Elimination of proliferating cells from stem cell-derived grafts

Abstract

Provided herein are methods and compositions for a suicide gene approach comprising an expression vector comprising a cell cycle-dependent promoter driving the expression of a suicide gene. Also provided herein are methods to render proliferative cells sensitive to a prodrug after transplantation but avoids expression of the suicide gene in post-mitotic cells, such as neurons.

1 .- 31 . (canceled) 
     
     
         32 . A method of cell replacement therapy for replacing cells that are known to be essentially non-dividing cells, the method comprising administering an effective amount of precursor cells that are not pluripotent stem cells, the precursor cells comprising expression vectors encoding a cell cycle-dependent promoter operably linked to a suicide gene coding sequence, wherein the suicide gene is cytomegalovirus (CMV) UL97 gene or mutant herpes simplex virus thymidine kinase (HSV-TK), and administering to the subject an amount of a prodrug that is activated by the suicide gene, the prodrug being administered in an amount effective to eliminate cycling precursor cells. 
     
     
         33 . The method of  claim 32 , wherein the precursor cells are neural precursor cells, cardiomyocyte precursor cells, or pancreatic precursor cells. 
     
     
         34 . The method of  claim 32 , wherein the subject is a mammal. 
     
     
         35 . The method of  claim 34 , wherein the mammal is a mouse, rat, non-human primate, or human. 
     
     
         36 . The method of  claim 32 , wherein the genome of the host cell comprises a genome essentially identical to the genome of the subject. 
     
     
         37 . The method of  claim 32 , wherein the essentially non-dividing cells to be replaced comprise dopaminergic cells and the host cells comprise dopaminergic neural precursor cells, defined as expressing tyrosine hydroxylase or dopamine active transporter. 
     
     
         38 . The method of  claim 32 , wherein the subject has Parkinson's disease. 
     
     
         39 . The method of  claim 32 , wherein the prodrug is penciclovir. 
     
     
         40 . The method of  claim 32 , wherein the prodrug is administered more than once. 
     
     
         41 . The method of  claim 32 , wherein the prodrug is administered after a sufficient period of time for the precursor cells to initiate differentiation. 
     
     
         42 . The method of  claim 41 , wherein the period of time is 3-6 days. 
     
     
         43 . The method of  claim 41 , wherein the period of time is 7-15 days. 
     
     
         44 . The method of  claim 32 , wherein the prodrug is administered by injection. 
     
     
         45 . The method of  claim 32 , wherein the precursor cells are further defined as neural precursor cells, cardiomyocyte precursor cells, endothelial precursor cells, pancreatic precursor cells, kidney precursor cells, oligodendrocyte precursor cells, hematopoietic precursor cells, myeloid precursor cells, mesenchymal precursor cells, retinal precursor cells, or osteoclast precursor cells. 
     
     
         46 . The method of  claim 45 , wherein the precursor cells are further defined as a neural precursor cells. 
     
     
         47 . The method of  claim 46 , wherein the neural precursor cells are further defined as expressing at least one of the markers selected from the group consisting of musashi, nestin, sox2, vimentin, pax6, and sox1. 
     
     
         48 . The method of  claim 32 , wherein the CMV-UL97 is mutant CMV-UL97. 
     
     
         49 . The method of  claim 32 , wherein the mutant HSV-TK is SR11, SR26, SR39, SR4, SR15, SR32, or SR53. 
     
     
         50 . The method of  claim 32 , wherein the cell cycle-dependent promoter is a Ki-67, PCNA, CCNA2, CCNB2, DLGAPS, or TOP2A promoter. 
     
     
         51 . The method of  claim 32 , wherein the cell cycle-dependent promoter is a synthetic cell cycle promoter. 
     
     
         52 . The method of  claim 32 , wherein the expression vectors further encode a selectable marker. 
     
     
         53 . The method of  claim 52 , wherein the selectable marker is encoded by an antibiotic resistance gene or a gene encoding a fluorescent protein. 
     
     
         54 . The method of  claim 32 , wherein the vectors are viral vectors. 
     
     
         55 . The method of  claim 54 , wherein the viral vectors are lentiviral vectors, adenoviral vectors, retroviral vectors, vaccinia viral vectors, adeno-associated viral vectors, herpes viral vectors, or polyoma viral vectors.