Biology subjects

Kelley, C. P.

Publications and source records attributed to Kelley, C. P..

3 recordsLinked to original sources

bioRxiv2025

Interventionally targeting somatic CAG expansions can be a rapid disease-modifying therapeutic avenue: Preclinical evidence.

Huntington disease (HD) is caused by inherited CAG expansions, which continue expanding somatically in affected brain regions to hasten disease onset and progression. Therapeutically diminishing somatic expansions is expected to be clinically beneficial. However, it is not known if interventionally modifying somatic CAG expansions will actually modify in vivo clinically-relevant phenotypes, what the therapeutic window is, or which phenotypes will be altered. Here we show that acute (6-week) delivery of the contraction-inducing slipped-CAG DNA ligand naphthyridine-azaquinolone to young (4-week-old) (CAG)120 HD mice, induces contractions throughout brain regions, improves motor function (locomotion, balance, coordination, muscle strength), molecular disease landmarks (mHTT aggregates, nuclear envelope morphology, nucleocytoplasmic mRNA transport, transcriptomic dysregulation, neuroinflammation), and neurodegeneration. Beneficial effects of modifying somatic expansions were also evident in muscle and blood, where blood CAG instability correlated with brain instability and blood serum had diminished levels of neurofilament light (a biomarker for neurodegeneration) -- offering blood as having elements of target engagement and efficacy. These data support that targeting somatic repeat expansions can be a rapid disease-modifying therapeutic avenue for HD and possibly other repeat expansion diseases. Our findings support an etiologic pathway interconnected to somatic CAG expansions that will inform the design of clinical trials expecting clinical benefit by modulating somatic expansions.

genetics↗

bioRxiv2021

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

Cas13 is a unique family of CRISPR endonucleases exhibiting programmable binding and cleavage of RNAs and is a strong candidate for eukaryotic RNA knockdown in the laboratory and the clinic. However, sequence-specific binding of Cas13 to the target RNA unleashes non-specific bystander RNA cleavage, or collateral activity, which may confound knockdown experiments and raises concerns for therapeutic applications. Although conserved across orthologs and robust in cell-free and bacterial environments, the extent of collateral activity in mammalian cells remains disputed. Here, we investigate Cas13d collateral activity in the context of an RNA-targeting therapy for myotonic dystrophy type 1, a disease caused by a transcribed long CTG repeat expansion. We find that when targeting CUGn RNA in HeLa and other cell lines, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis pathways. We also observe collateral effects when targeting other repetitive and unique transgenic sequences, and we provide evidence for collateral activity when targeting highly expressed endogenous transcripts. To minimize collateral activity for repeat-targeting Cas13d therapeutics, we introduce gRNA excision for negative-autoregulatory optimization (GENO), a simple strategy that leverages crRNA processing to control Cas13d expression and is easily integrated into an AAV gene therapy. We argue that thorough assessment of collateral activity is necessary when applying Cas13d in mammalian cells and that implementation of GENO illustrates the advantages of compact and universally robust regulatory systems for Cas-based gene therapies. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2021

Microtubule-based Transport is Essential to Distribute RNA and Nascent Protein in Skeletal Muscle

While the importance of RNA localization in highly differentiated cells is well appreciated, basic principles of RNA localization in skeletal muscle remain poorly characterized. Here, we develop a method to detect single RNA molecules and quantify localization patterns in skeletal myofibers, and we uncover a critical and general role for directed transport of RNPs in muscle. We find that RNAs are localized and translated along cytoskeletal filaments, and we identify the Z-disk as a biological hub for RNA localization and protein synthesis. We show that muscle development triggers complete reliance on the lattice-like microtubule network to transport RNAs and that disruption of microtubules leads to striking accumulation of RNPs and nascent protein around myonuclei. Our observations suggest that active transport may be globally required to distribute RNAs in highly differentiated cells and reveal fundamental mechanisms relevant to myopathies caused by perturbations to RNPs, microtubules, and the nuclear envelope. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/433059v2_ufig1.gif" ALT="Figure 1"> View larger version (88K): org.highwire.dtl.DTLVardef@1d801ddorg.highwire.dtl.DTLVardef@625623org.highwire.dtl.DTLVardef@54230org.highwire.dtl.DTLVardef@13c95be_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗