<i>HML-2 env</i> knockdown by AAV9-mediated miRNAs attenuates amyotrophic lateral sclerosis-like manifestations in mice

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease for which there is no cure. While the precise etiology of ALS remains elusive, growing evidence suggests a pathogenic role for human endogenous retrovirus-K (HERV-K) in ALS. Expression of HERV-K subtype HML-2 envelope protein in neurons causes neurotoxicity in vitro and induces ALS-like symptoms in mice. We investigated the use of the Adeno-Associated Virus-9 (AAV9)-mediated artificial microRNA (amiRNA) targeting the HML-2 env gene in an ALS mouse model. From an in vitro screen of amiRNAs targeting the HML-2 env gene three were chosen and inserted in tandem into an AAV9 vector and validated in vitro. This approach provided robust silencing of the transgene, with tandem amiRNA achieving robust reduction in gene and protein expression levels. Its therapeutic effectiveness was tested in an HML-2 Env transgenic mouse model in which the env gene is expressed under the neuron-specific thy1 promoter and develops an ALS-like phenotype. A single intracerebroventricular injection of AAV9 vector encoding the amiRNAs into the mice at postnatal day 1 effectively reduced HML-2 Env expression in the brain and spinal cord at 84 days post-injection which was the longest time point studied. Knockdown of HML-2 env decreased the loss of cortical and spinal motor neurons and alleviated muscle fiber degeneration and fiber type grouping. This led to improved motor function. Our results provide compelling evidence supporting the use of multiple amiRNAs delivered in an AAV9 vector for treating forms of ALS linked to HML-2.