CRISPR/Cas9: implication for modeling and therapy of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease, is a progressive neurodegenerative disease affecting motor neurons (MNs) in the spinal cord, brainstem, and motor cortex. The disease progresses rapidly, typically leading to death within 2-5 years of symptom onset due to muscle weakness, atrophy, paralysis, and respiratory failure. ALS is one of the most common adult motor neuron diseases, with a global prevalence of 2-3 per 100,000 individuals, and its incidence appears to be increasing. Current treatments, such as Riluzole, offer only modest benefits, extending survival by a few months. The etiology of ALS is complex, with most cases being sporadic (unknown origin). However, approximately 10-15% of cases have a family history, strongly suggesting a genetic component. Over 50 genes have been linked to ALS, either as causal factors or disease modifiers. The most studied genes include SOD1, C9orf72, TDP-43, and FUS, accounting for about 75% of familial ALS cases. Understanding the molecular pathways of these genes has revealed potential therapeutic targets. This review focuses on the development of ALS models and gene therapy approaches, particularly using CRISPR/Cas9 technology, aiming to translate these findings into effective clinical treatments.

This paper reviews the evolution of gene editing tools, starting with homologous recombination (HR) and non-homologous end joining (NHEJ), which had limited efficacy. The review then details three main gene editing technologies: Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the CRISPR/Cas system. ZFNs, discovered in the 1990s, utilize FokI endonuclease and zinc finger structures to target DNA sequences, but suffer from issues with specificity and cytotoxicity. TALENs offer improved specificity compared to ZFNs due to their unique DNA-binding domain recognizing a single nucleotide. However, their large size hinders delivery. The CRISPR/Cas system, particularly the type II CRISPR/Cas9 system, utilizes sgRNA and Cas9 endonuclease for precise DNA targeting, offering ease of use and high efficiency. While off-target effects and cytotoxicity are potential concerns, the CRISPR/Cas9 system remains the most prevalent gene editing technology due to its simplicity, scalability, and efficiency. The paper also discusses newer advancements like CRISPR single-base editors (ABEs and CBEs) and prime editing, which further enhance precision and reduce off-target effects.

The paper utilizes a comprehensive literature review methodology, systematically collecting and analyzing research articles focusing on gene editing technologies, specifically CRISPR/Cas9, and their applications in ALS modeling and therapy. The authors explore the evolution of gene editing methods, detailing the mechanisms and limitations of ZFNs, TALENs, and the CRISPR/Cas system. They then extensively review the development of various ALS models, including cell-based models (HT22, iPSCs, BV2, Neuro 2a, NSC-34, hESC, HeLa cells) and animal models (zebrafish, Drosophila, mice, rats), emphasizing the contributions of CRISPR/Cas9 in creating these models by targeting specific genes like SOD1, C9orf72, TDP-43, FUS, UBQLN2, VCP, TP73, MATR3, and CREST. The review further analyzes the therapeutic applications of CRISPR/Cas9 in ALS, focusing on studies targeting these genes, including in vivo studies and the use of iPSCs. Finally, the authors summarize ongoing phase III clinical trials for ALS, highlighting the challenges and the need for improved therapies and diagnostics. The review synthesizes findings from a wide range of studies to present a comprehensive overview of CRISPR/Cas9 technology in ALS research and therapy.

The review highlights the significant role of CRISPR/Cas9 technology in both modeling and treating ALS. In ALS modeling, CRISPR/Cas9 has enabled the creation of numerous cell-based and animal models which faithfully recapitulate aspects of the disease, facilitating investigation into disease mechanisms and potential therapies. Several studies using CRISPR/Cas9-mediated gene editing of SOD1, C9orf72, TDP-43, and FUS have shown promising results in reversing or ameliorating disease phenotypes in in vitro and in vivo ALS models. For example, CRISPR/Cas9-mediated disruption of SOD1 in mice led to decreased SOD1 protein expression, reduced muscle atrophy, and increased lifespan. In C9orf72, CRISPR/Cas9 has been used to excise the repeat expansion mutation, rescuing some ALS phenotypes in mouse models. Studies have also utilized CRISPR/Cas9 to correct mutations in FUS and TDP-43 in iPSC-derived motor neurons, rescuing some of the pathogenic features. However, the review also notes that the efficacy of these approaches depends on factors like the age of treatment in animal models and the efficiency of gene correction. The review also presents a comprehensive list of ongoing phase III clinical trials for ALS, underscoring the ongoing effort to find effective treatments for this devastating disease despite many trials failing to show meaningful results. The limitations of the existing clinical trials were also discussed, including issues with drug delivery and biomarkers.

The findings of this review underscore the potential of CRISPR/Cas9 as a powerful tool for both basic research and therapeutic development in ALS. The ability to precisely target and modify genes associated with ALS has greatly advanced our understanding of the disease's complex pathophysiology. The development of accurate disease models using CRISPR/Cas9 is crucial for preclinical testing of new therapies and identifying potential drug targets. The successful reversal or amelioration of disease phenotypes in several studies using CRISPR/Cas9-mediated gene editing demonstrates the feasibility of using gene therapy to treat ALS. However, it is essential to address limitations, such as off-target effects and efficient delivery systems, to ensure the safety and efficacy of CRISPR-Cas9-based therapies. The current lack of clinically effective treatments for ALS highlights the urgent need for further research. The significant number of ongoing clinical trials, as summarized in the review, reflects the continued drive to improve treatments and early diagnosis for ALS.

This review demonstrates the substantial contribution of CRISPR/Cas9 technology to ALS research. The generation of accurate disease models and the successful correction of disease-causing mutations in cellular and animal models show considerable promise for gene therapy. However, challenges remain in optimizing delivery methods and mitigating off-target effects. Future research should focus on developing more efficient and safe CRISPR-Cas9-based gene therapies for ALS, while also further refining disease modeling to improve drug discovery and development.

While the review comprehensively covers a substantial body of research, it acknowledges several limitations inherent in the current state of CRISPR/Cas9 technology and ALS research. These include off-target effects of CRISPR/Cas9, the size limitations of delivery vehicles, and the relatively low efficiency of homology-directed repair. Additionally, many studies using CRISPR/Cas9 in ALS models have treated young animals before disease onset, limiting the ability to fully assess the long-term efficacy in animals with established disease pathology. Furthermore, the translation of preclinical findings into effective clinical therapies remains a considerable challenge, as evidenced by the lack of success in many ALS clinical trials.