Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33-50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders.

Objectives Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, paralysis and death within five years of diagnosis. About 10% of cases are inherited, of which 20% are due to mutations in the superoxide dismutase 1 (SOD1) gene. Riluzole, the only FDA approved ALS drug, prolongs survival by only a few months. Experiments in transgenic ALS mouse models have shown decreasing levels of mutant SOD1 protein as a potential therapeutic approach. We sought to develop an efficient AAV mediated RNAi gene therapy for ALS. Methods A single stranded AAV9 vector encoding an artificial microRNA against human SOD1 was injected into the cerebral lateral ventricles of neonatal SOD1G93A mice and impact on disease progression and survival assessed. Results This therapy extended median survival by 50% and delayed hindlimb paralysis, with animals remaining ambulatory until the humane endpoint, which was due to rapid body weight loss. AAV9-treated SOD1G93A mice showed reduction of mutant human SOD1 mRNA levels in upper and lower motor neurons and significant improvements in multiple parameters including the numbers of spinal motor neurons, diameter of ventral root axons, and extent of neuroinflammation in the SOD1G93A spinal cord. Mice also showed previously unexplored changes in pulmonary function, with AAV9-treated SOD1G93A mice displaying a phenotype reminiscent of patient pathophysiology. Interpretation These studies clearly demonstrate that an AAV9-delivered SOD1-specific artificial microRNA is an effective and translatable therapeutic approach for ALS.

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The findings here show that intrastriatal infusion of an AAV9-GFP-miRHtt vector lowers mRNA expression of Htt in striatum by ∼50%, through a partial reduction in the number of copies of mutant Htt mRNAs per cell. These findings demonstrate at the neuronal level the variable levels of Htt mRNA expression in MSNs and the neuronal heterogeneity of RNAi dependent Htt mRNA knockdown.

Recombinant adeno‐associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long‐term gene expression. rAAVs have been used successfully as gene transfer vehicles in multiple animal models of CNS disorders, and several clinical trials are currently underway. rAAV vectors have been used at various stages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the mouse CNS, depending on the stage of development. In neonates, intravascular injections into the facial vein are often used. In adults, direct injections into target regions of the brain are achieved with great spatiotemporal control through stereotaxic surgeries. Recently, discoveries of new AAV vectors with the ability to cross the blood brain barrier have made it possible to target the adult CNS by intravascular injections. Curr. Protoc. Microbiol. 29:14D.5.1‐14D.5.18. © 2013 by John Wiley & Sons, Inc.