Lentiviral-mediated gene transfer into haematopoietic stem cells

Objectives. Lentiviral vectors can transduce nondividing cells, As most hae matopoietic stem cells (HSCs) are nondividing in vivo, lentiviral vectors a re promising viral vectors to transfer genes into HSCs, Design and Setting, We have used HIV-1 based lentiviral vectors containing the green fluorecent protein (GFP) gene to transduce umbilical cord blood C D34+ and CD34+/CD38- cells prior to transplantation into NOD/SCID mice. Results. High level engraftment of human cells was obtained and transgene e xpression was seen in both myeloid and lymphoid lineages. Bone marrow from the primary transplant recipients mice was transplanted into secondary reci pients. GFP expression was seen in both lymphoid and myeloid cells in the s econdary recipients 6 weeks posttransplantation. Human haematopoietic proge nitor colonies were grown from both primary and secondary recipients. Over 50% of the haematopoietic colonies in these recipients were positive for th e GFP transgene by PCR. Following inverse PCR, amplified fragments were seq uenced and integration of the vector into human genomic DNA was demonstrate d, Several vectors containing different internal promoters were tested in N OD/SCID mice that had been transplanted with transduced CD34+ and CD34+/CD3 8- cells, The elongation factor-1 alpha (EF-1 alpha) promoter gave the high est level of expression, both in the myeloid and lymphoid progeny of the en grafting cells. Conclusions, These data collectively indicate that candidate human HSCs can be efficiently transduced with lentiviral vectors and that the transgene i s highly expressed in their progeny cells.