CRISPR-Cas9 in Huntington’s Disease: Progress and Possibilities For Future Cure

In one of the earliest and most complete reports on Huntington’s disease, physician George Huntington described it very aptly as “an heirloom from generations away back in the dim past.” The iconic document titled “On Chorea” chronicles the melancholy legacy of Huntington’s Disease and how it affects individuals and families. 

Nearly two centuries on from when it was first documented, there is still no cure for Huntington’s. In this updated blog, let’s learn about this rare neurodegenerative disorder and the recent progress of scientists using CRISPR technology to generate more accurate disease models and novel therapies for Huntington’s disease. 

Huntington’s Disease: Symptoms, Diagnosis, Treatment, and Clinical Trials

Huntington’s disease is a rare, progressive neurodegenerative disease. In the United States, it affects 1 in every 10,000 individuals - an estimated 40,000 people in the US are affected by the disease, and approximately 16% are juvenile Huntington’s cases. Let’s explore Huntington’s disease symptoms, diagnosis, current treatment options, and clinical trials.

Symptoms of Huntington’s disease

Huntington’s usually affects people in their 30s, but in rare cases with juvenile Huntington’s disease, symptoms could appear much earlier in life (before an individual turns 20). Huntington’s disease impacts part of the basal ganglia in the brain called the striatum. It leads to the degeneration and death of striatal neurons as the disease progresses. 

Common symptoms of Huntington’s include the loss of motor function, chorea (involuntary movements), coordination disabilities, cognitive impairment, difficulty with speech or swallowing, and psychiatric disorders. The disease progresses for 10-25 years from when the symptoms first arise and is highly debilitating; patients in later stages can no longer care for themselves. 

Huntington’s disease diagnosis

The current diagnosis of Huntington’s disease includes a physical exam, a review of individual and family medical history, and neurological and psychiatric evaluation. Physicians may order brain imaging scans for structural and functional details. Predictive genetic testing can also help individuals evaluate their risk of developing the disease. 

Treatments for Huntington’s disease 

There is no cure available for Huntington's disease, and it is a progressive, irreversible condition. Current treatments help manage the symptoms by regulating neurotransmitters but cannot reverse or cure the disease. Neurotransmitters are chemical signals that neurons use to communicate with each other. Tetrabenazine (Xenazine), a commonly used drug in Huntington’s treatment, helps manage irregular involuntary movements by depleting a dopamine neurotransmitter. An array of antipsychotic medications also help alleviate related symptoms of chorea, hallucination, and delusion. 

Huntington’s disease clinical trials 

A myriad of approaches has been tested in clinical trials to improve treatment options for Huntington’s. Among those, antisense oligonucleotides (ASOs) are the most prominent. ASOs are short-length nucleotides (12-25 units) and block protein production from the targeted genes. 

In 2021, Roche halted a Phase III trial for Tominersen, an ASO drug designed to inhibit mutant huntingtin protein. Tominersen, also known as IONIS-HTTRx and RG6042, had shown promising results in the earlier phases of the trial. The study was halted in response to the advice from an independent data monitoring committee (IDMC). The company and the clinical investigators decided to stop the trial after weighing the benefits versus risks profile of the drug. 

Following this, another pharma company, Wave Life Sciences, also announced results from Phase 1b/2a trials for two drugs in the Huntington’s disease space. Based on the results of no significant reduction of mutant huntingtin protein in the patients, the company decided to discontinue the clinical trial. Several other past or ongoing trials in this space also include ASO-based therapeutics.

The Genetics of Huntington’s Disease

Huntington’s is a genetically inherited, autosomal dominant disease, meaning having a single copy of the mutated gene is sufficient to cause the disease. There’s a 50% chance of a person inheriting the condition if one of their parents is affected. Huntington’s disease has been pinned to a single gene called huntingtin (HTT), mapped to the short arm of chromosome 4.

Mutation in Huntingtin Gene

Human DNA is made up of building blocks called nucleotides. Each nucleotide comprises three subunits: sugar, phosphate, and a nitrogenous base. There are four known nitrogenous bases, cytosine (C), adenine (A), guanine (G), and thymine (T). 

The gene mutation underlying Huntington’s disease is very well understood. It involves the repetition of C, A, and G, commonly known as a trinucleotide repeat, in the first intron of the HTT gene. The hyperexpansion of CAG repeats (40 or higher) leads to the onset of Huntington’s symptoms. 

This CAG sequence codes for the amino acid glutamine (Q), and hyperexpansions of this trinucleotide repeat are also known to cause other neurological diseases, including spinal muscular atrophy (SMA). In fact, Huntington’s disease is actually part of a larger group of related neurological conditions known as polyglutamine (polyQ) diseases. 

The length of repetition correlates with the age of onset, and longer repeats would mean an individual starts to show signs early in their lifetime. Interestingly, the repeat sequence tends to amplify with successive generations within the family. Typically, at 28 or more copies, the mutation becomes unstable, is more likely to expand, and codes for a dysfunctional protein. However, having 35 or fewer copies of the repeat does not lead to the disease symptom. Only when the repeats are in the high 30s (36 or more) will the affected individuals be at a higher risk of developing Huntington’s.

The role of the huntingtin protein

The HTT gene codes for a protein known as huntingtin. However, the exact role of this protein is not well understood. Scientists do know that it plays a crucial role in early neuronal development and adult neuronal homeostasis in mice. The huntingtin protein is well conserved across species, from invertebrates to humans. A closer look at the human huntingtin protein structure (using a cryo-electron microscope!) revealed three different regions or domains of the protein, each with a unique function. 

The genetic mutation in HTT means that an abnormal huntingtin protein is produced. Unlike normal huntingtin protein, this mutant protein is misfolded and tends to aggregate and clump together; the misfolded protein accumulation appears toxic to neuronal cells. Future research will further our understanding of the protein and its role in neuronal development and Huntington’s disease pathophysiology. 

Huntington’s Disease CRISPR Research: Improved Disease Models

One of the ways in which CRISPR has revolutionized the field of neurodegenerative diseases is by allowing researchers to generate more accurate disease models in human cells, mice, and even large animals. This genetic manipulation aims to mimic the disease more closely, allowing scientists to better understand the pathogenesis and progression of the condition. It also enables drug discovery studies and preclinical research so that more effective therapies can be generated in the future. Let’s explore some key recent developments in CRISPR Huntington’s disease modeling.

CRISPR knock-in generates a porcine model of Huntington’s disease 

Scientists have used CRISPR Cas9 to generate a knock-in model of Huntington's disease in pigs by replacing the endogenous HTT gene with large CAG repeats. The resultant disease model closely recapitulated neurodegenerative symptoms in human patients, and symptoms were observed across generations, suggesting germline transmission. Using the CRISPR-Cas9 approach opened up opportunities to explore endogenous molecular changes and neuropathogenesis in Huntington's disease. 

CRISPR-Cas9 generates isogenic cell models of Huntington’s disease

In a separate study from the Olejniczak lab in Poland, researchers used CRISPR-Cas9 to create a series of human embryonic kidney (HEK) cell lines with different CAG repeats. This disease modeling effort can tremendously impact drug discovery and validation. In addition to this, they were also able to attenuate the mutant HTT gene in induced pluripotent stem cell models. 

CRISPR editing in Huntington’s disease neurosphere models

Led by researchers at South Korea’s Seoul National University Hospital, a recent study demonstrated the editing of neurosphere models of Huntington's disease with CRISPR-Cas9. Neurospheres are clusters of nerve stem cells grown in vitro to model diseases of the nervous system, such as Huntington's disease. Taken from R6/2 mice, a current Huntington's disease animal model, the neurospheres were grown and edited with CRISPR to delete the CAG expansion in HTT, then studied to explore the effect on disease phenotypes.

Developing Novel CRISPR Therapies for Huntington’s Disease

CRISPR-Cas9 is used for gene editing, and Huntington’s is caused by a genetic mutation. So, can CRISPR cure Huntington’s disease? Recent progress in the field suggests that the answer is yes: by treating the underlying cause of the disease - the genetic mutation - CRISPR can potentially cure the condition with a single treatment. 

Many scientists are currently using CRISPR-Cas9 and derivative technologies to develop novel therapies for Huntington’s disease. The two main CRISPR strategies involve either selectively knocking out the mutant HTT allele or correcting it by excising the CAG hyperexpansion, but other strategies have also recently been developed. If the aim is to delete or silence the mutant HTT allele, the therapy must be highly specific so it does not affect the wild-type allele, which is critical for normal brain function. Let’s look at the different CRISPR Huntington’s disease therapy methods.  

CRISPR-SaCas9 editing for Huntington’s alleviates symptoms in mice 

The Gaj lab at the University of Illinois, Urbana, reported an effective CRISPR-based targeted attenuation of mutant HTT gene in a mouse model of the disease. They used a Cas9 endonuclease from Staphylococcus aureus (SaCas9), much smaller than the conventional Streptococcus pyogenes-derived Cas9 (SpCas9). Using SaCas9 allowed easy packaging via a single adeno-associated viral (AAV) particle for in vivo delivery into the mouse striatum.

The SaCas9 coupled with a single guide RNA disrupted the mutation in vivo, alleviated motor deficit symptoms, and even increased lifespan in R6/2 mice, a particularly aggressive murine model for Huntington's disease. Results from this study are encouraging and point to the future use of CRISPR-Cas9 in treating Huntington's disease in clinical settings.

CRISPR-Cas9 genome editing to knock out the mutant huntingtin allele

At the University of Pennsylvania, Philadelphia, the Davidson lab demonstrated allele-specific precision editing of the huntingtin gene using CRISPR-Cas9 in patient-derived cells and mice. This was one of the early studies in the field and paved the way for CRISPR-Cas9 editing in preclinical models of Huntington’s. 

At Emory University, Georgia, the Li lab took a slightly different approach and used CRISPR-Cas9-mediated inactivation to attenuate the mutant HTT gene in a mouse model. They were able to reduce the neurotoxicity successfully in these mice via non-allele-specific CRISPR targeting.

Allele-specific HTT knockout using CRISPR-Cas9 and two sgRNAs

A 2022 paper from the Harvard Medical School demonstrated a clever method to knock out the mutated HTT gene without the risk of affecting the wild-type allele in induced pluripotent stem cells (iPSCs). The study utilized mutant-specific variation in the protospacer adjacent motif (PAM) sites that are necessary for CRISPR editing to target HTT in an allele-specific manner, ensuring functional knockout of the huntingtin protein by employing two sgRNAs. 

CRISPR-Cas9 gene editing to correct the HTT mutation

Following on from their earlier work creating a porcine model of Huntington's disease, researchers from Jinan University in China recently used this model to demonstrate that CRISPR-Cas9 editing can be used to correct the mutation in HTT, replacing the hyperexpansion with a normal CAG repeat.

Packaged into an AAV vector, the gene knock-in therapy was delivered to the pigs by either intracranial or intravenous injection. A single treatment resulted in the depletion of mutant huntingtin protein and a reduction in neurotoxicity and related symptoms. As pigs are the accepted large animal model of Huntington's disease, this pre-clinical research is very promising, and the therapy will hopefully progress into clinical trials. 

CRISPR-Cas13d RNA editing as a novel therapy for Huntington’s disease

A proof-of-concept study published in Nature Neuroscience in February 2023 revealed a completely different method for treating Huntington’s disease: CRISPR-based RNA editing. The mutant messenger RNA (mRNA) transcripts that are produced in Huntington’s disease have been found to significantly contribute to disease pathogenesis, making them a target for treatment. The collaboration between Johns Hopkins and UC San Diego used the Cas13d nuclease, which targets RNA and can be used to destroy specific mRNA transcripts. 

The study demonstrated a significant depletion of mutant transcripts in three different iPSC lines derived from Huntington's disease patients, each carrying a different number of repeats. Subsequent in vivo experiments in a mouse model of Huntington's disease demonstrated that treated mice had significantly improved motor function. Surprisingly, this positive effect continued for up to eight months in the mice, suggesting lasting therapeutic benefits. 

Huntington’s Disease and CRISPR: Future Therapies and Treatments

The hard work of scientists in labs around the world indicates that multiple Huntington’s disease therapies are possible using CRISPR technology. While it will take several more years for these therapies to progress through the development pipeline before they are tested in clinical trials, there is certainly hope for the future. More advanced disease models generated using CRISPR will also help us to better understand the disease and accelerate drug discovery research.

One of the key initiatives providing hope for Huntington’s and other neurological disorders is the Alliance for Therapies in Neuroscience (ATN), formed by the Innovative Genomics Institute (IGI) at Berkeley, the University of California, San Francisco, and Genentech. ATN is a long-term research partnership that involves cross-collaboration spanning multiple disciplines, such as CRISPR technology, clinical neuroscience, and biophysics. The overarching goal of this collaboration is to harness the power of CRISPR technology to study complex genetic interactions and molecular mechanisms underlying neurodegenerative disorders such as Huntington’s disease and hopefully treat them. 

We hope this updated blog helped fill you in on all the latest developments in Huntington’s disease CRISPR research. As researchers, clinicians, and pharma companies combine forces to understand and treat the disease, we look forward to a time when Huntington’s will no longer disrupt individual lives and families.