Chimeric proteins and methods of regulating gene expression

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.

1 - 29 . (canceled) 
     
     
         30 . A system for editing a target polynucleotide of a cell, the system comprising:
 (a) a chimeric receptor polypeptide (receptor) that undergoes a receptor modification upon binding to a ligand;   (b) a chimeric adaptor polypeptide (adaptor) that binds the receptor in response to the receptor modification;   (c) a gene modulating polypeptide (GMP) comprising an actuator moiety linked to a cleavage recognition site, wherein the actuator moiety is capable of editing the target polynucleotide of the cell, and wherein the actuator moiety includes a fusion polypeptide conferring (i) a nuclease activity and (ii) an additional activity selected from the group consisting of: a methyltransferase activity, a demethylase activity, a depurination activity, a pyrimidine dimer forming activity, a polymerase activity, and a hydrolase activity; and   (d) a cleavage moiety that cleaves the cleavage recognition site when in proximity to the cleavage recognition site,   wherein (1) the GMP forms a portion of the adaptor, and the cleavage moiety forms a portion of the receptor, or (2) the GMP forms a portion of the receptor, and the cleavage moiety forms a portion of the adaptor.   
     
     
         31 . The system of  claim 30 , wherein the GMP forms the portion of the adaptor, and the cleavage moiety forms the portion of the receptor, and the receptor releases the actuator moiety from the GMP of the adaptor by action of the cleavage moiety at the cleavage recognition site, to effect editing of the target polynucleotide in the cell. 
     
     
         32 . The system of  claim 30 , wherein the GMP forms the portion of the receptor, and the cleavage moiety forms the portion of the adaptor, and the adaptor releases the actuator moiety from the GMP of the receptor by action of the cleavage moiety at the cleavage recognition site, to effect editing of the target polynucleotide in the cell. 
     
     
         33 . The system of  claim 30 , wherein the actuator moiety is capable of complexing with the target polynucleotide of the cell. 
     
     
         34 . The system of  claim 30 , wherein the actuator moiety is capable of editing a single strand of the target polynucleotide of the cell. 
     
     
         35 . The system of  claim 30 , wherein the actuator moiety comprises a ribonucleic acid (RNA)-guided actuator moiety, and wherein the system further comprises a guide RNA that complexes with the actuator moiety. 
     
     
         36 . The system of  claim 30 , wherein the fusion polypeptide comprises a Cas protein that confers the nuclease activity. 
     
     
         37 . The system of  claim 36 , wherein the Cas protein exhibits a reduced nuclease activity as compared to a wild type Cas protein. 
     
     
         38 . The system of  claim 30 , wherein the receptor modification is a conformational change or chemical modification. 
     
     
         39 . The system of  claim 38 , wherein the receptor modification is phosphorylation. 
     
     
         40 . The system of  claim 30 , wherein the receptor modification comprises modification at multiple modification sites, and each modification site is effective to bind an adaptor. 
     
     
         41 . The system of  claim 30 , wherein the adaptor binds to an intracellular domain of the receptor upon the receptor modification. 
     
     
         42 . The system of  claim 30 , wherein the receptor comprises a G-protein coupled receptor or fragment thereof. 
     
     
         43 . The system of  claim 30 , wherein the target polynucleotide comprises genomic deoxyribonucleic acid (DNA) or RNA. 
     
     
         44 . The system of  claim 30 , wherein the actuator includes the fusion polypeptide that confers the nuclease activity and the polymerase activity. 
     
     
         45 . The system of  claim 30 , wherein the actuator includes the fusion polypeptide that confers the nuclease activity and the hydrolase activity. 
     
     
         46 . The system of  claim 30 , wherein the target polynucleotide is associated with a genetic disease or medical condition. 
     
     
         47 . The system of  claim 30 , wherein the cell is a lymphocyte. 
     
     
         48 . The system of  claim 30 , wherein the target polynucleotide encodes for a protein involved in immune cell regulation. 
     
     
         49 . The system of  claim 48 , wherein the protein involved in immune cell regulation is PD-1 or CTLA-4.