1055. High Levels of Human Factor IX Expression in Hemophilia B Mice after Adult Stem Cell-Based Gene Therapy Combined with Non- Myeloablative Conditioning

Abstract

Top of pageAbstract Various cell types have been targeted for expression of clotting factors in experimental models of hemophilia, with muscle and hepatocyte-directed gene transfer advancing to clinical trials. Hematopoietic stem cells (HSCs) are attractive alternative targets because of their self-renewing properties, their ability to generate blood cells over extended periods of time, and their ease of manipulation ex vivo. However, several hurdles must be overcome to make this approach safe and practical. These include the need to express sufficient amount of therapeutic gene products and minimize the risk of insertional mutagenesis and the toxicity of host conditioning to facilitate HSC engraftment. We have previously developed an erythoid cell-specific platform for long-term and systemic therapeutic protein delivery in vivo using an erythroid- specific lentiviral vector. We demonstrated in an immune competent C57BL/6 hemophilia B mouse model, that therapeutic levels of human factor IX (hFIX) can be expressed at low average vector copy (VC) number (250-350 ng/mL with under 0.5 VC per cell). In this study, we examined the level of erythroid-specific protein delivery obtained after engraftment following minimal conditioning, eventually in the presence of in vivo methylguanine methyltransferase (MGMT)-mediated drug resistance. Low to moderate levels of hFIX expression were achieved in control, lethally irradiated mice (average 100-200 ng/ml), 4-weeks post-transplantation. In non-myeloablated recipients conditioned with 400 cGy or Busulfan (20 mg/kg, given twice at 48 h and 24 h before engraftment), the levels of hFIX expression were ranged from negligible to low (0-20 ng/ml and 0 |[ndash]| 100 ng/ml, respectively). Four weeks after the first round of BG/BCNU (30 mg/kg and 5 mg/kg, respectively), a wide range of hFIX expression levels were achieved, reaching in some mice levels equivalent to 20% of physiological levels in humans. We are currently monitoring long-term hFIX expression, as well as the average VC number in blood cells and CFUs. Further rounds of drug selection are also in progress. In conclusion, our data suggest that it is possible to express high levels of a secreted protein from HSC-derived erythroid cells engrafted under minimal conditioning. The combined features of reduced intensity conditioning, reduced insertional mutagenesis due to low vector copy number requirement and erythroid-specific transgene expression, as well as long-term protein expression at therapeutic levels, increase the potential applicability of adult stem cell-based gene therapy in non-lethal disorders such as hemophilia.