HIGH-EFFICIENCY TRANSDUCTION OF HUMAN LYMPHOID PROGENITOR CELLS AND EXPRESSION IN DIFFERENTIATED T-CELLS

Gene therapy strategies for humans have been limited by low transducti on efficiencies and poor expression of retroviral vectors in different iated progeny cells carrying the transduced vector. Here we describe a strategy utilizing a cell surface reporter gene, murine thy-1.2, sele ctable by fluorescence-activated cell sorting (FACS), to achieve highe r gene marking efficiencies, Human CD34-positive cells were transduced by a murine retroviral vector bearing the thy-1.2 marker and pseudoty ped with vesicular stomatitis virus G protein, followed by FACS to enr ich for CD34-positive cells that express Thy-1.2 on the cell surface, Gene marking and expression after differentiation into thymocytes were assessed in a SCID-hu Thy/Liv mouse model for human lymphoid progenit or cell gene therapy, We found that virtually all of the differentiate d T-cell progeny were marked with vector sequences, It is of particula r importance that reconstitution with the selected cells resulted in e xpression of Thy-1.2 in up to 71% of donor-derived thymocytes. It is o f note that the donor derived thymocytes that did not express Thy-1.2 still harbored vector thy-1.2 sequences, suggesting repression of tran sgene expression in some cells during progenitor cell differentiation into thymocytes, These studies provide a proof of concept for efficien t expression of transgenes through T-lymphoid differentiation and a po tential basis for utilizing similar strategies in human gene therapy c linical trials.