Molecular features of a Huntington's disease knock-in minipig

Huntington's disease is caused by a CAG expansion in the HTT gene, leading to somatic repeat instability, alternative processing of HTT pre-mRNA, and mutant huntingtin protein production. To model these features, we generated a knock-in minipig (KI-85Q-HD) carrying a (CAG)82CAA(CAG)2 repeat in the endogenous HTT locus. To evaluate this, we quantified somatic expansion in various tissues using small pool- and bulk-PCR; detected HTT1a, an aberrantly spliced HTT transcript, using 3' rapid amplification of cDNA ends and quantitative PCR; and assessed mutant huntingtin protein isoforms using homogeneous time-resolved fluorescence assays. Moderate levels of tissue-specific and age-dependent somatic expansion were observed, highest in the caudate nucleus, kidney and spleen, and detectable in blood cells. We confirmed the presence of HTT1a transcripts terminating at a cryptic polyadenylation site in HTT intron 1, and detected soluble full-length mutant HTT and HTT1a proteins across brain regions and peripheral tissues, while aggregated HTT1a was only detected in the cortex. These results indicate that KI-85Q-HD minipigs exhibit molecular features of Huntington's disease at a pre-symptomatic stage and may serve as a platform for assessing therapeutic distribution and potency.