Sustained AAV-mediated Dystrophin Expression in a Canine Model of Duchenne Muscular Dystrophy with a Brief Course of Immunosuppression

Abstract

Adeno-associated virus-based vector (AAV)-mediated gene delivery has been successful in some animal models of human disease such as the mdx mouse model of human Duchenne muscular dystrophy (DMD). However, recent evidence of immune-mediated loss of vector persistence in dogs and humans suggests that immune modulation might be necessary to achieve successful long-term transgene expression in these species. We have previously demonstrated that direct intramuscular injection of AAV2 or AAV6 in wild-type random-bred dogs resulted in a robust immune response to capsid or capsid-associated proteins. We now demonstrate that a brief course of immunosuppression with a combination of anti-thymocyte globulin (ATG), cyclosporine (CSP), and mycophenolate mofetil (MMF) is sufficient to permit long-term and robust expression of a canine micro-dystrophin (c-micro-dys) transgene in the skeletal muscle of a dog model for DMD (canine X-linked muscular dystrophy, or cxmd dog) and that its expression restored localization of components of the dystrophin-associated protein complex at the muscle membrane. This protocol has potential applications to human clinical trials to enhance AAV-mediated therapies.

A canine minidystrophin is functional and therapeutic in mdx mice

“…13 We considered that the immune rejection of human minidystrophin in dog muscle tissues might be associated with species differences in the minidystrophin. The human minidystrophin and canine minidystrophin, used in our experiment, have a great percentage of difference in amino acids through the N terminus to the CR domain such as: N terminus (0.8%), hing1 (9.3%), rod1 (8.1%), rod2 (3.7%), rod 22 (6.1%), rod 23 (1.5%), rod 24 (6.4%), CR domain (0.3%), although they have the exactly same rods and hinges (Materials and methods).…”

“…[12][13][14] Our own preliminary experiment with a human minidystrophin gene in the GRMD dogs has met the same fate (unpublished data). Such robust cellular immune responses have not been seen previously in the mice and other rodents, suggesting that the GRMD dog is a more challenging model for gene therapy studies.…”

Immunity to adeno-associated virus vectors in animals and humans: a continued challenge

“…However, 3 months of immunosuppression with a combination of antithymocyte globulin, cyclosporine and MMF immediately following vector injection was sufficient to permit long-term and sustained expression of canine micro-dystrophin in the skeletal muscle in the same dog model. 92 Another anti-T-cell regimen was tested in a non-human primate model of AAV-mediated hF.IX administration via the hepatic artery. Since nonhuman primates did not develop capsid-specific CTLs regardless of the immunosuppression, the immunemodulating drug regimen could only be tested for safety.…”

Soluble TNF-α receptor secretion from healthy or dystrophic mice after AAV6-mediated muscle gene transfer

“…25 However, more important, the latter can be mastered by a short course of immunosuppression. 26 Owing to the immune response against the human chimera, we decided to conduct the same study using a fully murine chimeric protein (mTNFR-Is/mIgG1). As with muSeAP and hTNFR-Is/mIgG1 transgenes, we found a weaker expression in mdx as compared with B10 mice.…”