DEVELOPMENT AND OPTIMISATION OF A QUANTIGENE ASSAY TO MEASURE HTT TRANSCRIPTS LEVELS AND INVESTIGATE THE EFFICIENCY OF LOWERING HTT IN VIVO

Background Huntington’s Disease (HD) is neurodegenerative disorder caused by the expansion of a CAG repeat in the HTT gene. We have recently shown that incomplete splicing of exon 1 to exon 2 of the HTT (human) and Htt (mouse) mRNAs produces a polyadenylated transcript that contains exon 1 and a short sequence of intron 1, that is translated to an exon 1 HTT protein. Measurement of the abundance of these transcripts has relied on quantitative RT-PCR assays (qPCR), which are time consuming and involve many steps (i.e. RNA isolation, reverse transcription and amplification). Assays employing branched DNA technology to detect nucleic acids involve less steps, can be multiplexed and are commercially available. Aims The aim of this study was to develop and optimize a Quantigene assay to measure concomitantly all Htt or HTT transcripts in a timely and efficient manner. Methods/techniques We designed probes to develop multiplex assays for mouse Htt and human HTT. In each case, the location of the probe sets within exons, introns and the 3’UTR were designed to identify all possible Htt or HTT transcripts. The zQ175 knock-in HD mouse model at 2 months of age was used to validate the mouse multiplex assay and fibroblasts from HD patients and control samples were used for the human assay. Results/outcome We optimized our mouse Quantigene assay and show that it generates comparable data to the much more time-consuming qPCRs. We show that transcription of Httexon1 stops after the second polyA site within intron 1. We also measured differences in the Htt 3’UTR transcripts. The human multiplex Quantigene assay needs to be further optimized as to date, only the full length HTT transcript has been detected, in contrast to our qPCR data. Conclusions We have developed and optimized a multiplex Quantigene assay to rapidly measure all mouse Htt transcripts directly in tissue lysates. This assay can be used for investigating levels of the Htt transcripts in vivo after administration of therapeutic agents. This work is supported by the CHDI foundation.