Modulation of Somatic Repeat Expansion as a Therapeutic Approach to Huntington's Disease

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.