In utero gene delivery of AAV vectors for efficient treatment of muscle disorders.

摘要

Duchenne muscular dystrophy (DMD) is a devastating primary muscle disease with pathological changes in skeletal muscle that are ongoing at the time of birth. Progressive deterioration in striated muscle function in affected individuals ultimately results in early death due to cardiopulmonary failure. Since affected individuals can be identified prior to birth by prenatal genetic testing for DMD, gene replacement treatment can be started in utero . This approach offers the possibility of preventing pathological changes in muscle that begin early in life.;Previous studies with systemic in utero adenoassociated viral (AAV) vector serotype 1 gene delivery to embryonic day 16 (E-16) pups resulted in high levels of transduction in diaphragm and intercostal muscles, but no detectable levels in limb muscle. Recently newer AAV serotypes such as AAV8 have demonstrated widespread and high transgene expression in skeletal muscles and diaphragm by systemic delivery in adults and neonatal mice. In this study I tested AAV8 vector gene delivery by intraperitoneal administration in E-16 mice in utero. Using an AAV8 vector carrying a lacZ transgene, I observed high level transduction of diaphragm and more moderate transduction of multiple limb muscles and heart. Encouraged with these results I tested in utero gene transfer in the mdx mouse model of DMD, a minidystrophin gene driven by the human cytomegalovirus promoter was delivered systemically by an intraperitoneal injection to the fetus at embryonic day 16. Treated mdx mice studied at 9 weeks after birth demonstrated widespread expression of recombinant dystrophin in skeletal muscle, restoration of the dystrophin associated glycoprotein complex in dystrophin-expressing muscle fibers, improved muscle pathology, and functional benefit to the transduced diaphragm compared to untreated littermate controls. In order to further extend these studies, AAV9 carrying a minidystrophin gene was also tested. Robust expression in heart and muscles were seen at 4 weeks post treatment by in utero gene delivery. Furthermore robust heart expression persisted as long as 3 months post treatment. These results support the potential of AAV8 and AAV9 vectors to efficiently cross the blood vessel barrier to achieve systemic gene transfer to skeletal muscle in utero in a mouse model of muscular dystrophy, to significantly improve the dystrophic phenotype and to ameliorate the processes that lead to exhaustion of the skeletal muscle regenerative capacity.