6. In Vivo Adult Stem Cell Gene Transfer in Mice by In Situ Delivery of a Self-Inactivating Lentiviral Vector Using Intrafemoral Injection

Abstract

The potential of in vivo bone marrow stem cell gene transfer by bone cavity injection has not been explored. This approach could take full advantage of any source of stem cells present in bone cavity and avoid many of the difficulties encountered by ex vivo hematopoietic stem cell (HSC) gene transfer. To determine if efficient gene transfer can be achieved in HSC and mesenchymal stem cells (MSC) by in situ delivery of a HIV-based lent virus vector (LV), we intrafemorally injected into adult normal mice (Ly5.1) with a self-inactivating GFP-containing LV (at 2|[times]|106 IU/mouse). GFP-expressing peripheral blood leukocytes (PBL) were observed in both myeloid (up to 4%) and lymphoid subpopulations (up to 2%) 3-month post injection. The colony-forming cell (CFC) assay resulted in 4|[ndash]|5% of GFP-expressing CFUs with multilineage potentials 4-months after injection, which was consistent with 4-color FACS analysis data demonstrating up to 3.7% of GFP+ HSC/progenitors (Lin-c-kit+Sca1+). Higher frequencies of GFP-containing CFUs (up to 15.9%) were revealed by a colony-direct duplex PCR. To further evaluate HSC gene transfer, bone marrow from injected mice was transplanted into lethally irradiated adult C57BL/6 (Ly5.2) mice with successful engraftment demonstrated by 97.5-98.5% donor-derived PBL in all recipients 3-months post BMT. Four months after transplantation, we observed 8.4 to 17.7 % GFP-expressing CFUs, as well as 16 to 24% GFP-containing CFUs in all recipients. Vector integrants and their multi-clonality were further confirmed by linear-amplification mediated PCR (LAM-PCR) in both primary injected and secondary transplanted mice. We also demonstrated that GFP-expressing MSC retained multiple differentiation potential. Our data provide the first evidence that adult stem cells in bone marrow could be efficiently transduced in vivo by |[ldquo]|in situ|[rdquo]| vector administration. This may potentially open a door to a novel approach for treatment of human diseases.