I005 Mutant-specific lowering strategies for Huntington’s disease using HTT genetic variants

<h3></h3> Huntington’s disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. Given the dominant gain-of-function nature of this mutation and the essential role of HTT in normal development, there is a critical need for therapeutic strategies that selectively inactivate the mutant allele while preserving the normal one. Our research demonstrates a novel genetics-based precision medicine approach utilizing allele-specific CRISPR/Cas9 and antisense oligonucleotides (ASOs) to achieve this specificity. We developed a CRISPR/Cas9 strategy that targets patient-specific PAM-altering SNPs (PASs) to inactivate the mutant HTT allele. We identified suitable target sites in the largest HD genotype dataset by leveraging extensive knowledge of HTT gene haplotype structure and PAM-altering SNPs (PASs). Using dual guide RNAs (gRNAs) based on PASs, we achieved selective excision of DNA fragment, including the promoter region, transcription start site, and CAG expansion mutation of the mutant HTT gene. This excision prevented the generation of mutant HTT mRNA and protein, demonstrating complete and permanent inactivation of the mutant allele without affecting the normal allele. Further analysis identified PASs on exonic regions of common HTT haplotypes, allowing us to target a significant portion of the HD population with this approach. Specifically, SNP rs363099 on the 29th exon provided a promising target for approximately 20% of HD subjects with European ancestry, showing perfect allele specificity and efficient targeting in patient-derived cells. In parallel, we mapped haplotype-specific insertion-deletion variations (indels) to develop alternative mutant-specific strategies. Based on the 1000 Genomes Project data, our map revealed candidate indels for HTT targeting. Subsequent sequencing confirmed these sites and identified additional allele-specific indels. Treatment of patient-derived cells with ASOs targeting these indels with the most common HTT diplotype achieved complete mutant specificity. Those approaches hold significant promise for the development of safe and effective HTT-lowering therapeutics, particularly suited for early treatment in HD, thereby potentially benefiting a broad spectrum of individuals with this devastating disorder, including the Enroll-HD family.