Understanding the mechanism of repeat expansion has allowed Dr. Christopher E. Pearson and colleagues to target unique disease-associated mutagenic DNA structures as a potential therapeutic avenue.
Elvira Mukharryamova, Sornnujah Kathirgamanathan, and Tanvi Anadampillai
The progressive neurodegeneration (loss of brain cells) in individuals with Huntington Disease (HD) highlights the limits of modern medicine in relation to prognosis and cure. As a condition that worsens over time, HD individuals become entirely reliant on others for their daily living. The characteristic neurodegeneration in HD individuals is due to a curious mutation of DNA, called tandem repeat expansions, in the protein-coding gene HTT, which is involved in brain development. These repeat expansions consist of nucleotide sequence units, such as CAG in the case of HD, that occur in tandem (‘CAG CAG CAG…’). For example, healthy individuals carry a repeat tract lengths of 5-35 ‘CAG’ units in the HTT gene. Individuals with 35-39 copies are at an increased risk for HD, while those with 40 or more copies will develop HD earlier in life1 (Fig. 1A). Importantly, Pearson says “As patients age, the mutation continues in their brains and their disease worsens. For example, ‘THE CAT ATE THE FAT FAT RAT’ mutates to ‘THE CAT ATE THE FAT FAT FAT RAT,’ which eventually mutates to ‘THE CAT ATE THE FAT FAT FAT FAT FAT RAT,’ and so on.” The number of tandem repeats in functionally relevant genes – also referred to as repeat length – is negatively correlated with symptom age-of-onset and positively correlated with disease progression and severity (Fig. 1B). Generally, longer repeat lengths lead to an earlier age-of-onset with a more severe disease phenotype2. “Essentially, for therapy we would like to put that RAT on a diet, which should delay onset and slow progression”, says Pearson. Tandem repeat expansions also cause 69 other serious disorders3
When it comes to elucidating the underlying mechanisms of disease-associated repeat expansions, it is difficult to find someone with a higher level of expertise than Dr. Christopher E. Pearson – a Canada Research Chair in Disease-Associated Genome Instability, a Senior Scientist at The Hospital for Sick Children, and a Full-Professor at the University of Toronto. In a career that spans nearly three decades, Dr. Pearson has published 97 publications largely focusing on tandem repeat DNA sequences and the mechanism of disease-causing repeat expansion. Looking back on his decision to pursue what Dr. Pearson calls “dynamic mutations” back in 1993, he considers himself fortunate to have discovered something that has captured his curiosity and become increasingly relevant all these years.
The inspiring work of Dr. Pearson and his team has contributed greatly to our current understanding of repeat expansions. His recent publications featured here, have catapulted the field closer to developing a treatment that can potentially reverse repeat-associated neurodegenerative diseases.
Repeat expansions as a driver of disease
In molecular genetics, the adage “you can’t harvest what you haven’t planted” holds true. One cannot design a treatment for a complex genetic disorder without first understanding the molecular mechanisms of its pathogenicity4. In HD, the root cause of disease is the inheritance and ongoing expansion of tandem repeats, where the repeats expand throughout an individual’s life, causing symptoms to worsen2. Although the exact mechanism of expansion has remained elusive, several factors involved in repeat instability have been established. They include repeat length
, slipped-DNA structures, and the influence of DNA repair proteins5.
A distinguishing feature of disease genes with expanded repeats is the presence of unusual slipped-DNA structures. Slipped-DNAs form at expanded repeats when unwound DNA attempts to reanneal but does so incorrectly, “much like a mis-aligned zipper”, says Pearson (Fig. 2). Slipped-DNAs occur only if the gene contains a threshold number of tandem repeat units, where greater number of repeats enhances slip-out formation. Slipped-DNAs are critical because they act as mutagenic intermediates of instability by attracting DNA repair proteins, which ultimately drive further repeat expansion, which enhances slip-DNA formation…leading to a compounding cycle of expansion mutations. These DNA repair proteins introduce additional repeats through the error-prone attempts to repair the slipped-DNAs – in this manner, rather than protecting against mutation the repair proteins are driving mutations (Fig. 2)6.
Dr. Pearson remembers identifying slipped-DNAs by accident during his time as a post-doctoral fellow. He recalls thinking at that moment that these unusual structures must be important and might even be the key to novel therapeutics. Lo and behold, Dr. Pearson’s suspicions turned out to be right.
Overview of mutation-centric therapeutic targets
Multiple therapeutic approaches can target various downstream pathogenic aspects of HD, such as lowering the mutant repeat RNA transcript or mutant protein aggregates. Current approaches looking to treat repeat expansion disorders at the root-cause, the DNA mutation, have either targeted the repeat sequences themselves, or the DNA repair proteins involved in repeat expansions4. However, a significant limitation of these approaches is that they lack the specificity required to treat only the disease-causing gene in affected cells, while avoiding the normal gene and other off-target effects. Dr. Pearson provides the example of potentially targeting MSH3 or FAN1, DNA repair proteins that drive or supress CAG expansions6. Key features of these proteins is their DNA structure-specificity, meaning they only recognize and process unusual structures like slipped-DNAs. MSH3 and FAN1 can modulate repeat stability by either promoting or inhibiting repeat expansion5,7. Additionally, certain variations in the MSH3 and FAN1 genes can alter the age-of-onset and progression of various repeat expansion disorders, including HD. Taken together, altering levels of MSH3 or FAN1 could therapeutically modulate expanded pathogenic repeats. However, due to the involvement of MSH3 and FAN1 in maintaining the integrity of the entire genome through DNA repair, targeting these proteins would certainly affect their actions elsewhere beyond the mutant CAG tract. One can expect modulating the levels or activities of MSH3 and FAN1 will cause widespread DNA abnormalities, possibly resulting in cancer. This lack of specificity could be worrisome.
A novel molecule targets slip-out structures to reverse repeat expansion
Hoping to find an alternative therapeutic avenue that can address the challenge of specificity, Dr. Pearson and colleagues designed the small molecule DNA ligand Naphthyridine–Azaquinolone (NA). This molecule has a high degree of specificity to slip-out structures within expanded CAG repeats, effectively providing a means of differentiating between normal and pathogenic alleles, as well as the rest of the genome8. This feature of NA reduces its off-target effects and can be attributed to Dr. Pearson’s unique appreciation for the importance of structure-specificity: “Slipped-DNAs only form at the disease repeats that are long and unstable, this provides exact specificity of NA to only the disease gene.”
Although the discovery of a molecule that could recognize and bind pathogenic CAG repeats was exciting, Dr. Pearson admits that the group had no prior knowledge of whether this molecule could prevent repeat expansions, let alone induce contractions. He adds “It was a blind experiment…stabilized repeats would be good, contractions would be even better, but enhanced expansions would be really bad”. Subsequent work by Dr. Pearson and colleagues demonstrated that in addition to its binding specificity, NA stabilized and shortened the expanded repeats in affected brain cells. “We were ecstatic that NA induced CAG contractions in the brain to less than what the HD mice inherited”, explains Dr. Pearson. NA is believed to obstruct the processing of slip-out structures by FAN1, thus inducing CAG contractions, but details of this obstruction remain to be elucidated.
Dr. Pearson explains that in addition to having spectacular specificity, NA induces contractions in the majority of treated brain cells in HD mice. This is astounding feat considering that NA must cross both cellular and nuclear membranes to reach its target DNA. Moreover, Dr. Pearson and colleagues observed an improvement in motor coordination of these mice after only four weeks of treatment with NA9. Assuming the effects in mouse models can be translated into humans, the effectiveness of NA in treating repeat expansion disorders is extremely promising. Given the complexity and progressive degeneration of these conditions, NA’s rapid and effective onset of action, makes the molecule an attractive treatment option for HD individuals. While direct delivery to the central nervous system is an option, the ability for NA to cross the blood-brain barrier, which is unknown, would facilitate delivery. Further studies are needed to enhance delivery, and characterize this molecule’s tissue distribution and safety profile.
The Future of HD Therapeutics: Just Keep Fishing
According to Dr. Pearson, the first-of-its-kind approach of targeting slip-out-structures with NA has advanced the field of HD therapeutic development. However, as this approach is still in its infancy, whether NA will survive the “valley of death” – a term used to describe the hurdles of drug development – is still unknown. Dr. Pearson intends to continue improving the druggability and safety profile of NA up until its translation to the bedside: “We will do what we can to improve delivery and safety – we’re working on that now.”
Dr. Pearson’s team are investigating other potential therapeutic avenues centered upon targeting expansions – or in his terms, “fishing in multiple waters”. These approaches include identifying new DNA repair proteins involved in expansions, screening for inhibitors/modifiers of MSH3, FAN1 or other DNA repair proteins. Dr. Pearson emphasizes that “Fishing in multiple waters increases the likelihood that one of these approaches will cross the long, wide and deep valley of death” and go on to become an approved treatment for HD. Were more than one approach to succeed, combinatorial therapeutic regimens could be developed to further enhance patient outcomes. Despite current excitement and hope, Dr. Pearson acknowledges that crossing this valley is a long and challenging journey and credits the young, bright, and intelligent students and fellows in his lab for taking up the challenge.
The applicability of NA in treating repeat expansion disorders
Might the discovery of NA be applied to other repeat expansion disorders? That NA targets CAG slip-outs suggests it could act on the other 15 CAG-expansion disorders, including spinocerebellar ataxias and dentatorubral-pallidoluysian syndrome (DRPLA). Dr. Pearson and his team recently revealed that NA contracted CAG repeats and improved motor coordination in a mouse model of DRPLA8, validating the broad applicability of this approach.
Looking to the future, Dr. Pearson is expanding his focus to other repeat expansion disorders, such as amyotrophic lateral sclerosis, frontotemporal dementia, and schizophrenia. Dr. Pearson claims, “the likelihood that other repeat sequences causing other diseases are forming unusual mutagenic structures is extremely high, which is why we are searching for ligands to those”. As the field of repeat expansion disorders continues to advance, Dr. Pearson is ready to face new questions that will arise for him and his team to address.
Hoping to motivate young minds, Dr. Pearson concludes our interview by thoughtfully reminding us of the importance of pursuing interests, not career paths: “follow your nose, follow what excites your curiosity”.
1. Lu, X. H. & Yang, X. W. ‘ Huntingtin Holiday’ : Progress toward an Antisense Therapy for Huntington’s Disease. Neuron 74, 964–966 (2012).
2. Flower, M. D. & Tabrizi, S. J. A small molecule kicks repeat expansion into reverse. Nat. Genet. 52, 136–137 (2020).
3. Gall-Duncan, T., Sato, N., Yuen, R. K. C. & Pearson, C. E. Advancing genomic technologies and clinical awareness accelerates discovery of disease-associated tandem repeat sequences. Genome Res. 32, 1–27 (2022).
4. Malik, I., Kelley, C. P., Wang, E. T. & Todd, P. K. Molecular mechanisms underlying nucleotide repeat expansion disorders. Nat. Rev. Mol. Cell Biol. 22, 589–607 (2021).
5. Deshmukh, A. L. et al. FAN1, a DNA Repair Nuclease, as a Modifier of Repeat Expansion Disorders. J. Huntingtons. Dis. 10, 95–122 (2021).
6. Deshmukh, A. L. et al. FAN1 exo- not endo-nuclease pausing on disease-associated slipped-DNA repeats: A mechanism of repeat instability. Cell Rep. 37, 110078 (2021).
7. Porro, A. et al. FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats. Sci. Adv. 7, 1–13 (2021).
8. Hasuike, Y. et al. CAG repeat-binding small molecule improves motor coordination impairment in a mouse model of Dentatorubral–pallidoluysian atrophy. Neurobiol. Dis. 163, 105604 (2022).
9. Nakamori, M. et al. A slipped-CAG DNA-binding small molecule induces trinucleotide-repeat contractions in vivo. Nat. Genet. 52, 146–159 (2020).