341. Therapeutic Approach for SOD1-ALS Using AAV9 Delivered Artificial microRNAs

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurons. This results in progressive muscle weakness, atrophy, paralysis and death within five years of diagnosis. About 10% of cases are inherited – typically in a dominant manner – of which twenty percent are due to mutations in the superoxide dismutase gene (SOD1). Experiments in transgenic ALS mouse models that overexpress human SOD1 have shown that decreasing levels of mutant SOD1 protein alters, and in some cases eliminates, disease progression.We postulated that silencing SOD1 expression with a micro RNA (miR) would be therapeutic in ALS. We developed a single stranded AAV9 vector encoding GFP and a miR against human SOD1, driven by a CBA promoter, which we injected into the SOD1G93A ALS mouse at postnatal day one. Each mouse received 2ml into each lateral ventricle, for a total vector dose of 4e10 vector genomes.At four weeks post injection, hSOD1 mRNA was reduced by almost 50% at all three levels of the spinal cord. Transduction was visible in both motor neurons and astrocytes in the spinal cord as well as neurons in layer V of the motor cortex. Transduced cells, assessed by GFP RNA FISH, had a decrease in hSOD1 mRNA. This translated into a 50% extension in median survival of treated mice (206 days) compared to untreated (135 days) mice.To assess neuromuscular health during the duration of the experiment, we performed motor unit number estimates (MUNEs) and needle electromyography (EMG). Treated mice had mild or no muscle denervation as opposed to the severe denervation seen in untreated SOD1. In fact, the treated mice did not develop paralysis but instead had to be euthanized due to weight loss and a hunched posture.We also assessed neuropathology in the spinal cord and nerves in untreated and treated animals, at their respective endpoints. The treated mice showed no spinal cord motor neuron loss, while the untreated mice lost the majority of the motor neurons. The axonal integrity of the lumbar ventral roots was also improved in treated animals. Furthermore, there was no axonal degeneration in the sciatic nerves of the treated animals when analyzed at 120 days, the endpoint of untreated mice. Lastly, treated animals show delayed onset of astrogliosis and microgliosis, as observed by IHC for inflammatory markers GFAP and Iba1, and confirmed by RT-qPRC for genes upregulated in inflammation.In conclusion, we were successful at extending the lifespan of the SOD1G93A mouse by 50% with our high dose neonatal AAV9-miR, and our treated animals remain ambulatory and active until the humane endpoint with minimal or no signs of paralysis.