Amyotrophic lateral sclerosis (ALS), originally known as Lou Gehrig’s disease, is a rare neurological disorder leading to motor neuron degeneration and ultimately cell-death. ALS is fatal and characterized by a rapid degeneration process, which leads to paralysis and typically culminates in respiratory failure within 3-5 years following the onset of symptoms (Amado and Davidson 2021).
Globally, the prevalence of ALS ranges between 4.1 - 8.4/100,000 people. In the US, various population-based studies support a prevalence change from 3.7/100,000 in 2002 to 5.2/100,000 in 2015. Ethnicity, specifically of European descent, is linked to greater disease prevalence (Longinetti and Fang 2019).
ALS is commonly brought about by spontaneous mutations, with inherited or familial ALS accounting for up to ~10% of cases. Mutations in several genes are linked to ALS, including mutations in genes encoding superoxide dismutase 1 (SOD1), transactive response DNA-binding protein 43 (TDP-43), and C9ORF72 protein, among many others. Mutations in the SOD1 gene, which account for up to ~20% of familial and ~2% of sporadic ALS cases, lead to increased oxidative stress, intracellular aggregates, and mitochondrial damage (Deng et al. 2006). In contrast, mutations in the TDP-43 coding gene (TARDBP), which account for up to ~4% of familial and ~1% of sporadic ALS cases, result in aggregates, cytoplasm-mislocalized TDP-43, and abnormal RNA processing (Redler and Dokholyan et al. 2012). Lastly, C9ORF72 variants carrying G4C2 hexanucleotide repeat expansions have more recently been implicated in ALS and account for up to ~40% of familial and ~8% of sporadic cases. The pathogenesis associated with C9ORF72 variants is driven by loss and gain of function mechanisms and involves cytosolic aggregates of expanded RNAs and dipeptide repeat proteins (Smeyers et al. 2021).
Presently, there is no cure for ALS, nevertheless advances in understanding the genetic bases of the disease and improved gene editing technologies are opening new opportunities to develop effective therapeutic strategies (Amado and Davidson 2021).
Molecular mechanisms in ALS. “Figure 2. Amyotrophic lateral sclerosis (ALS) appears to be mediated by a complex interaction between molecular and genetic pathways. Reduced uptake of glutamate from the synaptic cleft, leading to glutamate excitotoxicity, is mediated by dysfunction of the astrocytic excitatory amino acid transporter 2 (EAAT2). The resulting glutamate-induced excitotoxicity induces neurodegeneration through activation of Ca2+-dependent enzymatic pathways. Mutations in the c9orf72, TDP-43 and fused in sarcoma (FUS) genes result in dysregulated RNA metabolism leading to abnormalities of translation and formation of intracellular neuronal aggregates. Mutations in the superoxide dismuates-1 (SOD-1) gene increases oxidative stress, induces mitochondrial dysfunction, leads to intracellular aggregates, and defective axonal transportation. Separately, microglia activation results in secretion of proinflammatory cytokines and neurotoxicity.” Retrieved without modification from van den Bos et al. 2019. https://creativecommons.org/licenses/by/4.0/
SOD1 knock-out mice fail to develop ALS, supporting that SOD1 mutant proteins gain toxic functions. In agreement with this model, mice overexpressing mutant forms of the human SOD1 sequence develop ALS pathologies (Deng et al. 2006). With this knowledge and relying on current advances in gene editing, as enabled by CRISPR/Cas9 tools, investigators aim to develop curative solutions for ALS.
Recent work by a team led by Dr. Teepu Siddique at The Ken and Ruth Davee Department of Neurology, Feinberg School of Medicine, Northwestern University, USA, took advantage of the hSOD1G93A ALS mouse model to test the efficacy and long term effects of CRISPR/Cas9 editing (Deng et al. 2021).
Aiming to achieve high SOD1 gene editing efficiency, Siddique’s team chose to develop transgenic mice expressing both SpCas9 and the hSOD1 targeting guide RNA (gRNA). By selecting this approach, the team ensured the expression of CRISPR/Cas9 and gRNA for the animal’s life span. Additionally, this approach enabled the team to address long-term safety issues related to potential off-target gene editing.
Deng and colleagues showed that ALS onset was suppressed in hSOD1G93A mice co-expressing Cas9/SOD1 gRNA. Mice remained free from ALS-related phenotypes, including the absence of protein inclusions, mitochondrial vacuoles, activated microglia, and astrocytosis, well past the expected time of disease onset documented for hSOD1G93A transgenic mice. Similarly, no motor neuron or muscle atrophy phenotypes were present in hSOD1G93A mice co-expressing Ca9/SOD1 gRNA. Importantly, constitutive expression of Cas9 did not lead to abnormal phenotypes, such as tumorigenesis or inflammatory disease. While investigators found a low frequency of off-target events, they conceded that more work is needed to characterize these outcomes fully.
Overall, as a proof of concept, these studies allowed Deng and colleagues to demonstrate the effectiveness of targeting mutant SOD1 genes with CRISPR/Cas9 tools to prevent the development of ALS phenotypes. Also, choosing an approach where Cas9 and SOD1 gRNA were constitutively expressed allowed investigators to optimize gene silencing, creating a great model where potential off-target and unwanted gene modifications could be evaluated.
