Fanconi Anemia (FA) is a rare genetic disorder of DNA repair that typically manifests with bone marrow failure. Allogeneic stem cell transplant is the only cure known for the bone marrow disorder. Unfortunately, many patients do not have matched family donors, and alternative donor transplant has been associated with considerable morbidity and mortality. For individuals without an appropriate donor, ex vivo genetic modification of autologous stem cells is a potential therapeutic strategy. However, one of the major hurdles in gene therapy for this condition is the increased sensitivity of FA stem cells to free-radical induced DNA damage during ex vivo culture and manipulation. To minimize this damage we have developed a brief transduction procedure for lentivirus vector mediated gene transfer for Fanconi Anemia complementation group A (FancA), and evaluated this method in bone marrow progenitors from patients with FA, and murine progenitors from FancA deficient mice. The lentiviral vector, RSCPFancA-sW (FancA-sW), was specifically developed for clinical studies, and has a synthetic wpre which has been modified for safety (does not express a partial woodchuck hepatitis virus X protein open reading frame) and most of the 3′ untranslated region of the FancA gene was removed. FancA −/− mice were preconditioned with cyclophosphamide (120 mg/kg) prior to injection of transduced syngeneic bone marrow. After two subsequent rounds of the same dose of cyclophosphamide, 3/5 mice demonstrated mitomycin C (MMC) resistance in cells from bone marrow or spleen via colony assay. Two mice had peripheral blood marking with the vector seen by quantitative PCR. In an initial mouse experiment using similar conditions, the estimated transduction efficiency by colony PCR was determined to be 31% with the average number of integrations per cell determined to be 1.8 by LAM-PCR. Bone marrow mononuclear cells (BM MNCs) from a patient with FancA were also transduced with FancA-sW. Very few colonies were obtained on methylcellulose, although cell clusters were observed in MMC-treated cultures derived from FancA-sW transduced BM MNCs, while no clusters or colonies formed from cells transduced with the control vector. In an attempt to increase progenitor survival by reducing oxidative stress, primary human FA bone marrow was transduced and placed in 5% oxygen in the presence of 1 nM NAC (N-Acetyl-L-Cysteine). Cells plated in methylcellulose under these conditions had 2–3 fold increased colony formation compared to conditions without NAC or in 21% oxygen (P < 0.03). FancA-sW transduced BM MNCs from a second FancA patient were plated under these conditions formed colonies and numerous cell clusters and demonstrated resistance to MMC compared to GFP transduced controls (P < 0.007). Furthermore, transduced BM MNCs in culture had increased survival in culture when exposed to MMC ( P < 0.004). In summary, a lentiviral vector with a functional FancA transgene was developed which achieves phenotypic correction of human and murine hematopoietic progenitors with the brief period of prestimulation and transduction that would be required to maintain survival of FA cells ex vivo in culture. Reduction of oxidative stress may enhance the viability and engraftment of fragile FA stem cells.

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