Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV-microdystrophin gene therapy in DMD patients

Adeno-Associated Viral Vector-Mediated Transgene Expression Is Independent of DNA Methylation in Primate Liver and Skeletal Muscle

Recombinant adeno-associated viral (rAAV) vectors can support long-term transgene expression in quiescent tissues. Intramuscular (IM) administration of a single-stranded AAV vector (ssAAV) in the nonhuman primate (NHP) results in a peak protein level at 2–3 months, followed by a decrease over several months before reaching a steady-state. To investigate transgene expression and vector genome persistence, we previously demonstrated that rAAV vector genomes associate with histones and form a chromatin structure in NHP skeletal muscle more than one year after injection. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays key role in transcriptional regulation. Among those, CpG hyper-methylation of promoters is a known hallmark of gene silencing. To assess the involvement of DNA methylation on the transgene expression, we injected NHP via the IM or the intravenous (IV) route with a recombinant ssAAV2/1 vector. The expression cassette contains the transgene under the transcriptional control of the constitutive Rous Sarcoma Virus promoter (RSVp). Total DNA isolated from NHP muscle and liver biopsies from 1 to 37 months post-injection was treated with sodium bisulfite and subsequently analyzed by pyrosequencing. No significant CpG methylation of the RSVp was found in rAAV virions or in vector DNA isolated from NHP transduced tissues. Direct de novo DNA methylation appears not to be involved in repressing transgene expression in NHP after gene transfer mediated by ssAAV vectors. The study presented here examines host/vector interactions and the impact on transgene expression in a clinically relevant model

Detecting early bone changes using in vivo micro-CT in ovariectomized, zoledronic acid-treated, and sham-operated rats

SummaryThis study monitored in vivo the effect on bone microarchitecture of initiating antiresorptive treatment with zoledronic acid in rats at 2 weeks following ovariectomy, an early phase at which major degenerative bone changes have been found to occur. The treatment still facilitated the full reversal of cancellous bone loss in rat tibia, highlighting the importance of the time point of initiation of antiresorptive treatment.IntroductionInjection of zoledronic acid in rats at time of ovariectomy has been found to fully preserve tibial bone microarchitecture over time, whereas injection at 8 weeks after ovariectomy has shown partial bone recovery. This study investigated the effect on microarchitecture of initiating antiresorptive treatment in the early phase following ovariectomy, at 2 weeks, a time point at which major degenerative changes in the bone have been found to occur.MethodsFemale Sprague-Dawley rats were divided into ovariectomized group, ovariectomized group treated with zoledronic acid, and sham-operated group. In vivo micro-CT scanning of rat tibiae and morphometric analysis were performed at 0, 2, 4, 8, and 12 weeks after ovariectomy, with zoledronic acid treatment beginning 2 weeks after ovariectomy. Data were first analyzed with repeated measures analysis of variance (longitudinal study design) and then without repeated measures (cross-sectional study design).ResultsThe ovariectomized group demonstrated dramatic bone loss, first detected at week 2. Conversely, at week 4, the zoledronic acid-treated group returned microstructural parameters to baseline values. Remarkable increases in bone parameters were found after 6 weeks of treatment and maintained similar to sham group until the end. The longitudinal study design provided earlier detection of bone changes compared to the cross-sectional study design.ConclusionsTreatment with zoledronic acid as late as 2 weeks after ovariectomy still facilitates the full reversal of cancellous bone loss in the rat tibia.E. Perilli, V. Le, B. Ma, P. Salmon, K. Reynolds and N. L. Fazzalar