Chimeric proteins and methods of regulating gene expression

• US 10,457,961 B2
• Filed: 11/08/2017
• Issued: 10/29/2019
• Est. Priority Date: 01/11/2016
• 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: Qi, Lei S., Dingal, P. C. Dave P.
• Primary Examiner(s): Goddard, Laura B
• Application Number:
  US15/806,756
• Publication Number:
  US 20180273980A1
• Time in Patent Office: 720 Days
• Field of Search: None
• CPC Class Codes: A61K 2039/5156 expressing foreign proteins
• CPC Class Codes: A61K 2039/5158 Antigen-pulsed cells, e.g. T-cells
• CPC Class Codes: A61K 39/0011 Cancer antigens
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)
• CPC Class Codes: H05K 999/99 dummy group

Abstract

The present disclosure provides systems, compositions and methods for regulating expression of a target polynucleotide in a cell. The systems, compositions and methods comprise a chimeric receptor polypeptide comprising a G-protein coupled receptor (GPCR) or a fragment thereof, a chimeric adaptor polypeptide, at least one actuator moiety and a cleavage moiety.

Claims

1. A system for regulating expression of a target polynucleotide in a lymphocyte, the system comprising:

(a) a chimeric receptor polypeptide (receptor) comprising a transmembrane receptor or a fragment thereof, that undergoes a receptor modification including a conformational change or chemical modification upon binding to its ligand, wherein the receptor comprises a cleavage moiety that cleaves a cleavage recognition site; and

(b) a chimeric adaptor polypeptide (adaptor) that binds the receptor in response to the receptor modification, wherein the adaptor comprises a gene modulating polypeptide (GMP) comprising an actuator moiety linked to the cleavage recognition site, and wherein the adaptor does not bind any extracellular ligand,

wherein the target polynucleotide encodes for a protein involved in immune cell regulation, and

wherein the receptor is activatable upon binding of the receptor to the ligand to recruit the adaptor to the receptor, and wherein the receptor releases the actuator moiety from the GMP of the recruited adaptor by action of the cleavage moiety at the cleavage recognition site, to effect regulating expression of the target polynucleotide in the lymphocyte.

2. The system of claim 1, wherein the receptor modification is phosphorylation.

3. The system of claim 1, wherein the actuator moiety is an RNA-guided actuator moiety, and wherein the system further comprises a guide RNA that complexes with the actuator moiety.

4. The system of claim 3, wherein the RNA-guided actuator moiety substantially lacks DNA cleavage activity.

5. The system of claim 1, wherein cleavage of the cleavage recognition site is effective to release the adaptor from the receptor that has undergone the receptor modification, rendering the receptor available for binding with another adaptor comprising a GMP.

6. The system of claim 4, wherein the RNA-guided actuator moiety is Cas9 or fragment thereof that substantially lacks DNA cleavage activity.

7. The system of claim 1, wherein the transmembrane receptor comprises a G-protein coupled receptor or fragment thereof.

8. The system of claim 1, wherein the adaptor is linked to at least one targeting sequence which directs transport of the adaptor to a specific region of the lymphocyte.

9. The system of claim 1, wherein the receptor is linked to at least one targeting sequence which directs transport of the receptor to a cellular membrane.

10. The system of claim 1, wherein the receptor or the adaptor is linked to a polypeptide folding domain.

11. The system of claim 1, wherein the actuator moiety comprises a repressor effective to decrease expression of the target polynucleotide.

12. The system of claim 1, wherein the protein involved in immune cell regulation is PD-1 and/or CTLA-4.

13. The system of claim 1, wherein the target polynucleotide comprises genomic DNA or RNA.

14. The system of claim 1, wherein the adaptor binds to an intracellular domain of the receptor upon the receptor modification.

15. The system of claim 1, wherein the actuator moiety is a DNA-guided actuator moiety and the system further comprises a guide DNA that complexes with the actuator moiety.

16. The system of claim 1, wherein the lymphocyte is a T cell.

17. The system of claim 1, wherein the lymphocyte is a B cell.

18. The system of claim 1, wherein the lymphocyte is a natural killer (NK) cell.

19. A method of regulating expression of a target polynucleotide in a lymphocyte, the method comprising:

(a) contacting a ligand to a chimeric transmembrane receptor polypeptide (receptor) comprising a cleavage moiety, wherein the receptor is modified upon contacting to the ligand, and wherein the receptor modification comprises a conformational change or a chemical modification;

(b) binding a chimeric adaptor polypeptide (adaptor) comprising a gene modulating polypeptide (GMP) to the receptor in response to the receptor modification, wherein the GMP comprises an actuator moiety linked to a cleavage recognition site, wherein the receptor is activatable upon binding of the receptor to the ligand to recruit the adaptor to the receptor, and wherein the adaptor does not bind any extracellular ligand; and

(c) releasing the actuator moiety from the GMP of the recruited adaptor by action of the cleavage moiety at the cleavage recognition site, to effect regulating expression of the target polynucleotide in the lymphocyte, wherein the target polynucleotide encodes for a protein involved in immune cell regulation.

20. The method of claim 19, wherein the receptor modification is phosphorylation.

21. The method of claim 19, wherein the actuator moiety is an RNA-guided actuator moiety that forms a complex with the target polynucleotide.

22. The method of claim 19, wherein the RNA-guided actuator moiety substantially lacks DNA cleavage activity.

23. The method of claim 19, further comprising (i) binding another adaptor to the receptor that has undergone the receptor modification, wherein the another adaptor comprises a GMP comprising an actuator moiety linked to a cleavage recognition site, and (ii) cleaving the cleavage recognition site of the another adaptor to release its actuator moiety.

24. The method of claim 19, wherein receptor modification comprises modification at multiple modification sites, and each modification site is effective to bind an adaptor.

25. The method of claim 19, wherein the actuator moiety is a Cas9 or a fragment thereof that substantially lacks DNA cleavage activity.

26. The method of claim 19, wherein the receptor comprises a G-protein coupled receptor or fragment thereof.

27. The method of claim 19, wherein the actuator moiety comprises a repressor effective to decrease expression of the target polynucleotide.

28. The method of claim 19, wherein the protein involved in immune cell regulation is PD-1 and/or CTLA-4.

29. The method of claim 19, wherein the adaptor binds to an intracellular domain of the receptor upon the receptor modification.

30. The method of claim 19, wherein the lymphocyte is a T cell.

31. The method of claim 19, wherein the lymphocyte is a B cell.

32. The method of claim 19, wherein the lymphocyte is a natural killer (NK) cell.