Droplet digital PCR assay to analyze allele-specific mRNA expression on HTT repeat expansion locus

Background: Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a mutation in the huntingtin gene (HTT), characterized by an expanded CAG trinucleotide repeat. At the time of writing no cure or disease-modifying treatments exist. Currently, the most explored investigational therapeutic strategy targets HTT gene expression, either lowering both alleles (total lowering) or selectively lowering the mutant allele. These approaches require reliable pharmacodynamic biomarkers to measure an allele-selective knockdown. However, allele specific quantification of wild-type and mutant HTT RNA or protein remains a challenge. Results: Here we optimized a droplet digital PCR (ddPCR) assay to distinguish between mutant and wild-type HTT (mHTT and wtHTT respectively) mRNA expression based on differential amplification of HTT mRNA molecules with different CAG repeat lengths under limited dNTP conditions. This assay, combined with our novel automated analysis pipeline reliably detects allele-specific expression in HD patient cell lines. We simulated various mHTT to wtHTT mRNA ratios by mixing RNA from respective homozygous cell lines to demonstrate the assay's accuracy under varying allele ratios. We also validated the assay's utility in 13 cell lines from HD patients and their family members. Additionally, we optimized a one-step RT-ddPCR method, offering a streamlined alternative to a two-step ddPCR method. We further confirmed the assay's clinical relevance by demonstrating allele-selective siRNA mediated HTT lowering in HD patient fibroblast cell lines. Conclusions: Our optimized ddPCR assay, with its pipeline for automated data analysis, enables the precise quantification of allele-selective HTT mRNA knockdown. The method does not require prior knowledge of patients' SNP genotypes, previously a prerequisite for assays aiming to determine the mHTT transcript expression in patient samples. Our HTT ddPCR assay is universally applicable regardless of patient genotype. The ability to accurately monitor allele-specific HTT mRNA expression levels holds great promise for developing effective treatments for Huntington's disease.