Short hairpin rna (shrna734) and use of same to positively select and eliminate genetically modified cells

Abstract

A potent short hairpin RNA (shRNA734) directed to human Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) improves the rate of gene-modified stem cell engraftment by a conditioning and in vivo selection strategy to confer resistance to a clinically available guanine analog antimetabolite, 6TG, for efficient positive selection of gene-modified stem cells. Uses for polynucleotides comprising the shRNA734 include methods for knocking down HPRT in a cell, for conferring resistance to a guanine analog antimetabolite in a cell, for producing selectable genetically modified cells, for selecting cells genetically modified with a gene of interest from a plurality of cells, for removing cells genetically modified with a gene of interest from a plurality of cells, and for treating a subject infected with HIV.

1 .- 16 . (canceled) 
     
     
         17 . A method for removing cells genetically modified with a gene of interest, the method comprising:
 (a) contacting a plurality of cells that comprises genetically modified cells, wherein the genetically modified cells have been modified with a polynucleotide comprising a nucleic acid sequence encoding a short hairpin ribonucleic acid molecule 734 (shRNA734) and the gene of interest, wherein the shRNA734 nucleic acid sequence is SEQ ID NO: 1 under conditions permitting expression of SEQ ID NO: 1; and   (b) removing the genetically modified cells from the plurality of cells, wherein the removing comprises treating the plurality of cells with methotrexate (MTX).   
     
     
         18 . (canceled) 
     
     
         19 . A method for treating a subject exhibiting an adverse side effect from a genetically modified cell, the method comprising administering methotrexate to the subject. 
     
     
         20 . The method of  claim 19 , wherein the genetically modified cell comprises a polynucleotide comprising a nucleic acid sequence encoding a short hairpin ribonucleic acid (shRNA) targeting HPRT. 
     
     
         21 . The method of  claim 20 , wherein the shRNA comprises short hairpin ribonucleic acid molecule 734 (shRNA734), wherein the shRNA734 nucleic acid sequence comprises SEQ ID NO: 1. 
     
     
         22 . The method of  claim 21 , wherein the methotrexate induces cell death in the genetically modified cell by inhibiting dihydrofolate reductase (DHFR). 
     
     
         23 . The method of  claim 21 , wherein the polynucleotide further comprises an expression control sequence. 
     
     
         24 . The method of  claim 23 , wherein the expression control sequence comprises a 5′ long terminal repeat (LTR) upstream of the shRNA734 and a 3′ LTR downstream of the shRNA734. 
     
     
         25 . The method of  claim 24 , wherein the polynucleotide further comprises a gene of interest disposed downstream of the 5′ LTR and upstream of the shRNA734. 
     
     
         26 . The method of  claim 19 , wherein the adverse effect is graft versus host disease. 
     
     
         27 . The method of  claim 19 , wherein the adverse effect is cytokine storm syndrome. 
     
     
         28 . The method of  claim 21 , wherein the shRNA734 is under control of a human 7SK RNA promoter. 
     
     
         29 . The method of  claim 28 , wherein the 7SK RNA promoter comprises a sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. 
     
     
         30 . The method of  claim 19 , wherein the genetically modified cell is a genetically modified T cell with a tumor specific T cell receptor or a chimeric antigen receptor. 
     
     
         31 . The method of  claim 19 , wherein the genetically modified cell is a hematopoietic stem/progenitor cell. 
     
     
         32 . The method of  claim 21 , wherein the genetically modified cell is from a purified population of genetically modified cells which have been contacted ex vivo with a guanine analog antimetabolite to select from unmodified cells.