Efficient Base Editing Strategies to Convert CAG to CAA Repeats and Diminish the Disease-Causing Mutation in Huntington's Disease

Core Concepts

Base editing strategies employing combinations of cytosine base editors and guide RNAs can efficiently convert CAG to CAA in the huntingtin gene without generating significant indels, off-target edits, or transcriptome alterations, demonstrating their feasibility and specificity as a potential therapeutic approach for Huntington's disease.

Abstract

The content discusses the development and evaluation of base editing strategies to convert CAG to CAA repeats in the huntingtin gene as a potential therapeutic approach for Huntington's disease (HD).

Key highlights:

1. Human genetic data show that the length of the uninterrupted CAG repeat, not the polyglutamine length, determines the age-of-onset in HD. Introducing CAA interruptions into the CAG repeat can delay disease onset.
2. The authors tested various combinations of cytosine base editors (CBEs) and guide RNAs (gRNAs) to efficiently convert CAG to CAA in the huntingtin gene without generating significant indels or off-target edits.
3. The CBE-gRNA strategies showed high levels of CAG-to-CAA conversion, with the patterns of conversion sites depending on the specific CBE and gRNA used. Some strategies also generated modest levels of duplicated CAA-CAG interruptions.
4. The base editing strategies did not alter huntingtin mRNA or protein levels, and RNAseq analysis confirmed the lack of significant transcriptome changes.
5. In HD knock-in mouse models, a candidate base editing strategy significantly decreased somatic CAG repeat expansion, which is a major driver of HD pathogenesis. Mice carrying CAA-interrupted repeats showed complete abolishment of repeat expansion.
6. The authors developed a HEK293 cell line carrying an expanded HTT CAG repeat to further characterize the allele specificity and molecular outcomes of the base editing strategies.

Overall, the results demonstrate the feasibility and therapeutic potential of base editing approaches to convert CAG to CAA repeats in the huntingtin gene for the treatment of Huntington's disease.

Stats

The length of the uninterrupted CAG repeat, not the polyglutamine length, determines the age-of-onset in Huntington's disease.
Introducing CAA interruptions into the CAG repeat can delay disease onset.
Base editing strategies converted 27.7% of CAG to CAA at the 2nd CAG position and up to 30% at other sites.
Somatic CAG repeat expansion in HD knock-in mice was significantly decreased by a candidate base editing strategy.
Repeat expansion was entirely abolished in HD knock-in mice carrying CAA-interrupted repeats.

Quotes

"Since the length of uninterrupted CAG repeat, not polyglutamine, determines the age-at-onset in HD, base editing strategies to convert CAG to CAA are anticipated to delay onset by shortening the uninterrupted CAG repeat."
"Notably, CAG repeat expansion was abolished entirely in HD knock-in mice carrying CAA-interrupted repeats, supporting the therapeutic potential of CAG-to-CAA conversion base editing strategies in HD and potentially other repeat expansion disorders."

Key Insights Distilled From

Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington's disease

by Choi,D. E., ... at www.biorxiv.org 04-28-2023

https://www.biorxiv.org/content/10.1101/2023.04.28.538700v1

Deeper Inquiries

How could the base editing strategies be further optimized to achieve higher conversion efficiencies and allele specificity in patient-derived cells and tissues?

To enhance base editing strategies for improved conversion efficiencies and allele specificity in patient-derived cells and tissues, several optimization approaches can be considered:

gRNA Design: Designing gRNAs with high specificity and efficiency is crucial. Utilizing bioinformatics tools to predict off-target sites and selecting gRNAs with minimal off-target effects can enhance allele specificity.

Base Editor Engineering: Continuous advancements in base editor engineering can lead to the development of more efficient and specific base editors. Fine-tuning the components of base editors, such as the deaminase domain and Cas protein, can improve editing precision.

Delivery Methods: Optimizing delivery methods for base editors to ensure efficient transfection or transduction in patient-derived cells is essential. Utilizing viral vectors or nanoparticle-based delivery systems can enhance the uptake and expression of base editing components.

Cell Type Optimization: Understanding the specific characteristics of patient-derived cells and tissues and optimizing base editing protocols accordingly can improve conversion efficiencies. Factors such as cell cycle stage, chromatin accessibility, and DNA repair mechanisms can impact editing outcomes.

Validation and Screening: Rigorous validation of base editing outcomes through deep sequencing and functional assays is necessary to assess conversion efficiencies and allele specificity. High-throughput screening methods can help identify optimal base editing conditions for different cell types.

Safety Considerations: Considering the potential off-target effects and long-term safety concerns, incorporating safety features into base editing systems, such as self-inactivation mechanisms or inducible expression systems, can mitigate risks associated with off-target editing.

What are the potential off-target effects and long-term safety concerns of using base editing for Huntington's disease treatment, and how can these be addressed?

Potential off-target effects and long-term safety concerns associated with base editing for Huntington's disease treatment include:

Off-Target Editing: Off-target editing can lead to unintended mutations in non-target genomic loci, posing risks to the overall genome integrity. Utilizing advanced bioinformatics tools for off-target prediction and experimental validation of potential off-target sites can help mitigate this risk.

Immunogenicity: The immune response to base editing components or edited cells can impact the safety and efficacy of the treatment. Conducting thorough immunogenicity studies and monitoring immune responses in preclinical and clinical settings can address this concern.

Genomic Stability: Prolonged expression of base editing components may affect genomic stability and cell viability over time. Implementing strategies for controlled expression of base editors, such as inducible systems or transient delivery methods, can minimize long-term safety risks.

Insertional Mutagenesis: If viral vectors are used for base editor delivery, the risk of insertional mutagenesis should be considered. Choosing non-integrating vectors or integrating them into safe genomic loci can reduce this risk.

Ethical Considerations: Ethical considerations regarding the use of genome editing technologies in human subjects, including informed consent, privacy, and equity in access to treatment, should be carefully addressed.

Addressing these concerns involves a comprehensive risk assessment, adherence to regulatory guidelines, continuous monitoring of treatment outcomes, and transparent communication with stakeholders.

Given the potential therapeutic benefits of CAG-to-CAA conversion, could similar base editing approaches be applied to other trinucleotide repeat disorders beyond Huntington's disease?

The therapeutic potential of CAG-to-CAA conversion in Huntington's disease suggests that similar base editing approaches could be applied to other trinucleotide repeat disorders, such as:

Spinocerebellar Ataxias (SCAs): SCAs are a group of neurodegenerative disorders caused by CAG repeat expansions. Base editing strategies targeting the expanded CAG repeats in genes associated with SCAs could potentially mitigate disease progression.

Myotonic Dystrophy (DM): DM is caused by CTG repeat expansions in specific genes. Adapting base editing techniques to convert CTG repeats to CAG repeats or interrupting the repeat structure could offer therapeutic benefits in DM.

Fragile X Syndrome: Fragile X Syndrome results from CGG repeat expansions in the FMR1 gene. Base editing to modify the CGG repeats or introduce interruptions in the repeat sequence may hold promise for treating this disorder.

Amyotrophic Lateral Sclerosis (ALS): Some forms of ALS are associated with hexanucleotide repeat expansions. Applying base editing to target and modify the expanded repeats could potentially address the underlying genetic cause of ALS.

By customizing base editing strategies to target the specific trinucleotide repeat sequences implicated in these disorders, similar therapeutic benefits to those observed in Huntington's disease may be achievable. However, careful consideration of the unique genetic and molecular characteristics of each disorder is essential for the successful application of base editing approaches.