An improved vector for high-level, consistent retroviral transgene expression in human thymocytes after competitive reconstitution from transduced peripheral blood stem cells

One problem in hematopoietic stem cell (HSC)-based gene therapy is the low- level, and often transient, transgene expression in progeny cells in vivo. Here we have evaluated retroviral vector designs for improved longterm in v ivo transgene expression levels in thymocytes recovered after transplantati on of gene-modified HSCs. First, several vector designs were screened in vi tro by single-cell analysis of transgene marking and expression to rapidly identify optimal vectors for sensitive tracking of marked cells. Next, usin g one optimal vector, we show that gene-modified HSCs can competitively rec onstitute thymopoiesis in SCID-hu thymus/liver mice, with transgene express ion detectable on 0-40% of marked donor thymocytes. Modified vector designs (termed MSCV-SAR and MoMLV-SAR), which enhance transgene expression in pri mary T cells in vitro, were shown here to improve in vivo transgene express ion levels per cell 12- to 14-fold (mean fluorescence intensity was 2175 fo r MSCV-SAR vs. 174 for LNGFRSN; %NGFR(+) donor(+) cells with high-level exp ression was 58% for MSCV-SAR vs. 4% for LNGFRSN). Importantly, 61% of graft s had high-level transgene expression on thymocytes with the MSCV-SAR vecto r versus 0% of grafts for LNGFRSN or MoMLV-SAR. Transgene expression was de monstrated in various stages of thymocyte differentiation and was consisten tly detected in early thymic progenitors. We suggest that the MSCV-SAR vect or described here is particularly advantageous for applications requiring h igh-level, consistent transgene expression in a diverse repertoire of T cel ls derived from gene-modified HSC grafts.