In vivo genome editing using novel AAV-PHP variants rescues motor function deficits and extends survival in a SOD1-ALS mouse model

CRISPR-based gene editing technology represents a promising approach to deliver therapies for inherited disorders, including amyotrophic lateral sclerosis (ALS). Toxic gain-of-function superoxide dismutase 1 (SOD1) mutations are responsible for ~20% of familial ALS cases. Thus, current clinical strategies to treat SOD1-ALS are designed to lower SOD1 levels. Here, we utilized AAV-PHP.B variants to deliver CRISPR-Cas9 guide RNAs designed to disrupt the human SOD1 (huSOD1) transgene in SOD1G93A mice. A one-time intracerebroventricular injection of AAV.PHP.B-huSOD1-sgRNA into neonatal H11Cas9 SOD1G93A mice caused robust and sustained mutant huSOD1 protein reduction in the cortex and spinal cord, and restored motor function. Neonatal treatment also reduced spinal motor neuron loss, neuromuscular junction (NMJ) denervation and muscle atrophy, diminished axonal damage and preserved compound muscle action potential throughout the lifespan of treated mice. SOD1G93A treated mice achieved significant disease-free survival, extending lifespan by more than 110 days. Importantly, a one-time intrathecal or intravenous injection of AAV.PHP.eB-huSOD1-sgRNA in adult H11Cas9 SOD1G93A mice, immediately before symptom onset, also extended lifespan by at least 170 days. We observed substantial protection against disease progression demonstrating the utility of our CRISPR editing preclinical approach for target evaluation. Our work not only uncovered key parameters (e.g., AAV capsid, Cas9 expression) that resulted in improved efficacy compared similar approaches but can also serve to accelerate drug target validation. Comparative gene expression profiling to quantify SOD1 suppression and off-target effects following AAV-PHP.B-U6-sgSOD1-5 treatment of a HeLa cell line stably expressing the CRISPR Cas9 nuclease. Total number of samples: 40. Samples were collected at two timepoints: 48 and 72 hrs post-transduction. There were three treatment groups: (1) untransduced, (2) transduced with sgLACZ (1000K MOI), (3) transduced with sgSOD1-5 (1000K MOI). Each treatment group had four biological replicates. In addition, samples from treatment groups 1 and 2 had two technical replicates, each.