Retroviral vectors based on Human Immunodeficiency Virus (HIV) and pseudotyped with vesicular stomatitis virus G (VSV-G) envelope glycoprotein can stably modify non-dividing hematopoietic stem cells. Ex vivo culture of vector particles and target cells is presumed to result in receptor mediated particle uptake by the cell and successful proviral integration, or degradation. However, recent evidence in dendritic cells suggests an alternate cellular fate for vector particles whereby they persist in exosomes and can be released to transduce secondary targets. We investigated the existence and consequences of such a pathway in other hematopoietic target cells after conventional ex vivo exposure. Murine bone marrow cells (5×10^5) were exposed to vector particles (2.5 ×10^6), washed twice, and placed alongside 293T fibroblasts (1×10^5). Direct, or transwell, co-culture resulted in GFP marking of 30% and 10% of secondary targets (ie. 293T cells), respectively. Transgene expression in 293T cells was stable over time in culture, abrogated by using integration-deficient particles, and reflected proviral integration, based on real-time PCR results. Cellular persistence of particles after primary exposure of murine whole bone marrow or SupT1 cells and secondary carryover were vector concentration dependent and pseudotype independent. Intriguingly, cell bound vector particles were selectively protected from serum inactivation, and the kinetics of secondary transduction suggested a prolonged particle half-life, when compared to particles cultured under cell-free conditions. Further, while direct protease exposure effectively eliminated vector infectivity, secondary transfer of particles from vector exposed, protease washed, cells was only partially inhibited, suggesting the uptake of particles in protease-inaccessible compartments of primary targets. When comparing vector exposures at 37 C versus 4 C (preventing particle uptake while permitting binding), again followed by protease treatment, secondary gene transfer was relatively greater at 37 C and increased with primary transduction duration, consistent with progressive intracellular particle retention. Indeed, deconvolution microscopy experiments to investigate the fate of tagged particles after brief vector exposure to target cells revealed intracellular persistence and perinuclear accumulation. To determine the relevance of these in vitro findings, we next performed studies in non-ablated murine recipients (n=30) that received ex vivo vector exposed, and washed, whole bone marrow or lineage depleted cells. To unambiguously demonstrate particle hand-off to recipient hematopoietic cells, we used CD45.1 donors and CD45.2 recipient animals. Results demonstrated long-term GFP marking by flow-cytometry (up to 9%) and by real-time PCR in recipient bone marrow and peripheral blood leukocytes. Additional immuno stains at sacrifice showed GFP-marked -CD45 negative- cells in liver and spleen tissue sections. Gain of replication competency in vector lots and in serum from recipient animals was excluded by p24 ELISA assay. Based on these results we propose an alternate fate for VSV-G lentivector particles, involving cellular retention after ex vivo exposure to primary hematopoietic target cells, subsequent release, and secondary transduction of susceptible target tissues. These findings may have implications for a range of applications using lentiviral vectors.
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Jun 30, 2008
Marjon Navis + 2
Open Access
