Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products

Abstract

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.

Efficient transduction of non-human primate motor neurons after intramuscular delivery of recombinant AAV serotype 6

“…Acute immune responses and elimination of transduced myofibers after the delivery of rAAV to skeletal muscle has been observed in canine models of muscular dystrophy. [14][15][16] Likewise, T cell-mediated immune responses have previously been observed after rAAV2/2 delivery to non-human primate muscle. 17 This immune response to rAAV in muscle can be circumvented through longterm immunosuppression in cynomolgus macaques.…”

“…As host immune responses have proven an important scale up issue from rodents to larger animals, [14][15][16] we have determined whether administration of the rAAV2/6 vector mediated cellular toxicity or an immune response in the monkeys. Infiltrating cells were observed in the injected muscle and corresponded to recruitment of both macrophages and T cells (Figure 4a and b).…”

A canine minidystrophin is functional and therapeutic in mdx mice

“…[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.…”

Progress in therapeutic antisense applications for neuromuscular disorders

“…9 In theory, exon skipping would be applicable to almost 80% 20 of all patients and AONs to induce the skipping of each dystrophin exon, except for the first and the last that have been identified. 31,32 As 70 and 25% of all deletions occur in the major (exons [45][46][47][48][49][50][51][52][53] and minor (exons 2-11) hotspots, respectively, and deletions are present in 65% of patients, 28 skipping of some exons is applicable to large groups of patients. The most notable example is exon 51 skipping, which is applicable to 13% of all patients (or 19% of all deletion patients).…”