Recombinant adeno-associated virus (rAAV)-mediated gene transfer is an attractive approach to the treatment of Duchenne muscular dystrophy (DMD). We investigated the muscle transduction profiles and immune responses associated with the administration of rAAV2 and rAAV8 in normal and canine X-linked muscular dystrophy in Japan (CXMD(J)) dogs. rAAV2 or rAAV8 encoding the lacZ gene was injected into the skeletal muscles of normal dogs. Two weeks after the injection, we detected a larger number of beta-galactosidase-positive fibers in rAAV8-transduced canine skeletal muscle than in rAAV2-transduced muscle. Although immunohistochemical analysis using anti-CD4 and anti-CD8 antibodies revealed less T-cell response to rAAV8 than to rAAV2, beta-galactosidase expression in rAAV8-injected muscle lasted for <4 weeks with intramuscular transduction. Canine bone marrow-derived dendritic cells (DCs) were activated by both rAAV2 and rAAV8, implying that innate immunity might be involved in both cases. Intravenous administration of rAAV8-lacZ into the hind limb in normal dogs and rAAV8-microdystrophin into the hind limb in CXMD(J) dogs resulted in improved transgene expression in the skeletal muscles lasting over a period of 8 weeks, but with a declining trend. The limb perfusion transduction protocol with adequate immune modulation would further enhance the rAAV8-mediated transduction strategy and lead to therapeutic benefits in DMD gene therapy.

Using murine models, we have previously demonstrated that recombinant adeno-associated virus (rAAV)-mediated microdystrophin gene transfer is a promising approach to treatment of Duchenne muscular dystrophy (DMD). To examine further therapeutic effects and the safety issue of rAAV-mediated microdystrophin gene transfer using larger animal models, such as dystrophic dog models, we first investigated transduction efficiency of rAAV in wild-type canine muscle cells, and found that rAAV2 encoding b-galactosidase effectively transduces canine primary myotubes in vitro. Subsequent rAAV2 transfer into skeletal muscles of normal dogs, however, resulted in low and transient expression of b-galactosidase together with intense cellular infiltrations in vivo, where cellular and humoral immune responses were remarkably activated.In contrast, rAAV2 expressing no transgene elicited no cellular infiltrations. Co-administration of immunosuppressants, cyclosporine and mycophenolate mofetil could partially improve rAAV2 transduction. Collectively, these results suggest that immune responses against the transgene product caused cellular infiltration and eliminated transduced myofibers in dogs. Furthermore, in vitro interferon-g release assay showed that canine splenocytes respond to immunogens or mitogens more susceptibly than murine ones. Our results emphasize the importance to scrutinize the immune responses to AAV vectors in larger animal models before applying rAAV-mediated gene therapy to DMD patients.

To explore stem cell therapy for Parkinson’s disease (PD), three adult rhesus monkeys were first rendered hemiparkinsonian by unilateral intracarotid 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) infusion. Five months postinfusion, they were given MRI-guided stereotaxic intrastriatal and intranigral injections of green fluorescent protein (GFP)-labeled cultures of dopaminergic neurons derived from human embryonic stem cells (DA-hES cells). The animals were immunosuppressed using daily oral cyclosporine (CsA). Three months later, viable grafts were observed at the injection sites in one animal, while no obvious grafts were present in the other two monkeys. The surviving grafts contained numerous GFP-positive cells that were positively labeled for nestin and MAP2 but not for glial fibrillary acidic protein (GFAP), NeuN, or tyrosine hydroxylase (TH). The grafted areas in all animals showed dense staining for GFAP, CD68, and CD45. These results indicated that xenografts of human stem cell derivatives in CsA-suppressed rhesus brain were mostly rejected. Our study suggests that immunological issues are obstacles for preclinical evaluation of hES cells and that improved immunosuppression paradigms and/or alternative cell sources that do not elicit immune rejection are needed for long-term preclinical studies.

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Autosomal recessive limb-girdle muscular dystrophy type 2D (LGMD 2D) is caused by mutations in the alpha-sarcoglycan gene (alpha-SG). The absence of alpha-SG results in the loss of the SG complex at the sarcolemma and compromises the integrity of the sarcolemma. To establish a method for recombinant adeno-associated virus (rAAV)-mediated alpha-SG gene therapy into alpha-SG-deficient muscle, we constructed rAAV serotypes 2 and 8 expressing the human alpha-SG gene under the control of the ubiquitous cytomegalovirus promoter (rAAV2-alpha-SG and rAAV8-alpha-SG). We compared the transduction profiles and evaluated the therapeutic effects of a single intramuscular injection of rAAVs into alpha-SG-deficient (Sgca(-/-)) mice. Four weeks after rAAV2 injection into the tibialis anterior (TA) muscle of 10-day-old Sgca(-/-) mice, transduction of the alpha-SG gene was localized to a limited area of the TA muscle. On the other hand, rAAV8-mediated alpha-SG expression was widely distributed in the hind limb muscle, and persisted for 7 months without inducing cytotoxic and immunological reactions, with a reversal of the muscle pathology and improvement in the contractile force of the Sgca(-/-) muscle. This extensive rAAV8-mediated alpha-SG transduction in LGMD 2D model animals paves the way for future clinical application.

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