Abstract
Unlike cell lines, human hematopoietic stem cells (HSCs) are less efficiently transduced with HIV-1 vectors, potentially limiting this approach. To investigate which step (internalization, reverse transcription, nuclear transport, and integration) limits lentiviral transduction, we evaluated the kinetics of lentiviral transduction in human CD34(+) cells. We transduced HeLa and CD34(+) cells with self-inactivating HIV-1 vector at low and tenfold higher multiplicity of infection (MOI) and evaluated vector amounts at various time points based on the rationale that if a given step was not limiting, tenfold greater vector amounts would be obtained at the tenfold higher MOI. We observed slower internalization (>60 min), a peak in reverse transcription at 24 hours, and completion of integration at 3 days in CD34(+) cells. In HeLa cells, there were approximately tenfold greater amounts at high MOI at all time points. When compared with HeLa cells, CD34(+) cells exhibited larger differences in vector amounts between high and low MOIs at 2-6 hours and a smaller difference at 12 hours to 10 days, revealing a limitation in human CD34(+) cell transduction around 12 hours, which corresponds to reverse transcription. In serial measurements of reverse transcription at 24 hours, vector amounts did not decrease once detected among CD34(+) cells. When using an HSC expansion medium, we observed less limitation for starting reverse transcription and more efficient transduction among CD34(+) cells in vitro and in xenografted mice. These data suggest that it is the initiation of reverse transcription that limits lentiviral transduction of human CD34(+) cells. Our findings provide an avenue for optimizing human CD34(+) cell transduction.