Effects of Cyclosporine A on Lentiviral Transduction of Mouse Hematopoietic Stem Cells and Transplantation Using Nonmyeloablative Conditioning.

Achieving high level transduction of murine hematopoietic stem cells (HSCs) using lentiviral vectors has been a challenge for many laboratories. We investigated the efficiency of lentiviral transduction of murine stem cell antigen-1⁺ (sca-1⁺) cells with and without cyclosporine A (CSA), which has previously been shown to increase the transduction efficiency of other types of murine cells. Sca-1⁺ cells were isolated from C57BL/6 mice and transduced with lentiviral vectors encoding green fluorescent protein (GFP) at various multiplicity of infections (MOI) and with various concentrations of CSA. Twenty-four hours after a single transduction, 1.5 x 10⁴ or 4.5 x 10⁴ cells were plated in methylcellulose containing the appropriate cytokines for progenitor cell growth, and colonies were counted on days 8–12. In the absence of CSA only 4± 2% of progenitor colonies were GFP⁺. However, when CSA (10 μM) was added within 3 hours of sca-1 cell isolation, transduction efficiency increased to 44 ± 6%. Although the transduction efficiency increased 10-fold, the number of progenitor colonies significantly decreased when CSA was added (up to 90% decrease). Lower concentrations of CSA (e.g. 1 μM) were less toxic to sca-1⁺ cells but resulted in inconsistent transduction efficiencies. We next determined the effects of CSA when applied at various times after sca-1 cell isolation. We found that the number of sca-1⁺ cells decreased within the first two days of culture but then begin to increase on day 3, and by day 7 there is a 7-fold increase compared to the number of cells originally isolated. Cells cultured with virus alone had an average increase of 3.5-fold on day 7, but only 3% of cells cultured in CSA survived to day 7. Cells cultured with both virus and CSA had no viability on day 7. However, by delaying the addition of virus and CSA until day 3, a 1.4-fold increase in sca-1⁺ cells was observed by day 7, which was achieved without affecting the efficiency of transduction. Sca-1⁺ cells were then transduced with the lentiviral vector in the presence of CSA and transplanted into transgenic sickle mice using a nonmyeloablative conditioning regimen that consisted of busulfan (25 mg/kg) administered on day -1 and costimulation blockade with CTLA-4Ig and anti-CD40 ligand administered on days 0, 2, 4, and 7. We were able to achieve donor engraftment levels of 98% with a 40% engraftment of gene-modified cells. These results show that using CSA in lentiviral transductions of murine HSCs can be an effective method for increasing overall transduction efficiency, and may aid in the use of lentiviral vectors in animal studies.