186. Phenotypic Correction of Hemophilic Mice Using Hematopoietic Stem Cells Transduced with an MSCV-Based Oncoretroviral Vector
Authors: Lixin Wang, Eric Dobrzynski, Ou Cao, Roland Herzog
Journal: Molecular Therapy (2004)

Skeletal muscle is an attractive target organ for gene transfer because of its abundance and accessibility. Upon gene transfer, myofibers are capable of synthesizing and secreting therapeutic gene products for systemic delivery, such as coagulation factor IX (F.IX). Adeno-associated viral (AAV) transfer of F.IX to muscle of subjects with severe hemophilia B (F.IX deficiency) has been evaluated in clinical trial. A concern for this approach is that the therapeutic transgene product may represent a neo-antigen, which may trigger an immune response. Experiments in animal models have demonstrated antibody formation to F.IX and other gene products after expression in muscle, while hepatic gene transfer often resulted in sustained expression without immune response. In order to study how immune responses to the transgene product develop in muscle gene transfer, we performed intramuscular (IM) injections of an AAV-2 vector expressing ovalbumin in BALB/c mice that were wild-type or transgenic for MHC II -restricted, ova-specific DO11.10 T cell receptor (TCR). Vector-dose dependent levels of systemic expression (≤30 ng/ml for 1 × 1012 vg/animal) were measured at week 2, which consistently diminished to undetectable by week 4. Mice formed low-titer anti-ova by week 6. Histochemical analyses revealed substantial inflammation, CD4+ and CD8+ lymphocytic infiltrate, and destruction of ova-expressing muscle fibers at 10 day in the injected leg of DO11.10 mice. At 30 days, extensive muscle regeneration was evident. Draining lymph nodes (LN) showed increase in CD69+CD4+TCR+ cells, indicating local T cell activation. Lymphocytes isolated from draining LN secreted high levels of IFN-γ (day 10) and IFN-γ and IL-10 (day 30) after in vitro stimulation with ova, reflecting Th1 and Th2 immunity. We observed transiently reduced T cell proliferation to ova for LN and spleen cells at day 10, but responsiveness was restored (and elevated for draining LN) by day 30. These data demonstrate a local immune response against ova resulting in a cytotoxic T lymphocyte (CTL) response that destroyed AAV-CMV-ova transduced muscle fibers. IM injection of AAV-CMV-ova in C57BL/6 mice again resulted in transient expression and rejection of transduced muscle fibers in an inflammatory immune response in wild-type animals. Splenocytes from these mice showed lysis of ova-expressing target cells in 51Cr-release assay. However, expression was sustained without inflammation in CD8-deficient mice, consistent with the interpretation that CD8+ T cells mediate CTL responses to ova in muscle gene transfer. In an analogous set of experiments, we found a robust Th1 and B cell response (IgG1 and IgG2a synthesis) to a human F.IX transgene product in draining LN of AAV-transduced murine muscle by day 14. ELISPOT and immunocapture assays showed a subsequent shift toward Th2 immunity, which appeared to be responsible for prolonged production of neutralizing antibody to hF.IX. Both systems illustrate activation of CD4+ T cells (Th1 cells early followed by Th2 cells), with B cell responses neutalizing F.IX expression and CD8+ CTL responses neutralizing ova expression. Therefore, local immune responses severely limit use of skeletal muscle for systemic protein delivery, if the transgene product represents a neo-antigen.