Efficient transduction of the peripheral nervous system (PNS) is required for gene therapy of acquired and inherited neuropathies, neuromuscular diseases and for pain treatment. We have characterized the tropism and transduction efficiency of different adeno-associated vectors (AAV) pseudotypes after sciatic nerve injection in the mouse. Among the pseudotypes tested, AAV2/1 transduced both Schwann cells and neurons, AAV2/2 infected only sensory neurons and AAV2/8 preferentially transduced Schwann cells. AAV2/8 expression in the sciatic nerve was detected up to 10 weeks after administration, the latest time point analyzed. The injected mice developed neutralizing antibodies against all AAVs tested; the titers were higher against AAV2/1 than AAV2/2 and were the lowest for AAV2/8, correlating with a higher transgene expression overtime. AAV2/8 coding for ciliary neurotrophic factor (CNTF) led to an upregulation of P0 and PMP22 myelin proteins, four weeks after transduction of injured sciatic nerves. Importantly, CNTF-transduced mice showed a significant increase in both GAP43 expression in sensory neurons, a marker of axonal regeneration, and the compound muscle action potential. These results prove the utility of AAV8 as a gene therapy vector for Schwann cells to treat myelin disorders or to improve nerve regeneration.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motoneurons, which is preceded by loss of neuromuscular connections in a "dying back" process. Neuregulin-1 (Nrg1) is a neurotrophic factor essential for the development and maintenance of neuromuscular junctions, and Nrg1 receptor ErbB4 loss-of-function mutations have been reported as causative for ALS. Our main goal was to investigate the role of Nrg1 type I (Nrg1-I) in SOD1 G93A mice muscles. We overexpressed Nrg1-I by means of an adeno-associated viral (AAV) vector, and investigated its effect by means of neurophysiological techniques assessing neuromuscular function, as well as molecular approaches (RT-PCR, western blot, immunohistochemetry, ELISA) to determine the mechanisms underlying Nrg1-I action. AAVNrg1-I intramuscular administration promoted motor axon collateral sprouting by acting on terminal Schwann cells, preventing denervation of the injected muscles through Akt and ERK1/2 pathways. We further used a model of muscle partial denervation by transecting the L4 spinal nerve. AAV-Nrg1-I intramuscular injection enhanced muscle reinnervation by collateral sprouting, whereas administration of lapatinib (ErbB receptor inhibitor) completely blocked it. We demonstrated that Nrg1-I plays a crucial role in the collateral reinnervation process, opening a new window for developing novel ALS therapies for functional recovery rather than preservation.
Canine adenovirus type 2 vectors (CAV-2) are promising tools to treat global central nervous system (CNS) disorders because of their preferential transduction of neurons and efficient retrograde axonal transport. Here we tested the potential of a helper-dependent CAV-2 vector expressing β-glucuronidase (HD-RIGIE) in a mouse model of mucopolysaccharidosis type VII (MPS VII), a lysosomal storage disease caused by deficiency in β-glucuronidase activity. MPS VII leads to glycosaminoglycan accumulation into enlarged vesicles in peripheral tissues and the CNS, resulting in peripheral and neuronal dysfunction. After intracranial administration of HD-RIGIE, we show long-term expression of β-glucuronidase that led to correction of neuropathology around the injection site and in distal areas. This phenotypic correction correlated with a decrease in secondary-elevated lysosomal enzyme activity and glycosaminoglycan levels, consistent with global biochemical correction. Moreover, HD-RIGIE-treated mice show significant cognitive improvement. Thus, injections of HD-CAV-2 vectors in the brain allow a global and sustained expression and may have implications for brain therapy in patients with lysosomal storage disease.
Different adeno-associated virus (AAV) serotypes efficiently transduce neurons from central and peripheral nervous systems through various administration routes. Direct administration of the vectors to the cerebrospinal fluid (CSF) could be an efficient and safe strategy. Here, we show that lumbar puncture of a nonhuman AAV leads to wide and stable distribution of the vector along the spinal cord in adult mice. AAVrh10 efficiently and specifically infects neurons, both in dorsal root ganglia (60% total sensory neurons) and in the spinal cord (up to one-third of α-motor neurons). As a proof of concept, we demonstrate the efficacy of AAVrh10 in a mouse model of diabetic neuropathy, in which intrathecal delivery of the vector coding for insulin-like growth factor (IGF-I) favored the release of the therapeutic protein into the CSF through its expression by sensory and motor neurons. IGF-I–treated diabetic animals showed increased vascular endothelial growth factor expression, activation of Akt/PI3K pathway, and stimulated nerve regeneration and myelination in injured limbs. Moreover, we achieved restoration of nerve conduction velocities in both sensory and motor nerves by AAVrh10, whereas we reached only sensory nerve improvement with AAV1. Our results indicate that intrathecal injection of AAVrh10 is a promising tool to design gene therapy approaches for sensorimotor diseases.
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