PROJECT SUMMARY
Huntington’s disease (HD) is caused by expanded trinucleotide repeats (CAG) in exon 1 of the huntingtin (HTT)
gene. Therapies lowering the downstream mutant HTT protein show limited clinical success. New evidence
reveals that repeat tract length in the HTT locus, not mutant HTT protein, correlates to disease onset/severity.
CAG repeat length is inherited, but further expands due to somatic instability, which contributes to HD
progression. Modulating somatic expansion is a key path toward treating HD. Somatic expansion occurs in non-
dividing cells like neurons when DNA repeats misalign after transcription, forming a slipped loop that activates
mismatch repair (MMR). In MMR, nuclease complexes help recognize the slipped loop and cut the non-slipped
strand to create a gap that is filled to expand the repeat. Polymorphisms in MMR complexes are linked to HD
onset, and knocking out or altering activity of MMR proteins block expansion or induce contraction in HD models.
Yet, the contribution of each MMR protein to CAG expansion, and the effect of their conditional CNS-specific
reduction on HD outcomes, is untested. Also, mechanisms favoring contraction over expansion are unknown.
This project seeks to define MMR complexes facilitating HTT CAG expansion/contraction using divalent small
interfering RNA (siRNA)—which induce potent, CNS-specific silencing of target genes—and antisense
oligonucleotides (ASOs)—which can disrupt specific protein-nucleic acid binding in the CNS.
Aim 1 will use divalent siRNA to evaluate the effects of MMR silencing on HTT CAG repeat expansion and HD
progression. Efficacies of siRNAs targeting each MMR protein have been validated in human and mouse cells.
Furthermore, one of these siRNAs was delivered to CNS of an HD mouse model, BAC-CAG (carries human HTT
with 120 CAG that undergo expansion), showing target MMR silencing and blocked somatic expansion 2 months
later. In Aim 1, divalent siRNA targeting each MMR enzyme will be injected into BAC-CAG mice. Target silencing
and HTT CAG repeat expansion will be measured 2 months later. Top siRNA that block expansion will be re-
injected into BAC-CAG mice, and the impact on motor behavior, ventricular size, and HD pathology will be
explored over 9 months. Aim 2 will develop HTT CAG-targeting ASOs to induce MMR-mediated contraction in
HD cells and mice. An initial panel of ASOs targeting HTT CAG repeats was screened in non-transformed HD
patient-derived fibroblasts (HDpFs) using a high-throughput format, and ASOs that increase contraction events
were identified. To improve contraction rates, ASO chemistries and lengths will be optimized and screened in
HDpFs using the same assay. HTT CAG repeat length/instability will be quantified over 40 days to identify leads.
Leads will be delivered to HDpFs, in combination with validated siRNA targeting each MMR protein, to identify
MMR proteins mediating ASO-induced contraction events. In parallel, in vivo efficacy of leads will be confirmed
in BAC-CAG mice. This work will reveal somatic expansion/contraction mechanisms, inform HD therapy design,
and provide the fellow with crucial training in therapeutic development, neurobiology, and bioinformatics.