RESUMO
Spinal muscular atrophy (SMA) is a neuromuscular disease mainly caused by mutations or deletions in the survival of motor neuron 1 (SMN1) gene and characterized by the degeneration of motor neurons and progressive muscle weakness. A viable therapeutic approach for SMA patients is a gene replacement strategy that restores functional SMN expression using adeno-associated virus serotype 9 (AAV9) vectors. Currently, systemic or intra-cerebrospinal fluid (CSF) delivery of AAV9-SMN is being explored in clinical trials. In this study, we show that the postnatal delivery of an AAV9 that expresses SMN under the control of the neuron-specific promoter synapsin selectively targets neurons without inducing re-expression in the peripheral organs of SMA mice. However, this approach is less efficient in restoring the survival and neuromuscular functions of SMA mice than the systemic or intra-CSF delivery of an AAV9 in which SMN is placed under the control of a ubiquitous promoter. This study suggests that further efforts are needed to understand the extent to which SMN is required in neurons and peripheral organs for a successful therapeutic effect.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Neurônios Motores/metabolismo , Neurônios Motores/virologia , Atrofia Muscular Espinal/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Animais , Modelos Animais de Doenças , Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética , Locomoção , Camundongos , Atrofia Muscular Espinal/tratamento farmacológico , Fenótipo , Prognóstico , Regiões Promotoras Genéticas , Medula Espinal/metabolismo , Medula Espinal/patologia , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Transdução Genética , Resultado do TratamentoRESUMO
One of the most promising therapeutic approaches for familial amyotrophic lateral sclerosis linked to superoxide dismutase 1 (SOD1) is the suppression of toxic mutant SOD1 in the affected tissues. Here, we report an innovative molecular strategy for inducing substantial, widespread, and sustained reduction of mutant human SOD1 (hSOD1) levels throughout the body of SOD1G93A mice, leading to therapeutic effects in animals. Adeno-associated virus serotype rh10 vectors (AAV10) were used to mediate exon skipping of the hSOD1 pre-mRNA by expression of exon-2-targeted antisense sequences embedded in a modified U7 small-nuclear RNA (AAV10-U7-hSOD). Skipping of hSOD1 exon 2 led to the generation of a premature termination codon, inducing production of a deleted transcript that was subsequently degraded by the activation of nonsense-mediated decay. Combined intravenous and intracerebroventricular delivery of AAV10-U7-hSOD increased the survival of SOD1G93A mice injected either at birth or at 50 days of age (by 92% and 58%, respectively) and prevented weight loss and the decline of neuromuscular function. This study reports the effectiveness of an exon-skipping approach in SOD1-ALS mice, supporting the translation of this technology to the treatment of this as yet incurable disease.
Assuntos
Esclerose Lateral Amiotrófica/genética , Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Superóxido Dismutase-1/genética , Idade de Início , Esclerose Lateral Amiotrófica/mortalidade , Esclerose Lateral Amiotrófica/fisiopatologia , Esclerose Lateral Amiotrófica/terapia , Animais , Modelos Animais de Doenças , Éxons , Ordem dos Genes , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Humanos , Camundongos , Camundongos Transgênicos , Atividade Motora/genética , Oligonucleotídeos Antissenso , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recuperação de Função Fisiológica , Superóxido Dismutase-1/metabolismo , Taxa de Sobrevida , Transdução GenéticaRESUMO
We have recently demonstrated the remarkable efficiency of self-complementary (sc) AAV9 vectors for central nervous system (CNS) gene transfer following intravenous delivery in mice and larger animals. Here, we investigated whether gene delivery to motor neurons (MNs) could also be achieved via intramuscular (i.m.) scAAV9 injection and subsequent retrograde transport along the MNs axons. Unexpectedly, we found that a single injection of scAAV9 into the adult mouse gastrocnemius (GA) mediated widespread MN transduction along the whole spinal cord, without limitation to the MNs connected to the injected muscle. Spinal cord astrocytes and peripheral organs were also transduced, indicating vector spread from the injected muscle to both the CNS and the periphery through release into the blood circulation. Moreover, we showed that i.m. injection of scAAV9 vectors expressing "survival of motor neuron" (Smn) in spinal muscular atrophy (SMA) mice mediated high survival motor neuron (SMN) expression levels at both the CNS and the periphery, and increased the median lifespan from 12 days to 163 days. These findings represent to date the longest extent in survival obtained in SMA mice following i.m. viral vector gene delivery, and might generate a renewed interest in the use of i.m. adeno-associated viruses (AAV) delivery for the development of gene therapy strategies for MN diseases.