A Modified Foamy Viral Envelope Enhances Gene Transfer Efficiency and Reduces Toxicity of Lentiviral FANCA Vectors in Fanca-/- HSCs.

Abstract 696

Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow failure, genomic instability, and a predisposition to malignancies. Stem cell gene transfer technology is a potentially promising therapy, however, we have previously shown that prolonged ex vivo culture of cells using gamma retroviruses, results in a high incidence of apoptosis and predisposes the surviving reinfused cells to hematological malignancy in a murine model of FA. Here, we developed a lentiviral vector encoding the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSV-G or a modified foamy virus (FV) envelope to correct murine Fanca-/- stem and progenitor cells. An overnight transduction protocol was utilized to minimize genotoxic stress due to extended ex vivo manipulations. Transduction and expression of hFANCA was confirmed by three classical functional and biochemical measures in vitro: improved survival of clonogenic progenitors in the presence of mitomycin C (MMC), correction of MMC-induced G2/M arrest, and by the restoration of Fancd2 mono-ubiquitination. Furthermore, in vivo competitive repopulation experiments demonstrated that the repopulating ability of Fanca-/- stem cells transduced with the lentivirus encoding hFANCA was equivalent to that of wild-type stem cells, and the genetically-corrected reconstituting Fanca-/- cells were resistant to MMC and TNF-αa. Importantly, while a significant toxicity was observed using the VSV-G envelope, the toxicity of the FV envelope to murine c-kit+ cells was limited.

In parallel experiments, human umbilical cord blood CD34+ cord blood cells were transduced with either a VSV-G- or FV envelope-pseudotyped lentivirus encoding the EGFP reporter gene. Transplantation of 4×10⁵ cells into NOD/SCID/gamma-chain^null yielded a peripheral blood human chimerism comparable to the untransduced control cells (∼30%) regardless of the envelope. However, a much higher gene transfer efficiency of CD34+ cells was observed prior to transplantation when the FV envelope was employed (∼60%), as compared to the VSV-G envelope (∼15%). Furthermore, a similar 4 fold increase in transduction efficiency was observed in peripheral blood and in progenitors isolated from the bone marrow of both primary and secondary long term reconstituted mice. Collectively, these data indicate that the lentiviral construct pseudotyped with FV envelope can efficiently correct murine FA HSC/progenitor cells in a short transduction protocol and that the modified foamy envelope offers significantly greater transduction efficiency at comparable titers in long term reconstituting human cells in a xenograft model. This envelope may offer significant advantages compared to VSV-G in moving forward to phase 1 clinical trials.