Muscle Function Recovery in Golden Retriever Muscular Dystrophy After AAV1-U7 Exon Skipping

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD). Widespread restoration of dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.

Duchenne muscular dystrophy gene therapy in the canine model

“…99 However, Wang et al detected a strong T cell response to AAV-1 capsid in normal dog muscle after injection of a much lower dose (5 · 10 11 vg) of a canine factor IX vector. 128 Two different groups reported cellular immune reaction to AAV-2 following direct muscle injection in normal dogs.…”

“…Vulin et al co-expressed two AONs (one for exon 6 skipping and the other for exon 8 skipping) in one vector using AAV-1 (AAV1-U7E6/8). 99 Exclusion of exons 6 and 8 results in an in-frame D6-8 transcript). Investigators performed local injection and forelimb perfusion in six 5-15-month-old dogs and multimuscle injection in the hindlimb of four 3-week-old puppies.…”

“…Further, specific muscle force was enhanced in treated puppies. 99 Le Guiner et al delivered the same set of AONs to fifteen 3-4-month-old juvenile GRMD dogs by forelimb perfusion. 100 Instead of AAV-1, the authors used AAV-8 (AAV8-U7E6/8).…”

“…In dogs with ‡ 40% dystrophin-positive myofibers, treatment also prevented progressive force decline. 100 Two independent groups examined AAV-6-mediated exon skipping in the heart of GRMD dogs using exactly the same U7E6/8 construct developed by Vulin et al 99,101,102 AAV-6 U7E6/8 was delivered by multiple transendocardial injection in young adult dogs (5-13 months old). Both groups achieved expected exon skipping and dystrophin restoration.…”

“…Vulin et al found that 20-50% dystrophin-positive cells might be sufficient to improve muscle force. 99 Le Guiner et al reported that a correction of 33%, 35%, and 40% of myofibers might result in histopathology amelioration, MRI improvement, and muscle function preservation, respectively. 100 Bish et al showed that a level of 15-20% of normal dystrophin might offer some heart protection.…”