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        Short-Term Culture of Human CD34+ Cells for Lentiviral Gene Transfer

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        Haematopoietic Stem Cells (HSCs) are attractive targets for the gene therapy. Upon ex vivo gene transfer and transplant, they may generate a progeny of gene-corrected cells potentially for a lifespan. The viral vectors most often used for HSC gene transfer are gamma-retroviral vectors (RVs) and HIV-derived lentiviral vectors (LVs). LVs have been proposed as improved tools for this task because they are able to transduce non-proliferating cells, while RVs are not. This implies that HSCs, which are mainly quiescent cells, need to be induced to proliferate in order to be transduced by RVs, whereas a prolonged stimulation is not needed for transduction by LVs. A short in vitro manipulation should reduce the risk of altering the characteristic biological properties of HSCs.
        We describe here methods for short-term ex vivo culture and gene transfer into human HSCs. Cord blood-derived HSC gene transfer can be tuned to limit the average level of vector integration or instead to maximize the frequency of transduction and extent of transgene expression, according to the absence or presence of cytokines. Mobilized peripheral blood-derived HSCs need cytokine stimulation to maintain viability and obtain adequate levels of gene transfer. Although HSCs can be transduced by LVs in short ex vivo culture, they display low permissiveness to the vector. Recently, we have demonstrated that this is because proteasome activity restricts LV gene transfer in HSCs. We developed and describe here strategies that effectively overcome this restriction. Finally, we also provide methods for the assessment of the transduction efficiency.
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