399. Transduction of Hematopoietic Stem Cells Using Polymer-Concentrated Ecotropic Retrovirus Coupled with In Vivo Selection of Gene-Modified Cells
Authors: Douglas W. McMillin, Natalia Landazuri, Bagirath Gangadharan, David Archer, Joseph Le Doux, H. Trent Spencer
Journal: Molecular Therapy (2004)

Steady advances have been made with respect to genetically modifying hematopoietic stem cells (HSCs) using recombinant retroviruses, but there is still the problematic need of generating high viral titers. Using ecotropic pseudotyped recombinant retrovirus, which is typically inactivated during concentration methods employing centrifugation, we show that recombinant virus can be rapidly concentrated using a combination of polymers, and HSCs are readily transduced using concentrated virus. In addition, HSCs transduced with polymer-concentrated virus at very low MOIs can be expanded in vivo using an antifolate regimen if HSCs are induced to cycle prior to selection. Recombinant ecotropic pseudotyped virus encoding eGFP was generated by transient transfection of GP2-293 producer cells, and viral supernatants were collected over 72 hours. Viral stocks were concentrated by incubating the retroviral supernatant with equal concentrations of polybrene and chondroitin sulfate C followed by low-speed centrifugation. The resulting pellet contains competent ecotropic virus and was resuspended in cell growth media. To determine if polymer-concentrated virus can be used to transduced HSCs, bone marrow was isolated from C3H mice, transduced with a single application of concentrated virus (MOI~1) and transplanted into lethally irradiated recipients. Total gene marking in transplanted animals averaged 30–35%, as measured by flow at 8 weeks post transplant, and stable integration is observed for over 16 weeks in all hematopoietic lineages. In addition, retroviral vectors encoding a drug resistant variant of dihydrofolate reductase (L22YDHFR) was concentrated and tested using in vitro progenitor assays. Approximately 40% of murine bone marrow progenitor cells were protected against concentrations of trimetrexate that completely eliminated growth of non-modified cells. Bone marrow transduced with concentrated virus at a low MOI (~0.5) and transplanted into lethally irradiated recipients results in 5–10% gene marking 8 weeks post transplant, as measured by real-time PCR. Mathematical models were generated to calculate the efficiency of in vivo selection using variants of DHFR and antifolate selection under conditions of steady-state hematopoiesis, which predicted multiple rounds of selection are required to achieve only modest in vivo selection. However, if HSCs are induced to cycle, it is predicted that significant selection can be achieved with only a few rounds of selection. Using 5-FU to induce cycling, we show that >6-fold increase in circulating gene-modified cells can be achieved with a single round of antifolate selection. These results show that by incorporating basic retroviral processing steps, HSCs can be efficiently transduced using polymer-concentrated ecotropic virus, resulting in high level stable expression. In addition, HSCs transduced with variants of DHFR can be selectively enriched if stem cells are induced to cycle prior to antifolate selection.