Marrow-Derived Stromal Cells as Gene Transfer Vehicles in a Murine Model of Hemophilia A.

Marrow-derived stromal cells (MSCs) are considered a promising cellular vehicle for the delivery of nucleic acid-based therapeutics. In preclinical studies using a murine model of hemophilia A, we studied the effectiveness of ex vivo retrovirally-modified MSCs at synthesizing therapeutic amounts of factor VIII (fVIII). Previously, we observed robust anti-fVIII immune responses following intraperitoneal or intravenous delivery of MSCs genetically-modified to express a B-domain-deleted high expression porcine fVIII (HEP-fVIII) transgene ([1][1]). The presence of anti-HEP-fVIII antibodies masked any potential HEP-fVIII in circulation and thus, it was not determined whether or not the MSCs persisted in vivo . Furthermore, transplantation of HEP-fVIII-expressing MSCs into wild-type C57BL/6 mice induced an anti-fVIII immune response that decreased the endogenous murine fVIII levels to <10% of normal between wks 2 and 3 post-transplant. Analysis of plasma samples from these mice demonstrated the presence of anti-fVIII immunoglobulin populations that displayed cross-reactivity with human and murine fVIII in an ELISA and inhibited human and murine fVIII activity in a modified Bethesda assay. Therefore, pre-existing tolerance does not prevent the generation of antibodies that bind and inhibit murine fVIII. To further study the expression of HEP-fVIII from MSCs in immuno-suppressed animals, hemophilia A mice were pretreated with 5.5 Gy total body irradiation (TBI) prior to receiving HEP-fVIII-expressing MSCs. Pre-transplant TBI facilitated the detection of circulating fVIII activity for up to 6 wks, compared to 5 days in non-irradiated mice with an observed peak fVIII activity of 20 units/ml, which is 20-fold higher than the normal human level. However approximately 6 wks post-transplant, these mice developed large abdominal tumors that displayed histology similar to non-malignant fibrosarcoma. PCR analysis confirmed the presence of the HEP-fVIII transgene in the tumor cells at ~38 copies per diploid genome equivalent. In contrast shortly after viral transduction, the MSCs were shown to contain ~2 transgene copies per diploid genome equivalent, which increased to 5 and subsequently to 19 during ~1yr of expansion in culture. These results suggest that time-dependent genome amplification occurred during ex vivo culture and possibly in vivo post-transplantation. Cultured tumor cell explants secreted high levels of fVIII at a rate of 12 units/106 cells/24 hr in serum-free medium. Together these data indicate that the genetically-modified MSCs comprised a significant proportion of the tumor mass. The observation of tumor formation in these mice is consistent with previous reports describing a transformed phenotype in human and murine MSCs that had been cultured extensively ex vivo ([2][2]–[4][3]). The current findings demonstrate that, although murine MSCs are amenable to ex vivo manipulations and are capable of efficient fVIII biosynthesis, the immunogenicity of MSC-produced fVIII and the potential for oncogenic transformation raise important concerns regarding the use of MSCs as vehicles for gene therapy. [1]: #ref-1 [2]: #ref-2 [3]: #ref-4