Gene Therapy for Fanconi Anemia in Seattle: Clinical Experience and Next Steps

One hallmark of the rare, monogenic disorder called Fanconi anemia (FA) is an accelerated decline in hematopoietic stem cells (HSCs) leading to bone marrow (BM) failure. Long-term treatment requires successful bone marrow transplant (BMT) from an unaffected donor. However, BMT success is limited if the donor is not a matched sibling and ~70% of FA patients lack such donors. Gene therapy could be an alternative, correcting the genetic defect in the patient9s own HSCs, and negating the need for a BMT donor. Based on lessons learned in previous FA gene therapy studies, we developed an optimized protocol for lentivirus (LV)-mediated FANCA gene transfer into HSCs from FA-A patients. This phase I clinical trial incorporates vector recommendations from the International FA Gene Therapy Working Group. Two patients have been treated on this trial to date (National Clinical Trials Registry ID: NCT01331018). This protocol harvests BM to collect unmanipulated HSC and does not include conditioning prior to cell infusion. Patient 1 is a 22-year old male with confirmed FA-A resulting from a splice variant in exon 22 of the FANCA gene (c. 1827-1 Gu003eA). Baseline ANC averaged 0.5-1.0 K/mcL and baseline platelet counts averaged 40 K/mcL. A total of 3.2 x 10 7 CD34+ cells were present in 1.1L of BM, but only 9.4 x 10 6 total CD34+ cells were successfully isolated by magnetic bead separation, due to low level CD34 expression. LV transduction at 10 infectious units (IU)/cell resulted in a vector copy number (VCN) of 0.33 per cell and 18.4% of colony-forming cells transduced. Patient 2 is a 10-year old male with confirmed FA-A resulting from gross deletion of exons 6-31 of the FANCA gene. Baseline ANC and platelets declined over a 4.5-year interval prior to gene therapy and were 0.67 K/mcL and 82 K/mcL, respectively, in the 6 months prior to treatment. A total of 400mL of BM was collected, containing a total of 30.6 x 10 6 CD34+ cells. To avoid excessive CD34+ cell loss, the CD34+ cell magnetic bead purification step was omitted and the entire red blood cell depleted BM product was subjected to LV transduction at 10 IU/cell. We observed a VCN of 1.83 per cell and 43% of colony-forming cells transduced, suggesting more efficient transduction of the mixed cell population. Both patients tolerated the harvest and infusion procedures but displayed low and declining levels of transduced cells in peripheral blood after infusion. For future subjects, use of pre-infusion conditioning may be required to achieve long-term engraftment in vivo . Interestingly, both patients have maintained stable blood cell counts since gene therapy. These data demonstrate that LV gene therapy in FA patients is safe and suggest that avoidance of direct CD34 selection is advantageous for transduction and gene transfer. However, one complicating factor is the volume of concentrated LV vector required to transduce the non-purified cell product infused in Patient 2. To address these barriers, we developed a modified clinical protocol which utilizes depletion of mature cell lineages including CD3+, CD14+, CD16+ and CD19+ cells. Using healthy donor bone marrow we demonstrate that this protocol efficiently depletes u003e85% of cells expressing each lineage marker, reducing the volume of LV vector required for gene transfer by 60-70%. Most importantly, we demonstrate that this protocol preserves u003e90% of CD34+ cells present in the starting bone marrow product, and that these cells are efficiently transduced and capable of engrafting in a xenotransplant model. This protocol is currently being implemented for subjects in the ongoing phase I trial. Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership. Kiem: Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.