Abrogated cryptic activation of lentiviral transfer vectors.

Despite significant improvements in lentivirus (LV) vector-based gene therapy there are still several safety risks using LV vectors including the potential formation of replication-competent LV particles. To address this shortcoming, we constructed a novel and safer gene transfer system using modified SIN-based LV gene transfer vectors. Central to our approach is a conditional deletion of the Ψ packaging signal after integration in the target genome. Here we demonstrate that after transduction of target cells, conventional SIN-based LV transfer vectors can still be mobilized. However mobilization is rendered undetectable if transductions are followed by a Cre/loxP-mediated excision of Ψ. Thus conditional elimination of the packaging signal may represent another advance in increasing the safety of LV vectors for gene therapeutic treatment of chronic diseases.

Safeguarding nonhuman primate iPS cells with suicide genes.

The development of technology to generate induced pluripotent stem (iPS) cells constitutes one of the most exciting scientific breakthroughs because of the enormous potential for regenerative medicine. However, the safety of iPS cell-related products is a major concern for clinical translation. Insertional mutagenesis, possible oncogenic transformation of iPS cells or their derivatives, or the contamination of differentiated iPS cells with undifferentiated cells, resulting in the formation of teratomas, have remained considerable obstacles. Here, we demonstrate the utility of suicide genes to safeguard iPS cells and their derivatives. We found suicide genes can control the cell fate of iPS cells in vitro and in vivo without interfering with their pluripotency and self-renewal capacity. This study will be useful to evaluate the safety of iPS cell technology in a clinically highly relevant, large animal model and further benefit the clinical use of human iPS cells.

Stable marking and transgene expression without progression to monoclonality in canine long-term hematopoietic repopulating cells transduced with lentiviral vectors.

Lentiviral gene transfer vectors have a number of potential advantages over gammaretroviral vectors including more efficient transduction of nondividing cells, a more favorable integration site profile, and the ability to accommodate large transgenes. Here, we present long-term follow-up data of animals that received lentivirus-transduced CD34-enriched cells. Six long-term surviving dogs were available for analysis. Transgene expression was analyzed from at least 12 months to more than 5 years after transplantation in peripheral blood cells and multiple cell lineages. All animals demonstrated long-term stable transgene expression in peripheral blood myeloid, lymphoid, and red blood cells as well as in platelets. Vector integration sites were analyzed by linear amplification-mediated polymerase chain reaction and showed a polyclonal repopulation pattern in all animals. There was no evidence of any development of monoclonality or leukemia in the animals. The stable long-term multilineage transgene expression, together with detection of the same integration site in myeloid and lymphoid cells, strongly suggests the transduction of long-term repopulating stem cells. Our data demonstrate safe and efficient transduction of multilineage long-term repopulating cells with lentiviral vectors and support the use of such vectors for gene therapy studies in patients.