974. Constitutive Expression of a Mutated Form of CTLA4Ig after Gene Transfer Is Well Tolerated and Prevents Immune Responses Against an Immunogenic Transgene in Nonhuman Primates
Authors: Alice Toromanoff, Marcelo Hill, Yan Cherel, Jack-Yves Deschamps, Olivier Gauthier, Bernard Vanhove, Fabienne Rolling, Philippe Moullier, Ignacio Anegon, Caroline Le Guiner
Journal: Molecular Therapy (2006)

rAAV-mediated gene transfer in skeletal muscle of large animal models can sustain long-term expression of transgene, but when applied to genetic diseases, the newly expressed therapeutic protein often induces a deleterious immune response. We investigated whether such immune response could be prevented by treatment with a mutant form of CTLA4Ig with higher affinity for B7 molecules (LEA29Y), known to induce potent immunosuppression and currently used, in its recombinant form (Belatacept®), in phase II/ III clinical development. Our laboratory has previously demonstrated that after intramuscular (IM) injection of a rAAV vector encoding for the cynomolgus erythropoietin (EPO) gene under the control of the (TLR9). In contrast to wild type mice, we found that TLR9doxycyclin-sensitive rtTA chimeric transactivator, 80% of nonhuman primates developed humoral and destructive cellular immune responses specific to rtTA, resulting in loss of EPO secretion. In this study, eight cynomolgus macaques received LEA29Y- encoding rAAV-1 and -8 vectors (3.5x1012 vector genome/kg) by IM or by limb perfusion. These injections were associated with a weak and transient oral immunosupression, in order to prevent anti- LEA29Y immune response in the first weeks after injection. LEA29Y reached steady-state levels two months post-rAAV delivery and was stably secreted thereafter. Depending on the rAAV serotypes and the route of administration (IM vs perfusion), LEA29Y levels varied between 10 to around 100 μg/ml. After LEA29Y reached plateau levels, animals were challenged with a rAAV-EPO-rtTA vector intramuscularly and EPO secretion was monitored for several months after repeated Dox inductions. Our preliminary data in three macaques suggested that a minimal level of LEA29Y is required to achieve long-term Dox-induced EPO secretion. We are currently evaluating EPO expression in the five other animals. The immune status of the injected macaques was also further analyzed. After immunisation, production of antibodies against SRBC and viral vector was inhibited in all individuals, but not in mock-injected controls. However, skin allograft rejection was similar to the control group, indicating conserved cellular immune responses. Importantly, no biological and no clinical adverse effect was so far (> one year) observed in the cohort. In conclusion, our study demonstrates that injection of LEA29Y- encoding rAAV is a safe and efficient method to prevent immune responses against transgene and viral vector in nonhuman primates. These results finally suggest (i) that preclinical evaluation of therapeutic transgenes in other large animal models would benefit from this simple and straightforward approach to block anti-transgene immune responses, providing that LEA29Y is able to block T cell proliferation in these other species, and (ii) that treatment with Belatacept® might overcome, when required, anti-transgene and anti-viral vector responses in human gene therapy trials.