Specificity assessment of allele selective Zinc Finger Protein Repressors in HD fibroblasts

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range, while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and at least 9 months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological, and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD. Four treatments (ZFP-A, ZFP-B, ZFP-C, control) were tested in HD fibroblasts (GM02151). For each treatment, 6 replicate transfections were conducted yielding 6 biological replicates. Cells were harvested 24 hours later and processed for total RNA isolation and Affymetrix sample Prep. The control treatment was a non-CAG targeted ZFP-TF. One control-treated replicate was excluded from the analysis based on an exploratory grouping analysis which identified it as an outlier.