B005 Characterisation of mouse embryonic fibroblast models of Huntington’s disease and their application as screening tools for therapeutic agents

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Background Mouse embryonic fibroblasts (MEFs) have been isolated from the widely studied Huntington’s disease (HD) mouse models zQ175 and YAC128. zQ175 knock-in mice contain a mutated version of human HTT exon 1 integrated into mouse Htt and carry approximately 175-200 CAGs. YAC128 transgenic mice express the full-length human HTT gene with an expanded CAG repeat. Both models undergo a process of alternative processing of their huntingtin genes that lead to the production of the Htt1a/HTT1a transcripts and pathogenic HTTexon1 protein. Aims To characterise the YAC128 and zQ175 MEF lines at the RNA and protein levels and their suitability as screening tools for therapeutic agents. Methods/Techniques QuantiGene assays were implemented to analyse the expression levels of full-length and intronic huntingtin transcripts and RNAscope probes designed to visualise these transcripts at a single molecular level. Pairwise combinations of huntingtin antibodies were optimised for HTRF/AlphaLISA assays to detect huntingtin isoforms. Results/Outcome The growth rates of both MEF models were assessed in several formats and PicoGreen assays were established as a proxy for cell number. QuantiGene analysis indicated the presence of Htt1a in zQ175 MEFs and HTT1a in YAC128 MEFs and RNAscope showed their distribution in YAC128 MEFs. Levels of full-length huntingtin and HTTexon1 proteins were measured by HTRF/AlphaLISA assays. These MEFs were successfully used to validate therapeutic agents (ASO, siRNA) targeting full-length or intronic huntingtin mRNAs. Conclusions These MEF lines provide a valuable tool to evaluate potential therapeutic effects of agents aimed at lowering huntingtin levels.

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