Human immunodeficiency virus type 1-derived lentivirus vectors bearing the vesicular stomatitis virus G (VSV-G) envelope glycoprotein demonstrate a wide host range and can stably transduce quiescent hematopoietic stem cells. In light of concerns about biosafety and potential germ line transmission, they have been used predominantly for ex vivo strategies, thought to ensure the removal of excess surface-bound particles and prevent in vivo dissemination. Studies presented here instead reveal prolonged particle adherence after ex vivo exposure, despite serial wash procedures, with subsequent transduction of secondary target cells in direct and transwell cocultures. We explored the critical parameters affecting particle retention and transfer and show that attachment to the cell surface selectively protects virus particles from serum complement-mediated inactivation. Moreover, studies with nonmyeloablated murine recipients show that transplantation of vectorexposed, washed hematopoietic cells results in systemic dissemination of functional VSV-G/lentivector particles. We demonstrate genetic marking by inadvertent transfer of vector particles and prolonged expression of transgene product in recipient tissues. Our findings have implications for biosafety, vector design, and cell biology research.
Adhesive interaction between chemokine receptor (CXCR) 4 and its ligand, stromal cell derived factor -1 alpha (SDF-1, CXCL12), is principally responsible for the homing of bone marrow progenitor cells to sites of hematopoiesis. Recent evidence in murine models suggests that engraftment and donor chimerism after transplantation are responsive to changes in progenitor CXCR4 expression. We hypothesized that this mechanism might also serve to overcome the repopulation deficiency in a murine model of Fanconi Anemia (FA), where an pro-apoptotic stem cell phenotype compromises overall repopulation efficiency. We investigated potential CXCR4 upregulation alternatively by cytokine stimulation, hypoxia, or CD26 (dipeptidylpeptidase IV) inactivation. Initial studies ascertained comparable baseline frequency and proliferation kinetics among c-kit, sca-1 and thy-1 expressing lineage depleted (lin−) subsets of bone marrow cells from wild-type (wt) and FA transgenic (Fancc−/−, ko) animals. Evaluation of CXCR4 expression by flow-cytometry revealed low baseline levels and substantial induction in sca-1, c-kit and thy-1 progenitors after stem cell factor and interleukin 3 supplemented culture, in both wt and ko animals. CXCR4 is also upregulated in response to hypoxia, and FA proteins are involved in oxidative stress signaling. We therefore reasoned that culture under hypoxic conditions may avoid some of the exaggerated apoptotic responses of FA hematopoietic cells during conventional culture and further improve Fancc−/− progenitor homing. Results in lin− bone marrow progenitor subsets from wt and Fancc−/− animals confirmed that CXCR4 could be upregulated following overnight hypoxia (1% O2 tension). No additional improvement was seen after extended culture. In murine competitive repopulation studies using CD45.1 and CD45.2 cells we confirmed the improved post transplantation chimerism in myeloablated recipients that received CD45.2/Fancc−/− cells cultured in low-O2 environment, compared to untreated wt cells. We also investigated the inactivation of cell surface peptidase CD26 (dipeptidylpeptidase IV) by Diprotin A (DA, Ile-Pro-Ile) which has recently been shown to improve progenitor homing. Our initial competitive transplantation experiments in cohorts of wt recipients confirmed improved chimerism from DA treated cells versus PBS control. In apparent contrast however, cells treated with DA after cytokine or hypoxia culture showed substantially decreased cell surface levels of CXCR4. This may suggest a more complex mechanism for the improved homing after DA exposure than previously thought. In summary, we show that Fancc−/− mice share similar progenitor compartment composition and dynamics in culture when compared with wt animals. Their hematopoietic cells express normal levels of CXCR4, and retain the ability to respond to stimulation by diverse stimuli with upregulation, resulting in improved donor engraftment and chimerism. These are the first reported studies to apply homing directed strategies to overcome the intrinsic qualitative defect in an established model of a stem cell repopulation deficiency (Fancc). The proposed strategies, individually or combined, may also offer a novel approach to overcome the repopulation disadvantages in other stem cell transplantation settings.
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