378. Improved Transduction of Canine X-Linked Muscular Dystrophy with rAAV9-Microdystrophin by Introducing Immune Tolerance
Authors: Hiromi Hayashita-Kinoh, Hironori Okada, Yuko N. Kasahara, Tomoko Chiyo, Kiwamu Imagawa, Katsuhiko Tachibana, Shin'ichi Takeda, Takashi Okada
Journal: Molecular Therapy (2016)

Background: Duchenne muscular dystrophy (DMD) is a congenital disease causing progressive deterioration of skeletal and cardiac muscles because of mutations in the dystrophin gene. Supplementation of dystrophin using rAAV is effective to improve pathogenesis of animal models of DMD. However, we have previously reported that local injection of rAAV2 or rAAV8 to canine skeletal muscles without immunosuppression resulted in insufficient transgene expression with potent immune responses. Here we used DMD dog (CXMDJ) to investigate three strategies of inducing immune tolerance to the rAAV vector and transgene expression with rAAV9-microdystrophin (rAAV-µDys). Methods: For fetal transduction, we tried two methods to induce immune tolerance against rAAV and microdystrophin. First, direct injection of rAAV into amniotic fluid at embryonic day 35 (oral ingestion of rAAV). Second, pregnant CXMDJ heterozygote with embryonic day 30 fetuses was injected with rAAV by intravenous injection (trans-placental rAAV transduction). Furthermore, for postnatal transduction, we tried mesenchymal stem cells (MSCs) pretreatment with rAAV transduction. Bone-marrow derived MSCs and rAAV9-Luciferase or rAAV9-µDys were intramuscularly or intravenously injected into the normal or CXMDJ dog at 8 weeks old. Seven days after injection, MSCs were systemically injected again. At 8 days after 1st injection, rAAV9-Luciferase or rAAV9-µDys was intramuscularly or intravenously injected into the same dog. To examine the immune response against rAAV, purified canine peripheral leukocytes were exposed to rAAV9 for 4 hours, and then IFN-γ expression was analyzed using qRT-PCR. Skeletal muscles of the rAAV-Luc or rAAV-µDys injected animals were sampled by biopsy for expression analysis at 4 weeks after rAAV injection. Results: Following the fetal transduction, expression of IFN-γ in the purified peripheral blood leukocytes after the rAAV exposure were not induced in both of the rAAV oral ingestion and trans-placental transduced dogs, suggesting the successful induction of immune tolerance against rAAV. rAAV-derived microdystrophin expression were confirmed by immunohistochemistry in the transduced affected dogs from additional rAAV injection in both methods. In normal or CXMDJ puppy, administration of rAAV-Luc or rAAV-µDys following MSCs treatment resulted in higher expression of transgene, compared to the rAAV transduction alone. Expression of IFNγ in the purified peripheral blood leukocytes after the rAAV exposure were not enhanced in the rAAV with MSCs, suggesting the immune suppressive effects of the MSCs. Conclusion: Our results demonstrate that induction of immune tolerance against rAAV and/or transgene can be achieved both by fetal or postnatal rAAV injections. These strategies would be effective approach to analyze the expression and function of transgene in vivo. These findings also support the future feasibilities of rAAV-mediated protein supplementation strategies.