Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, characterized by abnormalities of movement, cognition and emotion, with relentless progression until death ~20 years after disease onset. HD is caused by an expanded CAG repeat in exon 1 of the huntingtin (HTT) gene. Disease pathogenesis is largely a result of expression of the mutant transcript and protein, which have neurotoxic properties. Suppressing the expression of the mutant allele is therefore a promising therapeutic approach, thus far pursued in cell and animal models with antibody, oligonucleotide and siRNA strategies. As with any knockdown approach, especially in the CNS, problems of delivery, reversibility, and off-target effects using these methods remain unsolved. Surprisingly, the regulation of HTT expression has received little attention: the exact promoter region of HTT was just identified by Wang et al this year, and only a few transcription factors had been shown to regulate HTT expression. On the other hand, we have recently discovered a transcript, huntingtin antisense (HTTAS_v1), generated from the strand antisense to HTT at the HD locus. HTTAS_v1 down-regulates HTT expression, while its own expression is regulated by repeat length. We therefore hypothesize that small molecule-induced down regulation of HTT expression or upregulation of HTTAS_v1 expression may provide direct and powerful approaches to HD therapy, with the potential of avoiding some of the difficulties inherent in approaches to HTT suppression that require administration of exogenous nucleic acids. Here, we propose a pilot high throughput screen (HTS) of small compounds to suppress HTT expression, assayed by measuring the effect of the small molecules on the HTT and HTTAS_v1 promoters. This study will be performed in collaboration with the National Center for Advancing Translational Sciences (NCATS, Dr. Marc Ferrer, Team Leader).
In specific aim 1, we will engineer cell lines for use in HTS. First, we will first separately clone the optimal promoter regions of HTT and HTTAS_v1 into a coincidence reporter vector in which promoter activity can be measured by both Renilla and firefly luciferase simultaneously. The construct was developed at the NIH specifically for HTS assays. We will then generate stable cell lines (using the Flp-inTM T-RexTM system from Invitrogen) containing the HTTAS_v1 promoter or the HTT promoter fused to the reporter construct, as well as control lines.
In Specific Aim 2, NCATS will screen ~3800 compounds previously approved by the FDA for human use. We will test compounds that screen positive to determine their effect on endogenous levels of HTT in cell lines and in fibroblasts from HD patients. Our immediate goal is to validate methods that can then be applied to a large scale HTS of >400,000 small molecules. Our long term goal is to use this method to yield small molecules that can be used as therapy in HD.
Huntington's disease (HD) is a devastating adult-onset hereditary neurodegenerative disorder caused by a mutation in the gene huntingtin. There is no treatment that can stop the inexorable progression of the disease to death. We propose a pilot experiment to find small molecules that can directly or indirectly suppress expression of mutant huntingtin, with the potential of slowing or stopping the progression of the disease.