This abstract was selected by our Scientific Committee to pitch on the programme in the R2B session (PM).
Huntington’s disease (HD) is a neurodegenerative disease that is caused by an expansion of the CAG-repeat in the Huntingtin gene (HTT). The most profound effect of the mutant form of the HTT protein is the degeneration of striatal projection neurons, leading to progressive loss of motor and cognitive function. Targeting HTT mRNA is showing promising results in a Phase 1/2 trial in Huntington’s disease (HD) patients, while targeting the splicing machinery proved to lower HTT protein levels in an HD animal model. This indicates that (m)HTT mRNA can be considered as an important therapeutic target for HD. Phenotypic screening efforts focusing on HTT mRNA as target require a robust and high-throughput assay.
Here we aim to develop a highly sensitive ViewRNA assay able to detect total and allele-specific HTT mRNA levels in human embryonic stem cells (hESC) and in hESC-derived neurons. Using a probe set targeting HTT exons 42-47, we observed siRNA-mediated depletion of HTT mRNA in GEN020 hESCs, which was confirmed on the protein level. PTC compound-mediated knockdown of HTT mRNA demonstrated a concentration-dependent response as detected by ViewRNA and confirmed by RT-qPCR. To detect allele-specific HTT mRNA we barcoded HTT exon 12 in silico by introducing silent mutations. Specificity of the probe sets designed for wild-type (wt)HTT exon 12 and the barcoded (bc) version was confirmed in GEN020 hESC transiently expressing either wt or bc HTT exon 12 mRNA. Moreover, endogenous HTT mRNA levels were only detected with the wt exon 12 probe set. Although this probe set targets a significantly smaller region within HTT than the exon 42-47 probe set, similar siRNA and compound-mediated HTT mRNA depletion was detected for this custom-designed probe set.
Taken together, we demonstrated that total and allele-specific HTT mRNA expression levels can be robustly detected using ViewRNA technology in hESCs. Our future efforts focus on further optimization of the ViewRNA protocol for high-throughput assays and transferring these assays from hESCs to hESC-derived neurons. Barcoding of exon 12 by gene editing is performed to generate a hESC line to be able to screen for endogenous HTT mRNA modulation in an allele-specific manner.