Biology subjects

McCallister, T. X.

Publications and source records attributed to McCallister, T. X..

4 recordsLinked to original sources

bioRxiv2025

Treatment of Huntington's disease with a pan-HTT-targeting CRISPR nuclease

Huntingtons disease (HD) is an inherited neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene, which leads to a mutant protein that destroys neurons in the brain. Despite intense effort, there remains no approved disease-modifying therapy for HD. Here we develop a pan-HTT-targeting CRISPR-Cas9 system that, when delivered to the striatum of R6/2 and YAC128 mice by AAV5, lowered mutant HTT mRNA and protein by 55-80% via its induction of frameshift-inducing indel mutations in HTT exon 1. Cas9 targeting improved motor coordination and locomotor activity, decreased anxiety-like deficits, reduced clasping and weight loss, limited striatal atrophy, and decreased the formation of intranuclear inclusions immunoreactive for the mutant HTT protein. In Hu21/21 mice, which carry the wild-type human HTT gene in lieu of the mouse ortholog, Cas9 lowered the HTT protein by 44% but induced no measurable behavioral deficits and had no adverse effect on neuronal viability, though its targeting was associated with neuroinflammation. Altogether, our results demonstrate the ability for a newly developed pan-HTT-targeting Cas9 system to affect HD-related phenotypes across models and provides insights into its tolerability.

bioengineering↗

bioRxiv2023

A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

An abnormal expansion of a GGGGCC hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we developed a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform effectively curbed the expression of the GGGGCC repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci and reversed transcriptional deficits. This high-fidelity Cas13 variant possessed improved transcriptome-wide specificity compared to its native form and mediated efficient targeting in motor neuron-like cells derived from a patient with ALS. Our results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.

bioengineering↗

bioRxiv2022

CRISPR base editing of cis-regulatory elements enables target gene perturbations

CRISPR technology has demonstrated broad utility for controlling target gene expression; however, there remains a need for strategies capable of modulating expression via the precise editing of non-coding regulatory elements. Here we demonstrate that CRISPR base editors, a class of gene-modifying proteins capable of creating single-base substitutions in DNA, can be used to perturb gene expression via their targeted mutagenesis of cis-acting sequences. Using the promoter region of the human huntingtin (HTT) gene as an initial target, we show that editing of the binding site for the transcription factor NF-{kappa}B led to a marked reduction in HTT gene expression in base-edited cell populations. We found that these gene perturbations were persistent and specific, as a transcriptome-wide RNA analysis revealed minimal off-target effects resulting from the action of the base editor protein. We further demonstrate that this base-editing platform could influence gene expression in vivo as its delivery to a mouse model of Huntingtons disease led to a potent decrease in HTT mRNA in striatal neurons. Finally, to illustrate the applicability of this concept, we target the amyloid precursor protein, showing that multiplex editing of its promoter region significantly perturbed its expression. These findings demonstrate the potential for base editors to regulate target gene expression.

bioengineering↗

bioRxiv2019

β-HPV 8E6 Dysregulates the Hippo Signaling Pathway and Induces Aneuploidy

Beta genus human papillomaviruses ({beta}-HPVs) are associated with cutaneous squamous cell carcinomas (cSCCs) in a subset of immunocompromised patients. Although {beta}-HPVs are not necessary for tumor maintenance, they are hypothesized to destabilize the genome in the early stages of cancer development. Supporting this idea, {beta}-HPVs 8E6 protein attenuates p53 accumulation after failed cytokinesis. This paper identifies the mechanism of this abatement. We show {beta}-HPV 8E6 dysregulates the Hippo signaling pathway (HP). It increases pro-proliferative gene expression, enhances TEAD activity and promotes cell growth. {beta}-HPV 8E6 also reduces LATS activation and p53-mediated apoptosis following unsuccessful division of mitotic cells. These phenotypes are dependent on {beta}-HPV 8E6 binding and destabilizing a cellular histone acetyltransferase, p300. Despite circumventing apoptosis, {beta}-HPV 8E6 caused increased senescence after unsuccessful cytokinesis. We linked this lack of growth to the viral proteins inability to prevent cytoplasmic sequestration of the HP transcription factor, YAP. We also show that increased telomerase reverse transcriptase activity (a common alteration in cSCCs) acts synergistically with {beta}-HPV 8E6 to promote cellular proliferation after abortive cytokinesis. While {beta}-HPV 8E6 promoted aneuploidy on its own, this genome destabilization is amplified in cells that do not divide after mitosis. Although our group and others have previously described inhibition of DNA repair, to the best of our knowledge this marks the first time that a {beta}-HPV protein has been connected to chromosome level changes in the cellular genome. This represents a substantial escalation in the known genome destabilizing properties likely to occur during a {beta}-HPV infection.\n\nIMPORTANCEThere is mounting evidence that {beta}-HPVs contribute to cSCCs development in immunocompromised populations. They may also augment UVs mutagenic potential, increasing cancer risk in the general population. We demonstrate that {beta}-HPV 8E6 dysregulates the Hippo signaling pathway (HP). HP regulates cell growth and apoptosis in response to a myriad of stimuli, including failed cytokinesis. {beta}-HPV 8E6 attenuates phosphorylation of the HP kinase, LATS, decreasing some but not all downstream signaling events. This allows binucleated cells to avoid apoptosis, however they succumb to senescence. We show that {beta}-HPV 8E6 synergizes with a common cSCC mutation (telomerase activation) to avoid both apoptosis and senescence. We did not find any telomerase immortalized {beta}-HPV 8E6 expressing cells that were not aneuploid after aberrant cytokinesis. This represents a substantial escalation in {beta}-HPV E6s known mutagenic potential.

cancer biology↗