CRISPR in Amyotrophic Lateral Sclerosis: Disease Modeling and Hope for a Cure

Professor Stephen Hawking amazed the world not only because of his intellect but the fact that he lived and worked for five decades with a disease that was predicted to kill him within two years: amyotrophic lateral sclerosis, or ALS.

Many people will be aware of amyotrophic lateral sclerosis because of the work of Professor Hawking or the music of famed guitar virtuoso and ALS patient Jason Becker. The condition also garnered international attention in the last decade owing to the viral ice bucket challenge that swept social media in an effort to raise funds for ALS research. But few outside the ALS community are familiar with the details and impact of this devastating neurodegenerative disease.

Significant advances in CRISPR gene editing technology and a slew of resulting studies have recently renewed hope for ALS patients and their families. In this blog, we’re discussing ALS symptoms, diagnosis, and treatment options, the current state of ALS research and clinical trials, and exploring the impact of CRISPR technology in ALS research, including improved disease modeling and the potential for curative gene therapies.

There are a range of symptoms of ALS. Typical early signs of the disease include weakness in hands, feet, or legs, often accompanied by involuntary muscle twitches and spasms. Slurred speech, trouble swallowing, and other movement difficulties are also common symptoms.

The progressive nature of ALS means that these symptoms become worse over time as the muscles atrophy. In the later stages of the disease, all basic motor functions are impaired, including breathing, speaking, and eating. Progression of the disease is also frequently accompanied by cognitive and behavioral changes.

Compared to other neurodegenerative diseases, the progression of ALS is rapid. A majority of patients die within two years of the first onset of symptoms, however, some people with ALS can live for 10 years or longer. The lifespan of patients is now known to be correlated with the age of onset - people who are younger at the time of onset tend to live longer. Stephen Hawking, for example, was 21 when he discovered he had ALS, and he lived for more than 50 years after his diagnosis.

Unfortunately, there is no single test, such as a biomarker-based blood test, that can accurately diagnose ALS. Doctors may first perform blood tests or take a urine sample or muscle biopsy to rule out other possible causes of symptoms. Other tests may include physical exams, MRI of the brain and spinal cord, electromyography (EMG) to measure the electrical activity of the muscle fibers, and nerve conduction studies (NCS).

Current treatment options for ALS are extremely limited, and no therapies are able to repair damaged neurons or prevent disease progression. The two drugs that are available can slow the decline of ALS patients or prolong survival by short timeframes. Other treatments involve physical therapy and rehabilitation, and support for communication and breathing.

Amyotrophic lateral sclerosis is an incredibly complex disease and in many ways is not well understood, making it all the more difficult to treat. While 90% of ALS cases are sporadic and have unknown causes, the remaining 10% are caused by genetic mutations and are hereditary, known as familial ALS (fALS). Let’s explore the genetic and environmental factors that contribute to ALS.

While there are more than 50 genes implicated in ALS, mutations in only a few genes have been confirmed to cause the disease, including chromosome 9 open reading frame 72 (C9orf72), cytosolic Cu/Zn superoxide dismutase (SOD1), TAR DNA binding protein 43 (TARDBP), and fused in sarcoma (FUS). More recently, another gene, serine palmitoyltransferase long chain base subunit 1 (SPTLC1), has also been confirmed to cause a rare childhood form of ALS. More genetic causes for fALS are being discovered as scientists continue to study the disease.

Mutations in C9orf72 are responsible for between 35% and 45% of fALS cases. The mutation is a hexanucleotide GGGGCC repeat hyperexpansion, located in the first intron of the gene. In healthy individuals, there are typically 20 or fewer GGGGAA repeats, however, in fALS patients, the hyperexpansion can contain hundreds of repeats. This hyperexpansion disrupts the promoter region that would normally control the transcription of the C9orf72 gene. While it is unclear exactly how the mutation causes disease onset, overexpression and accumulation of the protein produced by C9orf72 can contribute to cellular dysfunction.

The SOD1 gene encodes a protein called Cu, Zn, superoxide dismutase, which is a neuroprotective enzyme. As many as 170 different mutations in the SOD1 gene can cause fALS, disrupting cellular homeostasis through a variety of mechanisms. These mechanisms are currently not well understood, as even patients carrying the same SOD1 mutation can have differences in disease phenotype.

TARDBP encodes the DNA/RNA binding TDP-43 protein, which is able to move between the nucleus and the cytoplasm of cells and plays a key role in the splicing, transport, and translation of RNA. It is also known to be involved in important post-translational modifications such as phosphorylation and ubiquitination. Up to 50 different mutations in TARDBP can cause fALS; both the lack of TDP-43 and the overexpression of TDP-43 within cells can contribute to the disease.

The function of the FUS gene and the protein it produces are not well understood, although it is known to contribute to RNA splicing and trafficking, DNA repair, and cellular stress responses. There have been more than 50 FUS mutations described that contribute to fALS, particularly juvenile fALS. These mutations cluster within specific regions of the gene, and typically cause mislocation of FUS protein within cells, contributing to degeneration of neurons.

In 2021, scientists discovered that mutations in the SPTLC1 gene cause juvenile-onset fALS. The protein encoded by this gene is an enzyme involved in the biosynthesis of sphingolipids, which play a role in membrane biology and regulate cell function. Several SPTLC1 mutations were confirmed to cause juvenile fALS in the study and research is ongoing.

In a major breakthrough, a 2023 paper revealed a previously unknown cause of ALS: paternally expressed gene 10 (PEG10). PEG10 is a fragment of an ancient retrovirus that integrated into the human genome millions of years ago. Since it can no longer replicate, the gene does not cause disease, but is still passed on from generation to generation. This domesticated retrotransposon has previously been shown to be involved in placental development in mammals.

Interestingly, however, when the PEG10 protein is overexpressed in the wrong areas - such as the spinal cord - it interferes with the expression of axon remodeling genes, preventing nerve cells from communicating properly. This overexpression of PEG10 protein seems to occur due to a previously identified mutation in the ubiquilin-2 gene (UBQLN2), a proteasome shuttle that normally regulates the expression of domesticated retrotransposons. It’s likely that PEG10 will now be developed as a biomarker for ALS because of its overabundance in ALS patients.

The vast majority of ALS cases are sporadic, and the cause is unknown. However, there are environmental factors that are thought to place people at higher risk of developing the disease. These factors include exposure to toxins such as lead, and smoking, particularly in women. Individuals who have served in the military are also at a higher risk of developing ALS, although it is unclear exactly why this is the case.

Compared to previous genome engineering technologies, CRISPR-Cas9 can be used to edit DNA with relative ease, particularly in primary cells such as stem cells and neurons. This has been crucial in ALS research, where accurate disease models are desperately needed in order to both understand the cause and progression of the condition, identify novel drug targets, and test new treatment options before they progress into human trials.

Published in late 2022 by researchers from Stanford University School of Medicine, a key study used CRISPR-Cas9 to perform a genome-wide screen in human cells. The paper was able to identify genes that code for lysosomal vacuolar ATPase (v-ATPase) that can lower levels of ataxin-2, a protein involved in ALS. Since several small molecule inhibitors of v-ATPase are already approved to treat other conditions, this is likely to yield positive results for ALS patients within relatively short timeframes.

A notable recent study provided a streamlined workflow for CRISPR editing in human induced pluripotent stem cells (iPSCs), introducing ALS genetic mutations such as those in SOD1 and FUS, as well as correcting them. Edited iPSCs can be then differentiated into clinically relevant motor neuron cells to provide accurate disease models.

A CRISPR mouse model of ALS was generated in 2018, by knocking in the human ALS mutation to the mouse TARDBP gene. The resulting TDP-43^Q331K mice were used to characterize the role of the mutation in ALS and how it affects the expression of other neurodegeneration-associated genes.

A 2019 CRISPR ALS study used human iPSCs to study the mechanisms by which TARDBP mutations and aberrant TDP-43 cause ALS. The authors found that TDP-43 malfunction impairs the secretion of brain-derived neurotrophic factor (BDNF) via altered splicing of Sortilin, a trafficking receptor. They also corrected the same mutation in patient-derived iPSCs.

CRISPR has enormous potential to provide curative therapies by treating the underlying cause of genetic disease. It has now been used in many fALS studies to demonstrate that the genetic mutations that cause the disease can be either corrected to restore normal function or knocked out to eliminate the production of toxic proteins. Let’s explore some of the breakthrough CRISPR ALS research papers that have used these approaches to create novel gene therapies.

A proof-of-concept Nature study published in 2022 demonstrated that the GGGGCC hyperexpansion in C9orf72 can be excised using CRISPR-Cas9. The authors employed an AAV vector to deliver the editing components to primary cortical neurons of ALS mice in vitro, and then to the brains of the same mouse models in vivo. They also tested their strategy in patient iPSC-derived motor neurons and brain organoids, rescuing disease phenotypes.

CRISPR-Cas9 has shown promise in deleting mutated SOD1 in a mouse model of ALS. A 2020 Nature paper used the smaller Cas9 variant from Staphylococcus aureus (SaCas9) and delivered it to the mice via an AAV vector along with an sgRNA targeting the gene. This strategy was able to delete the gene, improving the lifespan of the mice by over 50%.

Another CRISPR ALS study used a similar method in the same mouse model, reporting that mutant SOD1 protein was reduced significantly in the spinal cord of the mice. The mice displayed improved motor function, reduced muscle atrophy, delayed disease onset, and increased survival.

CRISPR base editing systems have also been used to knock out the mutated SOD1 gene. The authors of a 2020 Molecular Therapy study employed an intein-mediated trans-splicing system to deliver cytidine base editors (CBEs) to a mouse model of ALS, introducing a nonsense mutation to knock out the mutant gene. Treated mice showed reduced muscle atrophy and increased neuromuscular function.

It is also possible to use CRISPR-Cas9 to correct ALS mutations, such as those in the FUS gene. A 2018 Nature Communications article demonstrated this approach in patient iPSC-derived motor neurons, reporting that DNA ligation defects in the cells that contribute to neurodegeneration can be rescued by CRISPR-mediated knock-in to correct the mutation.