Mutant Huntingtin (mHTT) Clearance Mechanisms — Therapeutic Target Validation

SUMMARY

# Mutant Huntingtin (mHTT) Clearance Mechanisms — Therapeutic Target Validation ## Background and Rationale Huntington's disease (HD) is caused by CAG repeat expansion in the huntingtin gene, producing mutant huntingtin (mHTT) protein that undergoes pathological processing and aggregation. The heterogeneous nature of mHTT species—including full-length protein, N-terminal fragments of varying sizes, soluble oligomers, and insoluble aggregates—presents a complex therapeutic challenge. Current evid

METHODOLOGY NOTES

Phase 1 (Weeks 1-4): Generate human iPSC-derived striatal and cortical neurons from HD patients (n=6 lines, 18-72 CAG repeats) and controls (n=3 lines). Culture neurons for 8-12 weeks to achieve mature mHTT expression patterns. Phase 2 (Weeks 5-8): Perform comprehensive mHTT species characterization using sequential biochemical fractionation (RIPA, urea, formic acid extraction), followed by Western blotting with species-specific antibodies (anti-HTT N-terminus, polyQ-specific, aggregate-preferring) and quantitative mass spectrometry. Phase 3 (Weeks 9-16): Implement therapeutic interventions in 96-well format with n=8 replicates per condition: autophagy enhancers (rapamycin 100nM, trehalose 100mM), proteasome activators (PA28γ overexpression), and huntingtin-lowering ASOs (10μM). Monitor mHTT species levels at 24h, 72h, 1 week, and 2 weeks post-treatment using automated high-content imaging and biochemical analysis. Phase 4 (Weeks 17-20): Assess functional outcomes including neuronal vi