Pre-Transplant Integration Site Diagnostics in Hematopoietic Stem Cell Gene Transfer.

Abstract 3581

Poster Board III-518

We and others have previously shown that insertional activation of cellular genes caused by integrated retroviral vectors can lead to clonal dominance and malignant transformation. Pre-transplant diagnostics of vector flanking sequences and subsequent elimination of those clones that carry potentially dangerous integration sites prior to transplantation would dramatically improve the safety of clinical gene therapy regimens. Such a strategy requires efficient transduction of few or individual stem cells, their in vitro amplification and highly sensitive integration site determination before transplantation.

To define optimal time points for transduction and ascertain the transplantability of ex vivo expanded murine stem cell clones, single CD45⁺Lin⁻Rho⁺SP cells isolated from bone marrow of male C57BL/6J (B6J) mice were cultivated for 8-10 days in the presence of IL11, SCF and Flt3-L. 10% of the sorted cells formed clones in vitro. In 28% ± 5% of these clones, the first division occurred during the first 48 hours after sorting, another 32% ± 8% divided up to 72 hours after sorting and additional 33% ± 7% up to 96 hours after sorting. 7% ± 4% had undergone their first division at a later time point. To examine the transplantability after ex vivo expansion, individual cell clones (containing 12 to >600 cells) were transplanted together with 10⁵ carrier cells into lethally irradiated sex-mismatched syngeneic mice. The presence of donor-derived cells in peripheral blood of 20 transplanted mice was analyzed by Y-chromosome specific PCR. 55% of the ex vivo expanded clones contributed to post-transplant hematopoiesis. 25% of these clones exhibited long-term activity for >6 months after transplantation. Interestingly, only cell clones that had undergone their first division 48-96 hours after cell sorting contributed to long-term post-transplant hematopoiesis.

For transduction, individual stem cell clones were spinoculated for 60 minutes with a GFP encoding lentiviral vector (MOI 100-5000). 5 days after transduction, 50% of cells generated by each clone were harvested, lysed and analyzed by LAM-PCR and integration site sequencing. After an additional 3 days, single clones were transplanted together with 10⁵ carrier cells into lethally irradiated congeneic B6.SJL-Ptprc^aPepc^b/BoyJ mice. Four weeks after transplantation, in 30% of these mice ≥0.4% CD45.1+ cells derived from single cell clones were detected in the peripheral blood. In 50% of these mice, the transduced clones contributed to myelopoiesis as well as lymphopoiesis for more than 24 weeks after transplantation, demonstrating that the longterm hematopoietic stem cell potential was retained after single cell marking and expansion.

These results demonstrate that single stem cell gene transfer and subsequent expansion is possible to allow integration site determination. Long-term stem cells with defined lentiviral integration sites can be selected for transplantation. In summary, we provide proof of concept that pre-transplant diagnostics of integration sites is feasible to increase the safety of gene therapy by eliminating stem cell clones from transplants that carry unwanted integration sites.