Discovery of a Biomarker and Lead Small Molecules to Target r(GGGGCC)-Associated Defects in c9FTD/ALS

Abstract

Summary A repeat expansion in C9ORF72 causes frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). RNA of the expanded repeat (r(GGGGCC)exp) forms nuclear foci or undergoes repeat-associated non-ATG (RAN) translation producing “c9RAN proteins”. Since neutralizing r(GGGGCC)exp could inhibit these potentially toxic events, we sought to identify small molecule binders of r(GGGGCC)exp. Chemical and enzymatic probing of r(GGGGCC)8 indicate it adopts a hairpin structure in equilibrium with a quadruplex structure. Using this model, bioactive small molecules targeting r(GGGGCC)exp were designed and found to significantly inhibit RAN translation and foci formation in cultured cells expressing r(GGGGCC)66 and neurons trans-differentiated from fibroblasts of repeat expansion carriers. Finally, we show that poly(GP) c9RAN proteins are specifically detected in c9ALS patient cerebrospinal fluid. Our findings highlight r(GGGGCC)exp-binding small molecules as a possible c9FTD/ALS therapeutic, and suggest c9RAN proteins could potentially serve as a pharmacodynamic biomarker to assess efficacy of therapies that target r(GGGGCC)exp.

Poly(GP), neurofilament and grey matter deficits in C9orf72 expansion carriers

“…We showed that CSF poly(GP) has high power to differentiate C9orf72 expansion carriers and noncarriers, consistent with previous reports 10, 11, 14. Interestingly, poly(GP) levels were undetectable in three C9orf72 expansion carriers, who had various clinical presentations, including a presymptomatic individual, a bvFTD patient, and a PPA patient.…”

“…Disease‐modifying therapies advancing toward clinical trials include antisense oligonucleotides (ASOs) and small molecules that target G 4 C 2 transcripts and consequently reduce G 4 C 2 RNA foci and DPR proteins in C9orf72 patient‐derived cell models and animal models 9, 10, 11, 12, 13. In parallel with the rapid development of these potential therapeutics, biomarkers that measure target engagement, disease onset, and disease progression must be established for clinical trials to be successful.…”

“…Previous studies suggest that poly(GP) is a promising marker of target engagement. This protein is detectable in the cerebrospinal fluid (CSF) of presymptomatic and symptomatic C9orf72 expansion carriers,10, 11, 14 and poly(GP) levels in CSF from (G 4 C 2 ) 66 ‐expressing mice correlate with ASO‐induced decreases in G 4 C 2 RNA expression, RNA foci burden, and DPR protein levels within their brain 11. In addition, neurofilament light chain (NfL) is a potential marker of disease severity and progression for ALS, FTD, as well as other neurodegenerative diseases, including Alzheimer's disease 15, 16, 17.…”

Development of Therapeutics for C9ORF72 ALS/FTD-Related Disorders

“…Remarkable advances have already been achieved in this direction, and they are presented in detail in this review. Briefly, antisense oligonucleotides/RNA interference sequences have been used to stimulate the elimination of pathological C9ORF72 transcripts [16,21,22] and the small compounds that are able to link the secondary structure of the repeat sequence, which can thereby limit the accumulation of RNA foci and toxic DPRs [23]. Indeed, short hairpin (sh) RNA or similar nucleotidic sequences, either naked or delivered with a viral vector, can be used for the same purpose.…”

Clogging information flow in ALS

“…Assays for the abundance of the disease-associated dipeptides could be used in a clinical trial setting to monitor the efficacy of potential disease-modifying therapies that target the repeat RNA expansion. These approaches could include small molecules, as shown by Su et al (10), and antisense oligonucleotides (6,11,12). More focus on understanding the molecular mechanisms of RAN translation could reveal new and unexpected therapeutic strategies.…”