Abstract Body

Repeat associated non-AUG (RAN) proteins have been found in a growing number of neurological diseases. We recently showed that targeting RAN proteins using antibodies or decreasing RAN translation with PKR-K296R or metformin improved behavior and increased motor neuron survival in C9orf72 ALS/FTD BAC transgenic mice. These data demonstrate RAN proteins play a central role in C9orf72 ALS/FTD and identify novel strategies to treat RAN-protein diseases.

We now show novel RAN proteins accumulate in spinocerebellar ataxia type 1, 2, 3, 6 and 7 brains. The growing number of RAN-protein diseases and the high percentage (~50%) of repetitive DNA in the human genome raises the possibility that RAN proteins contribute to additional common neurodegenerative diseases. We developed a novel approach that enables identification of microsatellite expansion mutations directly from patient samples. This Cas9-based repeat enrichment and detection (dCas9READ) method, uses RAN-protein aggregate signatures and deactivated clustered regularly interspaced short palindromic repeat associated protein 9 (dCas9) to pull-down candidate RAN-protein producing expansion mutations. dCas9READ works on the principle that expansion mutations provide additional binding sites for repeat-containing single guide RNA (sgRNA)-dCas9 complexes compared to normal alleles. Repeat expansions are enriched using biotin-streptavidin and identified by sequencing. dCas9READ successfully enriched C9orf72 G4C2 (n=4) and DM2 CCTG (n=4) expansion mutations and their corresponding flanking sequences directly from the genomic DNA of all eight patient samples but none of the six expansion-negative controls. These data establish dCas9READ as a novel tool that can be used to identify novel RAN protein diseases that may respond to metformin or other therapies that target RAN translation.