A009 Characterising the role of HTT transcription in somatic CAG repeat expansion

<h3>Background</h3> Ongoing somatic expansion of the HTT CAG repeat occurs throughout a Huntington’s disease (HD) patient’s lifetime, particularly in the striatum and cortex, and correlates with disease severity, progression, and onset. This suggests somatic expansion plays a vital role in HD pathogenesis and it’s hypothesised that DNA repair and transcription drive CAG expansion in post-mitotic neurons, in the absence of DNA replication. <h3>Aims</h3> This study investigated the role of transcription in CAG repeat expansion using human U2OS cells with inducible or repressed transcription of pathogenic HTT exon 1. <h3>Methods</h3> A doxycycline-inducible construct expressing HTT exon 1 with 118 CAG repeats was stably integrated into a single locus in U2OS cells, allowing controlled inducible transcription of HTT exon 1 in response to increasing doxycycline dose. To assess expansion in the absence of transcription, a construct with transcriptional repressors flanking the HTT exon 1 reading frame was engineered. <h3>Results</h3> Increasing doxycycline concentration caused a corresponding increase in exon 1 transcript and protein levels, and a stepwise increase in CAG repeat instability. No expression was observed in transcriptionally repressed cells regardless of doxycycline dose, however low-level instability persisted. <h3>Conclusions</h3> These findings suggest HTT transcription influences CAG repeat expansion. The low-level instability observed despite transcriptional repression suggests other processes, such as DNA repair or replication in these mitotic U2OS cells, also contribute to expansion. We will further assess somatic expansion after HTT transcriptional repression in HD iPSC-derived neurons. These data may provide mechanistic insights into somatic expansion and reinforce therapeutic approaches targeting HTT transcription.