Chimeric Proteins and Methods of Regulating Gene Expression

• US 20180273980A1
• Filed: 11/08/2017
• Published: 09/27/2018
• 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.
• Granted Patent: US 10,457,961 B2
• Time in Patent Office: Days
• 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 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 chimeric receptor polypeptide comprises a cleavage moiety that cleaves a cleavage recognition site; and

(b) a chimeric adaptor polypeptide that binds the chimeric receptor polypeptide in response to the receptor modification, wherein the chimeric adaptor polypeptide comprises a gene modulating polypeptide (GMP) comprising an actuator moiety linked to the cleavage recognition site;

wherein the cleavage moiety cleaves the cleavage recognition site when in proximity to the cleavage recognition site and in response to receptor binding to the ligand, and wherein upon cleaving the cleavage recognition site, the actuator moiety regulates expression of the target polynucleotide, wherein the target nucleotide encodes for an immune related protein.

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

6. 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.

8. The system of claim 6, wherein the RNA-guided actuator moiety substantially lacks DNA cleavage activity.

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

11. The system of claim 8, wherein the RNA-guided actuator moiety is Cas9 or fragment thereof that substantially lacks DNA cleavage activity.

14. The system of claim 1, wherein the transmembrane receptor comprises a G-protein coupled receptor.

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

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

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

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

115. The system of claim 1, wherein the immune related protein is PD-1 and/or CTLA-4.

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

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

118. 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.

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

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

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

2-4. -4. (canceled)

7. (canceled)

10. (canceled)

12-13. -13. (canceled)

15-18. -18. (canceled)

20-22. -22. (canceled)

24-26. -26. (canceled)

29. 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 comprising a cleavage moiety, wherein the chimeric transmembrane receptor is modified upon contacting to the antigen, and wherein the receptor modification comprises a conformational change or a chemical modification;

(b) binding a chimeric adaptor polypeptide comprising a gene modulating polypeptide (GMP) to the chimeric transmembrane receptor polypeptide in response to the receptor modification, wherein the GMP comprises an actuator moiety linked to a cleavage recognition site; and

(c) cleaving the cleavage recognition site with the cleavage moiety to release the actuator moiety, wherein upon said release, the actuator moiety regulates expression of the target polynucleotide in the lymphocyte, wherein the target nucleotide encodes for an immune related proteins.

33. The method of claim 29, wherein the receptor modification is phosphorylation.

34. The method of claim 29, wherein the actuator moiety is an RNA-guided actuator moiety that forms a complex with the target polynucelotide.

36. The method of claim 29, wherein the RNA-guided actuator moeity substantially lacks DNA cleavage activity.

37. The method of claim 29, further comprising (i) binding another chimeric adaptor polypeptide to the chimeric transmembrane receptor polypeptide that has undergone the receptor modification, wherein the another chimeric adaptor polypeptide comprises a GMP comprising an actuator moiety linked to a cleavage recognition site, and (ii) cleaving the cleavage recognition site of the another chimeric adaptor polypeptide to release its actuator moiety.

38. The method of claim 29, wherein receptor modification comprises modification at multiple modification sites, and each modification site is effective to bind a chimeric adaptor polypeptide.

39. The method of claim 29, wherein the actuator moiety is a Cas9 or a fragment thereof that substantially lacks DNA cleavage activity.

42. The method of claim 29, wherein the chimeric transmembrane receptor polypeptide comprises a G-protein coupled receptor or fragment thereof.

45. The method of claim 29, wherein the actuator moiety comprises a repressor effective to decrease expression of the target polynucleotide.

122. The method of claim 29, wherein the immune related protein is PD-1 and/or CTLA-4.

123. The method of claim 29, wherein the chimeric adaptor polypeptide binds to an intracellular domain of the chimeric transmembrane receptor polypeptide upon the receptor modification.

124. The method of claim 29, wherein the lymphocyte is a T cell.

125. The method of claim 29, wherein the lymphocyte is a B cell.

126. The method of claim 29, wherein the lymphocyte is a natural killer (NK) cell.

30-32. -32. (canceled)

35. (canceled)

40-41. -41. (canceled)

43-44. -44. (canceled)

46-114. -114. (canceled)