Design and Production of Retro- and Lentiviral Vectors for Gene Expression in Hematopoietic Cells
Successful retroviral gene transfer into hematopoietic cells has been demonstrated in a number of small and large animal models and clinical trials. However, severe adverse events related to insertional muta-genesis in a recent clinical trial for X-linked severe combined immunodeficiency reinforced the need to develop novel retroviral vectors with improved biosafety. Improvements include the use of self-inactivating (SIN) vectors as well as improvements in vector design. This chapter describes the basic design of gamma-retroviral and lentiviral SIN vectors that utilize a split-packaging system and includes a description of the various cloning modules frequently used in the design of such vectors that impact biosafety, titer, and transgene expression. In addition, this chapter describes the methods used for high titer vector production using calcium phosphate transfection both at research scale and at large scale for clinical application using a closed system bioreactor.
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