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. Overall design: 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.

基于CRISPR（Clustered Regularly Interspaced Short Palindromic Repeats）的基因编辑技术，为包括肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）在内的遗传性疾病提供了极具前景的治疗递送方案。毒性功能获得型超氧化物歧化酶1（superoxide dismutase 1, SOD1）突变约占家族性ALS病例的20%，因此当前针对SOD1相关性ALS的临床治疗策略均以降低SOD1表达水平为核心目标。本研究利用腺相关病毒（adeno-associated virus, AAV）-PHP.B变体，递送靶向敲除SOD1G93A小鼠中人源SOD1（huSOD1）转基因的CRISPR-Cas9向导RNA（guide RNA, gRNA）。对新生期H11Cas9 SOD1G93A小鼠单次脑室内注射AAV.PHP.B-huSOD1-sgRNA后，可在皮层与脊髓中实现突变型人源SOD1蛋白的强效且持久的下调，并恢复小鼠的运动功能。新生期干预还可减少脊髓运动神经元丢失、神经肌肉接头（neuromuscular junction, NMJ）去神经支配与肌肉萎缩，减轻轴突损伤，并在治疗小鼠的整个生命周期中维持复合肌肉动作电位。接受治疗的SOD1G93A小鼠获得了显著的无病生存期，寿命延长超过110天。值得注意的是，在症状发作前即刻对成年H11Cas9 SOD1G93A小鼠单次鞘内或静脉注射AAV.PHP.eB-huSOD1-sgRNA，同样可使小鼠寿命延长至少170天。本研究观察到对疾病进展的显著保护作用，证实了本CRISPR编辑临床前策略用于靶点验证的实用性。本研究不仅明确了相较于同类编辑策略可提升效能的关键参数（如AAV衣壳、Cas9表达模式），还可为加速药物靶点验证提供重要参考。总体实验设计：对稳定表达CRISPR-Cas9核酸酶的海拉细胞系（HeLa）开展AAV-PHP.B-U6-sgSOD1-5处理后，通过比较基因表达谱量化SOD1抑制效果与脱靶效应。总样本量共计40份，样本于转导后48小时与72小时两个时间点收集。实验共设置3个处理组：(1) 未转导对照组；(2) 转染sgLACZ组（感染复数multiplicity of infection, MOI=1000K）；(3) 转染sgSOD1-5组（MOI=1000K）。每个处理组设置4个生物学重复；此外，组1与组2的样本各增设2个技术重复。