RESUMO
Replacing or editing disease-causing mutations holds great promise for treating many human diseases. Yet, delivering therapeutic genetic modifiers to specific cells in vivo has been challenging, particularly in large, anatomically distributed tissues such as skeletal muscle. Here, we establish an in vivo strategy to evolve and stringently select capsid variants of adeno-associated viruses (AAVs) that enable potent delivery to desired tissues. Using this method, we identify a class of RGD motif-containing capsids that transduces muscle with superior efficiency and selectivity after intravenous injection in mice and non-human primates. We demonstrate substantially enhanced potency and therapeutic efficacy of these engineered vectors compared to naturally occurring AAV capsids in two mouse models of genetic muscle disease. The top capsid variants from our selection approach show conserved potency for delivery across a variety of inbred mouse strains, and in cynomolgus macaques and human primary myotubes, with transduction dependent on target cell expressed integrin heterodimers.
Assuntos
Capsídeo/metabolismo , Dependovirus/metabolismo , Evolução Molecular Direcionada , Técnicas de Transferência de Genes , Músculo Esquelético/metabolismo , Sequência de Aminoácidos , Animais , Capsídeo/química , Células Cultivadas , Modelos Animais de Doenças , Células HEK293 , Humanos , Integrinas/metabolismo , Macaca fascicularis , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/terapia , Multimerização Proteica , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/uso terapêutico , RNA Guia de Cinetoplastídeos/metabolismo , Recombinação Genética/genética , Especificidade da Espécie , TransgenesRESUMO
INTRODUCTION: X-linked myotubular myopathy (XLMTM), a devastating pediatric disease caused by the absence of the protein myotubularin, results from mutations in the MTM1 gene. While there is no cure for XLMTM, we previously reported effects of MTM1 gene therapy using adeno-associated virus (AAV) vector on muscle weakness and pathology in MTM1-mutant dogs. Here, we followed 2 AAV-infused dogs over 4 years. METHODS: We evaluated gait, strength, respiration, neurological function, muscle pathology, AAV vector copy number (VCN), and transgene expression. RESULTS: Four years following AAV-mediated gene therapy, gait, respiratory performance, neurological function and pathology in AAV-infused XLMTM dogs remained comparable to their healthy littermate controls despite a decline in VCN and muscle strength. CONCLUSIONS: AAV-mediated gene transfer of MTM1 in young XLMTM dogs results in long-term expression of myotubularin transgene with normal muscular performance and neurological function in the absence of muscle pathology. These findings support a clinical trial in patients. Muscle Nerve 56: 943-953, 2017.