The most common genetic form of amyotrophic lateral sclerosis (ALS), called C9FTD/ALS, poses significant challenges to therapeutic development. It is caused by a repeat expansion in the first intron of the C9ORF72 gene that is composed of hundreds to thousands of tandem GGGGCC repeats. No consistently unique sequence features, other than the repeat expansion itself, are available for targeting since normal alleles also possess a handful of the repeats. Proposed mechanisms of disease all begin with repeat RNA, which is transcribed from this genomic locus. The expanded tandem repeat-containing RNA, or xtrRNA, is implicated in multiple disease mechanisms and can be further translated into repetitive polypeptides that are known to be toxic. These molecular events contribute to motor neuron degeneration in ALS and their complexity further complicates identification of promising pharmacologic drug targets. While the molecular disease mechanisms that lead to pathology will take years to understand, one immediate path to a potential treatment is preventing production of the xtrRNA itself. Here, we propose to exploit the nucleotide composition and size of the C9ORF72 repeat expansion to allele-selectively reduce transcription. Reducing or blocking transcription of one C9ORF72 allele is expected to be relatively safe since complete knock-out in mice only shows mild immunologic effects and no overt signs of neurological disease. To slow or block transcription of the C9ORF72 xtrRNA, we propose to select and screen a small library of molecules that are known to impede transcription, including several previously investigated as therapeutics for other diseases. In addition, to test our hypothesis and perform a more thorough systematic analysis, we will synthesize several new molecules. These molecules meet a number of criteria that are deemed necessary for success of the approach. Using in vitro and cell-based assays, we will identify small molecules that can slow or block transcription in a C9ORF72 repeat expansion-dependent manner. These molecules are expected to reduce focal aggregates of xtrRNA in patient-derived cells and allele-selectively block the C9ORF72 disease-associated allele. We will also characterize the ability of these molecules to affect the expression of other genes, as well as transcription by mitochondrial RNA polymerase, both common sources of cellular toxicity with this class of drugs. If successful, this study should identify one or more small molecules for continued development, including thorough mechanistic studies, molecular binding simulations, and animal model testing.
The leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), both progressive neurological disorders with no effective treatments, is a repeat expansion mutation in the C9ORF72 gene. The mutation results in the production of repeat expansion RNA that initiates the molecular cascade of pathology, thereby causing disease. This study will investigate a common class of small molecules for their therapeutic potential to block production of C9ORF72 repeat expansion RNA.