BACKGROUND:
Gene therapy strategies to modify human stem cells hold great promise for curing many human diseases. However, previous clinical studies have met with limited success, largely due to the low engraftment of gene modified stem cells. One strategy to overcome this challenge involves engineering stem cells in which HPRT expression is knocked down, thereby facilitating the selection of genetically modified cells by conferring resistance to a guanine analog antimetabolite.
While efforts have been made to disrupt HPRT, there remains a need for more effective materials and methods to directly target the HPRT gene.
INNOVATION:
The invention meets these needs and others by providing a polynucleotide comprising a short hairpin ribonucleic acid molecule 734 (shRNA734) and methods of using same. The invention provides a small RNA based chemoselection strategy that can be used for improving the engraftment of genetically modified cells for stem cell based gene therapy strategies.
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.