Generation of genetically engineered precursor T cells from human umbilical cord blood using an optimized alpharetroviral vector platform.

Retroviral engineering of hematopoietic stem cell-derived precursor T cells (preTs) opens the possibility of targeted T cell transfer across HLA-barriers. Alpharetroviral vectors exhibit a more neutral integration pattern thereby reducing the risk of insertional mutagenesis. Cord blood-derived CD34+ cells were transduced and differentiated into preTs in vitro. Two promoters, elongation-factor-1-short-form, and a myeloproliferative-sarcoma-virus variant in combination with two commonly used envelopes were comparatively assessed choosing green fluorescent protein or a third generation chimeric antigen receptor (CAR) against CD123 as gene of interest. Furthermore, the inducible suicide gene iCaspase 9 has been validated. Combining the sarcoma virus-derived promoter with a modified feline endogenous retrovirus envelope glycoprotein yielded in superior transgene expression and transduction rates. Fresh and previously frozen CD34+ cells showed similar transduction and expansion rates. Transgene positive cells did neither show proliferative impairment nor alteration in their lymphoid differentiation profile. The sarcoma virus-derived promoter only could express sufficient levels of iCaspase 9 to mediate dimerizer-induced apoptosis. Finally, the CD123 CAR was efficiently expressed in CD34+ cells and proved to be functional when expressed on differentiated T cells. Therefore, the transduction of CD34+ cells with alpharetroviral vectors represents a feasible and potentially safer approach for stem cell-based immunotherapies for cancer.Molecular Therapy (2016); doi:10.1038/mt.2016.89.