An improved vector for high-level, consistent retroviral transgene expression in human thymocytes after competitive...

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 long-term in vivo transgene expression levels in thymocytes recovered after transplantation of gene-modified HSCs. First, several vector designs were screened in vitro by single-cell analysis of transgene marking and expression to rapidly identify optimal vectors for sensitive tracking of marked cells. Next, using one optimal vector, we show that gene-modified HSCs can competitively reconstitute thymopoiesis in SCID-hu thymus/liver mice, with transgene expression detectable on 0-40% of marked donor thymocytes. Modified vector designs (termed MSCV-SAR and MoMLV-SAR), which enhance transgene expression in primary T cells in vitro, were shown here to improve in vivo transgene expression levels per cell 12- to 14-fold (mean fluorescence intensity was 2175 for MSCV-SAR vs. 174 for LNGFRSN; %NGFR(+) donor(+) cells with high-level expression was 58% for MSCV-SAR vs. 4% for LNGFRSN). Importantly, 61% of grafts had high-level transgene expression on thymocytes with the MSCV-SAR vector versus 0% of grafts for LNGFRSN or MoMLV-SAR. Transgene expression was demonstrated in various stages of thymocyte differentiation and was consistently detected in early thymic progenitors. We suggest that the MSCV-SAR vector described here is particularly advantageous for applications requiring high-level, consistent transgene expression in a diverse repertoire of T cells derived from gene-modified HSC grafts.