Base editing as a therapeutic strategy for somatic repeat expansion diseases

Abstract

CRISPR-Cas base editing of trinucleotide repeats shows promise in reducing somatic repeat expansions in Huntington’s disease and Friedreich’s ataxia, offering a potential new therapeutic strategy.

Matuszek et al. have significantly advanced the study of genetic neurodegenerative disorders by employing base editing techniques to reduce somatic repeat expansions linked to Huntington’s disease (HD) and Friedreich’s ataxia (FRDA). Their research, published in Nature Genetics,¹ showcases several robust base editing methodologies that precisely modify trinucleotide repeats (TNRs), which serve as the primary causative factors of these disorders.

Specific expansions of TNRs cause both HD and FRDA: HD is linked to the HTT gene, while FRDA involves the FXN gene. These expansions have a range of pathologic mechanisms including the production of toxic proteins, leading to neurological decline. Given that treatments employing oligonucleotides and small molecules have not effectively addressed these root, genetic causes, the findings from this study are particularly promising.

This study highlights significant progress in somatic cell genome editing that the HD and FRDA communities have eagerly anticipated. For HD, it raises the essential question of whether directly editing the CAG repeats can mitigate the harmful effects of the mutant Huntingtin (mHTT) gene. Interrupting long stretches of CAG repeats by a CGG repeat can change the stability of the TNR. Research from the Liu and Mouro-Pinto labs offers compelling evidence that focusing on CAG arrays is a practical therapeutic strategy within preclinical models (Figure 1), noting that simply reducing overall mHTT mRNA might be less impactful than directly modifying the CAG DNA repeats.

Figure 1. Treating neurodegenerative diseases like Huntington’s disease (HD) and Friedreich’s ataxia (FRDA) by direct CRISPR base editing of trinucleotide repeats (TNRs).

Cytosine base editing (CBE) makes critical changes in the mutant HTT, while adenine base editing (ABE) made critical changes in mutant FXN causing FRDA. Within 24 weeks, mice treated with adeno-associated viral vector-9 (AAV9) delivering a base editor resulted in a reduction of harmful repeat expansions within the brain by about 2–4 repeats in murine models of HD and 5–7 repeats in murine models of FRDA. The full dataset presented in Matuszek et al.

The team employed a variety of base editing enzymes, optimized through directed evolution, achieving efficient editing of TNR alleles (Figure 1). Comprehensive whole genome sequencing and assessments of off-target effects in human and mouse models provided crucial insights for future therapeutic development. Importantly, the research showed a substantial reduction in repeat expansions in mouse models (Figure 1), highlighting significant therapeutic promise. The purity of the repeats can cause instability during transcription of the repeat region: even one CGG in many CAG repeats can delay expansion.

A major strength of this study lies in its holistic approach by combining models within patient-derived cells and in mice. The team meticulously performed optimization of the editing strategy in human cell lines, testing of lead strategies in patient-derived cells, and then in vivo delivery in mice via clinically-relevant, adeno-associated viral vectors (AAVs). The team’s utilization of multiple base editing enzymes and thoroughly characterizing genomic outcomes establishes a solid foundation for future investigations. Merging the findings for HD and FRDA into a single article emphasizes the shared technological advancements and analyses, reinforcing the broader applicability of this method.

Despite the promise of this technology, several hurdles must be overcome before it can be used clinically. The current study lacks functional evidence demonstrating the cessation or reversal of disease traits in these model systems, which is crucial for establishing therapeutic efficacy. Additionally, a thorough safety evaluation of base editing is imperative, particularly regarding off-target effects and the risk of unintended genomic modifications.

In particular, further preclinical development could mitigate the genome-wide effects arising from the single-strand DNA breaks, or nicks, associated with base editing techniques. Promising strategies include transient nonviral delivery systems^2–4 with multiple dosing and self-restricting strategies^5,6 for viral delivery systems. More cell type-specific promoters could be utilized to limit editing activity to select tissues or cell types, sparing especially sensitive cells like those in the eyes and heart. Careful analysis of patient-derived samples or tissue-engineered models could provide valuable guidance.⁷

In conclusion, this team’s research represents a significant advancement in developing therapies for HD and FRDA. These studies demonstrate the power of allele-specific editing, indicating that complete restoration of the healthy allele sequence may not be needed. Instead, therapeutic effects from editing the pathogenic allele, through G•C>A•T or A•T>G•C transversions, indels, deletions, or inversion could have significant therapeutic effects. For instance, the very first in vivo somatic cell editing trial, BRILLIANCE, in the eye featured deletions and inversions without fixing the CEP290 point mutation. While challenges remain, patients with TNR somatic expansions may see the prospect of a new clinical trials in the coming years, utilizing a variety of editing techniques, including the base editing ones elegantly designed by Matuszek et al. Ongoing research with this versatile platform will likely uncover further insights into possible therapeutic targets for halting TNR somatic expansion.