682. Collection and Genetic Correction of Fanconi Complement Type A Hematopoietic Stem Cells: Results of Two Pilot Studies
Authors: P.F. Kelly, B. Balcik, K. Bohn, R. Mueller, T. Schuesler, L. Reeves, T. Leemhuis, R. Harris, S.M. Davies, F.O. Smith, C. von Kalle, D.A. Williams
Journal: Molecular Therapy (2006)

Fanconi anemia (FA) is a genetic syndrome characterized by the uniform development of aplastic anemia. Current therapy for patients lacking HLA-identical sibling hematopoietic stem cell (HSC) donors are not optimal. We have opened two phase I clinical trials to determine if potentially useful numbers of CD34+ cells can be collected early in the course of the disease (collection study) and if these cells, once corrected, can engraft without cytoreduction and demonstrate proliferative advantage in vivo over un-corrected cells (gene transfer study). To date, 7 FA patients have undergone a 20 ml/kg bone marrow harvest (BMH) with an average of 1.9x106 CD34+ cells/kg (range 0.3–2.9x106 CD34+ cells/kg) collected, suggesting that collection of adequate numbers of cells is possible but challenging, even early in the disease. In the gene transfer study, 3 FA patients with genotype A (FAA) have enrolled, meeting eligibility criteria of FAA, no evidence of malignancy and a minimum of 1x105 viable CD34+ cells/kg for ex vivo culture and gene transfer. BMHs from the 3 patients (2 fresh and one previously cryopreserved) were CD34+ cell selected. Despite collection before significant pancytopenia, an average of only 5x105 CD34+ cells/kg (range 1.5–10x105 CD34+ cells/kg) was purified from these 3 cases representing 10% of the expected yield from normal individuals. These cells underwent ex vivo gene transfer using cytokine prestimulation in serum-free medium followed 2 exposures to a GALV-MSCV-FANCA vector. Transduction efficiency of the final product determined by real-time PCR analysis of progenitors averaged 48% (range 40–62%). Equivalent correction of mitomycin C hypersensitivity in progenitor cells confirmed this analysis at the functional level. Nucleated cell recovery after ex vivo manipulation was 82–110% of input nucleated cells using freshly harvested bone marrow derived CD34+ cells (N=2). However, despite good CD34+ cell recovery and viability after CD34+ selection, only 6% of input nucleated cells were recovered utilizing CD34+ cells purified from the previously cryopreserved bone marrow and these cells were not re-infused. In the two patients who did receive gene corrected cells, the total cell dose re-infused was 2.5–3.5x105 nucleated cells/kg, reflecting the low number of initial CD34+ cells placed in culture. FAA vector sequences were detected in the PB of one patient early post-infusion (+4 weeks) but none was detected after 8 weeks. Despite no long term detection of the corrective gene in the PB and BM, both patients exhibited a transient but marked improvement (10–20% increase) in their peripheral blood hemoglobin (> 6 months) and platelets counts (2–3 months). The data demonstrates that gene transfer efficiency in the clinical setting has been significantly improved, however the collection of adequate numbers of HSC may be the critical barrier to the success of genetic correction attempts in FA.