RESUMO
A growing body of research supports the development of recombinant adeno-associated viral (rAAV) vectors for delivery of gene expression cassettes to striated musculature as a method of treating severe neuromuscular conditions. However, it is unclear whether delivery protocols that achieve extensive gene transfer in mice can be adapted to produce similarly extensive gene transfer in larger mammals and ultimately patients. Consequently, we sought to investigate methodological modifications that would facilitate rAAV-mediated gene transfer to the striated musculature of canines. A simple procedure incorporating acute (i) occlusion of limb blood flow, (ii) exsanguination via compression bandage, and (iii) vector "dwell" time of <20 minutes, markedly enhanced the transduction of limb muscles, compared with a simple bolus limb infusion of vector. A complementary method whereby vector was infused into the jugular vein led to efficient transduction of cardiomyocytes and to a lesser degree the diaphragm. Together these methods can be used to achieve transgene expression in heart, diaphragm, and limb muscles of juvenile dogs using rAAV6 vectors. These results establish that rAAV-mediated gene delivery is a viable approach to achieving systemic transduction of striated musculature in mammals approaching the dimensions of newborn humans.
Assuntos
Vasos Sanguíneos/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Fibras Musculares Esqueléticas/metabolismo , Fosfatase Alcalina , Animais , Vasos Sanguíneos/efeitos dos fármacos , Ciclosporina/farmacologia , Diafragma/metabolismo , Cães , Proteínas Ligadas por GPI , Vetores Genéticos/genética , Membro Posterior/metabolismo , Humanos , Terapia de Imunossupressão , Imunossupressores/farmacologia , Isoenzimas/genética , Isoenzimas/imunologia , Veias Jugulares/metabolismo , Camundongos , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacologia , Miocárdio/metabolismoRESUMO
Skeletal muscles are frequently analyzed for composition of phenotypically distinct myofibers, as a functional determinant. We describe an improved myofiber phenotyping procedure, involving cryosection co-incubation with fluorophore-labeled myosin heavy-chain (MyHC)-isoform-specific antibodies. This technique identifies multiple fiber "types" on a single section, thereby reducing reagents and processing, and offers side-by-side comparison of samples from multiple species including mice. These advances are valuable for studying the physiological attributes of skeletal muscle in health and disease.