Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice

Abstract

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expanded CAG repeats in the huntingtin (HTT) gene. Although several palliative treatments are available, there is currently no cure and patients generally die 10-15 y after diagnosis. Several promising approaches for HD therapy are currently in development, including RNAi and antisense analogs. We developed a complementary strategy to test repression of mutant HTT with zinc finger proteins (ZFPs) in an HD model. We tested a "molecular tape measure" approach, using long artificial ZFP chains, designed to bind longer CAG repeats more strongly than shorter repeats. After optimization, stable ZFP expression in a model HD cell line reduced chromosomal expression of the mutant gene at both the protein and mRNA levels (95% and 78% reduction, respectively). This was achieved chromosomally in the context of endogenous mouse HTT genes, with variable CAG-repeat lengths. Shorter wild-type alleles, other genomic CAG-repeat genes, and neighboring genes were unaffected. In vivo, striatal adeno-associated virus viral delivery in R6/2 mice was efficient and revealed dose-dependent repression of mutant HTT in the brain (up to 60%). Furthermore, zinc finger repression was tested at several levels, resulting in protein aggregate reduction, reduced decline in rotarod performance, and alleviation of clasping in R6/2 mice, establishing a proof-of-principle for synthetic transcription factor repressors in the brain.

Delivering a disease-modifying treatment for Huntington's disease

“…Furthermore, ZFN-based strategies have also reached the clinical phase for the treatment of glioblastoma targeting the glucocorticoid receptor gene as part of a T cell-based cancer immunotherapy (ClinicalTrial.gov Identifier: NCT01082926). In the specific case of HD, it has recently been demonstrated that zinc finger proteins (ZFP) are able to silence effectively muHTT, without affecting the expression of wtHTT, in vitro and in the R6/2 mouse brain (approximately 40% reduction in muHTT) [116]. In this study, ZFP repressors were delivered intraparenchymally using an AAV delivery system, resulting in significant improvements in HD-related neuropathology and motor deficits [116].…”

“…In the specific case of HD, it has recently been demonstrated that zinc finger proteins (ZFP) are able to silence effectively muHTT, without affecting the expression of wtHTT, in vitro and in the R6/2 mouse brain (approximately 40% reduction in muHTT) [116]. In this study, ZFP repressors were delivered intraparenchymally using an AAV delivery system, resulting in significant improvements in HD-related neuropathology and motor deficits [116]. Although these technologies are still in their infancy, they hold great promise not only for HD, but also other monogenic disorders.…”

Enabling functional genomics with genome engineering

“…The precise genetic repression of detrimental genes may be a useful strategy to mitigate pathology, such as repressing the huntingtin GUIDE-seq Digenome-seq (Kim et al 2015) BLESS (Ran et al 2015) DNase-seq/ATAC-seq gene for Huntington's disease (Garriga-Canut et al 2012). Similarly, targeted activation of aberrantly silenced endogenous loci may provide therapeutic benefits (Lara et al 2012).…”

Orphan drugs in development for Huntington's disease: challenges and progress

“…23 Long ZFPs have been prepared to bind CAG repeats in a selective way and to be expressed in an HD cell-line Burgunder model, with the effect of a decreased expression of the longer allele, and this was also achieved after striatal delivery using an adenoassociated virus system. 24 There are major hurdles in the path toward HTT gene silencing in humans; they include finding the appropriate delivery process, choice of target and tissue volume to be included, and timing of treatment along the course of evolution. Modes of application may include stereotactic intrastriatal delivery in the form of injections or infusions, which can be combined with convection-enhancing processes or with carriers facilitating diffusion, intraventricular injections or continuous delivery with pumps, and viral vectors or other cargo systems with selective brain targeting.…”