Utilizing CRISPR/Cas9 gene editing to treat Amyotrophic Lateral Sclerosis disease: An overview
Utilizing CRISPR/Cas9 gene editing to treat Amyotrophic Lateral Sclerosis disease: An overview
Behnam Molavi,1,*
1. Department of Biology, Islamic Azad University, Tehran Medical Sciences Branch, Tehran, Iran.
Introduction: Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a progressive neurodegenerative disorder that damages motor neurons (MNs) in the spinal cord, brainstem, and motor cortex. The disease typically progresses rapidly, leading to muscle weakness, atrophy, paralysis, respiratory failure, and eventually death within 2–5 years of symptom onset. ALS is among the most prevalent adult motor neuron diseases, affecting approximately 2–3 people per 100,000 worldwide. The increasing incidence of ALS in recent years is partly due to improvements in diagnostic capabilities. Despite these advancements, there is currently no cure. Existing treatments, such as Riluzole, extend survival by about three months but offer limited benefits, underscoring the urgent need for more effective therapies.
While most ALS cases are sporadic with unclear causes, around 10–15% of cases are familial, suggesting a genetic component. More than 50 genes have been associated with ALS, including those with causal or modifying mutations. Key genes often studied in ALS research, such as superoxide dismutase (SOD1), chromosome 9 open reading frame 72 (C9orf72), TAR DNA-binding protein 43 (TDP43), and RNA binding protein fused in sarcoma (FUS), are linked to about 75% of familial ALS cases. Advances in understanding the disease mechanisms have been driven by research into the molecular pathways affected by these genes, revealing potential new therapeutic targets. Consequently, ALS models and gene therapies targeting these mutations are being explored to develop effective treatments.
Gene editing, particularly using CRISPR/Cas9, presents a promising approach for treating ALS by correcting pathogenic mutations. This technology is increasingly being adopted in clinical research. CRISPR/Cas9 is favored for its simplicity and cost-effectiveness compared to other genome-editing methods such as transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs). This review focuses on the evolution of gene editing tools, emphasizing CRISPR/Cas9 for gene correction and the development of disease models and gene therapies for ALS.
Methods: Searched in 3 databases; PubMed, Scopus and Web of Science with the related terminol¬ogy of ALS, CRISPR and Lou Gehrig's disease. Also, relevant articles from 2020 until now are reviewed to mention gene therapy for ALS
Results: Recent research has explored the use of cellular models to correct genetic defects associated with ALS using CRISPR-Cas9 technology. This study reports the results are highly accurate, with corrections being approximately 99% precise. The analysis of on-target and off-target effects shows GC content ranging from 40-60%, as measured by the RNA/DNA GC Content Calculator. This finding is significant for the effectiveness of single-guide RNAs (sgRNAs) used in CRISPR-Cas9 treatments.
The targeted region in the mutant SOD1 gene is located near the start codon, with the CRISPR-Cas9 system inducing double-strand breaks, indicated by black highlights on the CAG repeats and orange or yellow highlights on the sgRNA cassettes. Additional validation includes the measurement of minimum free energy (MFE), which assesses the structural stability of sgRNAs using the Mfold web server and RNA structure webserver. These tools predict the most stable structures of oligonucleotides at 39°C, providing graphical representations of MFE structure, the thermodynamic ensemble of RNA structures, and centroid structures. These results suggest that CRISPR-Cas9 offers a promising therapeutic approach for ALS.
Encouraged by the success of CRISPR technology and its suitable sgRNAs, researchers and pharmaceutical companies are exploring this strategy for future treatments. CRISPR's ability to modify gene function and alter DNA sequences opens new possibilities for correcting genetic disorders. This technology has already proven efficient for site-specific genome editing in single cells and whole organisms. The study highlights CRISPR as a potential future treatment for genetic disorders like SBMA.
Despite these promising developments, these methods cannot fully capture the complexity of biological systems in living organisms. Further research involving animal models and human clinical trials is essential to validate the safety and efficacy of CRISPR-Cas9 as a treatment for ALS.
Conclusion: This review examines recent research utilizing CRISPR/Cas9-mediated gene correction to explore the pathophysiology of ALS through patient-derived iPSCs. Despite its simplicity and broad applicability, CRISPR/Cas9 technology has certain limitations. A significant concern is the risk of off-target effects, which occur when Cas9 and sgRNA inadvertently target non-specific DNA sites due to reduced specificity. Additionally, the efficiency of gene correction via the HDR mechanism is low (<1%). To improve HDR rates, strategies such as timed delivery with cell cycle synchronization and inhibition of key NHEJ molecules are necessary.
Since the identification of mutant SOD1 as a genetic cause of ALS in 1993, over 20 genes associated with ALS have been discovered. However, more than 80% of ALS patients lack identifiable genetic variants. This suggests that ALS may involve various mechanisms and genetic causes.
The integration of genetic information, advanced CRISPR/Cas9 genome engineering techniques, and in vitro disease modeling with iPSCs is expected to advance the identification of disease-causing mutations and deepen our understanding of ALS pathology. This approach holds the potential to thoroughly investigate ALS mechanisms, paving the way for the development of effective treatments and ultimately a cure for the disease.