Search

Micro-dystrophins are highly promising candidates for treating Duchenne muscular dystrophy, a lethal muscle disease caused by dystrophin deficiency. Here, we report robust disease rescue in the severe DBA/2J-mdx model with a neuronal nitric oxide synthase (nNOS)-binding micro-dystrophin vector. 2 × 1013 vector genome particles/mouse of the vector were delivered intravenously to 10-week-old mice and were evaluated at 6 months of age. Saturated micro-dystrophin expression was detected in all skeletal muscles and the heart and restored the dystrophin-associated glycoprotein complex and nNOS. In skeletal muscle, therapy substantially reduced fibrosis and calcification and significantly attenuated inflammation. Centronucleation was significantly decreased in the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles but not in the quadriceps. Muscle function was normalized in the TA and significantly improved in the EDL muscle. Heart histology and function were also evaluated. Consistent with the literature, DBA/2J-mdx mice showed myocardial calcification and fibrosis and cardiac hemodynamics was compromised. Surprisingly, similar myocardial pathology and hemodynamic defects were detected in control DBA/2J mice. As a result, interpretation of the cardiac data proved difficult due to the confounding phenotype in control DBA/2J mice. Our results support further development of this microgene vector for clinical translation. Further, DBA/2J-mdx mice are not good models for Duchenne cardiomyopathy.

The ultimate goal of muscular dystrophy gene therapy is to treat all muscles in the body. Global gene delivery was demonstrated in dystrophic mice more than a decade ago using adeno-associated virus (AAV). However, translation to affected large mammals has been challenging. The only reported attempt was performed in newborn Duchenne muscular dystrophy (DMD) dogs. Unfortunately, AAV injection resulted in growth delay, muscle atrophy and contracture. Here we report safe and bodywide AAV delivery in juvenile DMD dogs. Three ∼2-m-old affected dogs received intravenous injection of a tyrosine-engineered AAV-9 reporter or micro-dystrophin (μDys) vector at the doses of 1.92-6.24 × 10(14) viral genome particles/kg under transient or sustained immune suppression. DMD dogs tolerated injection well and their growth was not altered. Hematology and blood biochemistry were unremarkable. No adverse reactions were observed. Widespread muscle transduction was seen in skeletal muscle, the diaphragm and heart for at least 4 months (the end of the study). Nominal expression was detected in internal organs. Improvement in muscle histology was observed in μDys-treated dogs. In summary, systemic AAV gene transfer is safe and efficient in young adult dystrophic large mammals. This may translate to bodywide gene therapy in pediatric patients in the future.

Adeno-associated virus serotype-8 and 9 (AAV-8 and 9) are the leading candidate vectors to test bodywide neonatal muscle gene therapy in large mammals. We have previously shown that systemic injection of 2-2.5×10(14) viral genome (vg) particles/kg of AAV-9 resulted in widespread skeletal muscle gene transfer in newborn dogs. However, nominal transduction was observed in the heart. In contrast, robust expression was achieved in both skeletal muscle and heart in neonatal dogs with 7.14-9.06×10(14) vg particles/kg of AAV-8. To determine whether superior cardiac transduction of AAV-8 is because of the higher vector dose, we delivered 6.14×10(14) and 9.65×10(14) vg particles/kg of AAV-9 to newborn puppies via the jugular vein. Transduction was examined 2.5 months later. Consistent with our previous reports, we observed robust bodywide transduction in skeletal muscle. However, increased AAV dose only moderately improved heart transduction. It never reached the level achieved by AAV-8. Our results suggest that differential cardiac transduction by AAV-8 and AAV-9 is likely because of the intrinsic property of the viral capsid rather than the vector dose.

Duchenne muscular dystrophy (DMD) is a progressive, muscle wasting disorder that affects all muscles in the body. An effective gene therapy for DMD will require efficient whole body muscle transduction. It was recently demonstrated that a single intravenous injection of adeno-associated virus (AAV) can lead to safe, bodywide muscle gene transfer in adolescent dogs affected by the canine model of DMD (cDMD) (Yue et al. 2015 Hum Mol Genet). Here we evaluated systemic gene therapy in three 3.5-m-old cDMD dogs using a novel canine codon-optimized micro-dystrophin vector. Transcriptional regulation is controlled by the muscle-specific CK8 promoter and a synthetic polyadenylation signal. All experimental subjects received transient immune suppression. One dog was administrated with 5×1013 viral genome (vg) particles/kg of the vector. Two dogs received 1×1014 vg particles/kg of the vector. All dogs tolerated injection well. Blood biochemistry (weekly in the first four weeks and biweekly thereafter) was unremarkable. Growth curve was nominally disturbed during the immunosuppression regimen, but recovered thereafter. Biopsy at 1,3 and 6 months after injection revealed widespread micro-dystrophin expression in 50-80% myofibers. The dystrophin-associated glycoprotein complex, including neuronal nitric oxide synthase (nNOS), was restored. While limited in sample size, muscle damage usually seen in young adult untreated dogs (inflammation, fibrosis, calcification) were rarely observed. CD4+, CD8+, and regulatory T cells were minimally detected. Night activity monitoring showed a trend of improvement. Limb muscle force (both forelimb and hind limb) was significantly enhanced compared to that of pre-injection. Our data suggest that systemic AAV micro-dystrophin therapy may translate to large mammals afflicted by DMD (Supported by Solid GT, NIH, DOD, Jesse's Journey).