Transduction of human CD34(+)CD38(-) bone marrow and cord blood-derived SCID-repopulating cells with third-generation lentiviral vectors

The major limitations of Moloney murine leukemia virus (MoMLV)-based vector s for human stem cell applications, particularly those requiring bone marro w (BM) stem cells, include their requirement for mitosis and retroviral rec eptor expression. New vectors based upon lentiviruses such as HIV-1 exhibit properties that may circumvent these problems. We report that novel third- generation, self-inactivating lentiviral vectors, expressing enhanced green fluorescent protein (ECFP) and pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G), can efficiently transduce primitive human repopula ting cells derived from human BM and cord blood (CB) tested by the SCID-rep opulating cell (SRC) assay. Highly purified CD34(+)CD38(-) CB or BM cells w ere efficiently transduced (4-69%) and stably expressed in ECFP for 40 days in culture following infection for only 24 h without fibronectin, polybren e, or cytokines. Nonobese diabetic/severe combined immune-deficient (NOD/SC ID) mice transplanted with transduced cells from either CB or BM donors wer e well engrafted, demonstrating maintenance of SRC during the infection pro cedure. Serially obtained femoral BM samples indicated that the proportion of EGFP(+) cells within both myeloid and lymphoid lineages was maintained o r even increased over time, averaging 42.3 +/- 6.6% for BM donors and 23.3 +/- 7.2% for CB at 12 weeks. Thus, the third-generation lentivectors readil y transduce human CB and BM stem cells, under minimal conditions of ex vivo culture, where MoMLV-based vectors are ineffective. Since CB is inappropri ate for most therapeutic applications, the efficient maintenance and transd uction of BM-derived SRC during the short infection procedure are notable a dvantages of lentivectors.