A CTG repeat-selective chemical screen identifies microtubule inhibitors as selective modulators of toxic CUG RNA levels.

A CTG repeat expansion in the DMPK gene is the causative mutation of myotonic dystrophy type 1 (DM1). Transcription of the expanded CTG repeat produces toxic gain-of-function CUG RNA, leading to disease symptoms. A screening platform that targets production or stability of the toxic CUG RNA in a selective manner has the potential to provide new biological and therapeutic insights. A DM1 HeLa cell model was generated that stably expresses a toxic r(CUG)480 and an analogous r(CUG)0 control from DMPK and was used to measure the ratio-metric level of r(CUG)480 versus r(CUG)0. This DM1 HeLa model recapitulates pathogenic hallmarks of DM1, including CUG ribonuclear foci and missplicing of pre-mRNA targets of the muscleblind (MBNL) alternative splicing factors. Repeat-selective screening using this cell line led to the unexpected identification of multiple microtubule inhibitors as hits that selectively reduce r(CUG)480 levels and partially rescue MBNL-dependent missplicing. These results were validated by using the Food and Drug Administration-approved clinical microtubule inhibitor colchicine in DM1 mouse and primary patient cell models. The mechanism of action was found to involve selective reduced transcription of the CTG expansion that we hypothesize to involve the LINC (linker of nucleoskeleton and cytoskeleton) complex. The unanticipated identification of microtubule inhibitors as selective modulators of toxic CUG RNA opens research directions for this form of muscular dystrophy and may shed light on the biology of CTG repeat expansion and inform therapeutic avenues. This approach has the potential to identify modulators of expanded repeat-containing gene expression for over 30 microsatellite expansion disorders.

Early-Onset Parkinson's Disease: A Novel Deletion Comprising the DJ-1 and TNFRSF9 Genes.

Patients with mutations in DJ-1 have early-onset Parkinson's disease and slow progression. Here we describe a Turkish family with a large deletion in the neighboring genes DJ-1 (del exons 1-5) and TNFRSF9 (del exons 1-6), raising the question if TNFRSF9 is a possible disease modifier.

Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells.

CRISPR-Cas13 RNA endonucleases show promise for programmable RNA knockdown. However, sequence-specific binding of Cas13 unleashes non-specific bystander RNA cleavage, or collateral activity, raising concerns for experiments and therapeutic applications. Although robust in cell-free and bacterial environments, collateral activity in mammalian cells remains disputed. We investigate Cas13d collateral activity in a therapeutic context for myotonic dystrophy type 1, caused by a transcribed CTG repeat expansion. We find that, when targeting CUGn RNA in mammalian cells, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis. Collateral effects also occur when targeting abundant endogenous transcripts. To minimize collateral activity for repeat-targeting approaches, we introduce GENO, an adeno-associated virus-compatible strategy that leverages guide RNA processing to control Cas13d expression. We argue that thorough assessment of collateral activity is necessary when applying Cas13 in mammalian cells and that GENO illustrates advantages of compact regulatory systems for Cas-based gene therapies.