TRANSDUCTION OF PLURIPOTENT HUMAN HEMATOPOIETIC STEM-CELLS DEMONSTRATED BY CLONAL ANALYSIS AFTER ENGRAFTMENT IN IMMUNE-DEFICIENT MICE

Gene transduction of pluripotent human hematopoietic stem cells (HSCs) is necessary for successful gene therapy of genetic disorders involvi ng hematolymphoid cells, Evidence for transduction of pluripotent HSCs can be deduced from the demonstration of a retroviral vector integrat ed into the same cellular chromosomal DNA site in myeloid and lymphoid cells descended from a common HSC precursor, CD34(+) progenitors from human bone marrow and mobilized peripheral blood were transduced by r etroviral vectors and used for long-term engraftment in immune-deficie nt (beige/nude/XID) mice, Human lymphoid and myeloid populations were recovered from the marrow of the mice after 7-11 months, and individua l human granulocyte-macrophage and T-cell clones were isolated and exp anded ex vivo. Inverse PCR from the retroviral long terminal repeat in to the flanking genomic DNA was performed on each sorted cell populati on, The recovered cellular DNA segments that flanked proviral integran ts were sequenced to confirm identity, Three mice were found (of 24 in formative mice) to contain human lymphoid and myeloid populations with identical proviral integration sites, confirming that pluripotent hum an HSCs had been transduced.