comprising administering the pharmaceutical composition of claim 1 , to a subject in need thereof.
8. A method of introducing a Cas9 protein into a cell, the method comprising contacting the cell with the composition of claim 1 , under conditions suitable for the Cas9 protein to enter the cell, thereby introducing the Cas9 protein into the cell.
9. The composition of claim 6 , wherein the cytidine deaminase is selected from the group consisting of APOBEC1, AID, and ACF1/ASE deaminases.
10. The composition of claim 1 , wherein the deaminase comprises an adenosine deaminase.
11. The composition of claim 10 , wherein the adenosine deaminase comprises an ADAT family deaminase.
12. The composition of claim 1 , wherein the Cas9 protein is fused to the deaminase via a linker.
13. The composition of claim 12 , wherein the linker comprises an amino acid sequence selected from the group consisting of SGSETPGTSESATPES (SEQ ID NO:3), SGSETPGTSESA (SEQ ID NO:4), SGSETPGTSESATPEGGSGGS (SEQ ID NO:5), VPFLLEPDNINGKTC (SEQ ID NO:6), GSAGSAAGSGEF (SEQ ID NO:7), SIVAQLSRPDPA (SEQ ID NO:8), MKIIEQLPSA (SEQ ID NO:9), VRHKLKRVGS (SEQ ID NO:10), GHGTGSTGSGSS (SEQ ID NO:11), MSRPDPA (SEQ ID NO:12), GGSM (SEQ ID NO:13), GGS, and (GGS) n , wherein n is from 1 to 10.
14. The composition of claim 1 , wherein the Cas9 protein fused to the deaminase comprises the structure:
[NH 2 ]-[Cas9]-[deaminase]-[COOH] or
[NH 2 ]-[deaminase]-[Cas9]-[COOH].
15. The composition of claim 1 further comprising a nuclear localization signal (NLS) fused to the Cas9 protein or the deaminase.
16. The composition of claim 15 , wherein the nuclear localization signal comprises the amino acid sequence MAPKKKRKVGIHRGVP (SEQ ID NO: 47).
17. The composition of claim 15 , wherein the NLS is C-terminal of the deaminase.
18. The composition of claim 15 , wherein the NLS is C-terminal of the Cas9 domain.
19. The composition of claim 15 , wherein the Cas9 protein fused to the deaminase comprises the structure:
[NH 2 ]-[NLS]-[Cas9]-[deaminase]-[COOH] or
[NH 2 ]-[NLS]-[deaminase]-[Cas9]-[COOH].
20. The method of claim 8 further comprising confirming that the deaminase has penetrated the cell.
21. The method of claim 8 , wherein the cell is in a subject, and the contacting is done in vivo.
22. The composition of claim 2 , wherein at least at least 70% of the cells are viable following administration of the composition to the cells.
23. The composition of claim 2 , wherein at least at least 80% of the cells are viable following administration of the composition to the cells.
24. The composition of claim 2 , wherein at least at least 90% of the cells are viable following administration of the composition to the cells.
25. The composition of claim 1 , wherein the Cas9 protein comprises a wild type Cas9 protein or a Cas9 variant that comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 44.
26. The composition of claim 1 , wherein the Cas9 protein comprises a Cas9 nickase.
27. The composition of claim 1 , wherein the Cas9 protein comprises a nuclease-inactivated Cas9 (dCas9) that comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 45 and comprises a D10A and/or a H840A substitution as compared to the amino acid sequence of a wild type Cas9.
28. The composition of claim 6 , wherein the cytidine deaminase is an APOBEC1 deaminase.
29. The composition of claim 6 , wherein the cytidine deaminase is an AID deaminase.
30. The composition of claim 6 , wherein the cytidine deaminase is an ACF1/ASE deaminase.