Abstract Details

C2 - Epigenetic Editing and RNA Editing

1174: CRISPR Epigenetic Reprogramming of Myokines as Therapy for Alzheimer’s Disease

Type: Poster Session

Poster Board Number: 1174

Presentation Details

Session Title: Thursday Posters: Epigenetic Editing and RNA Editing






Background. Alzheimer’s disease (AD) is a neurodegenerative condition imposing a significant burden on healthcare systems globally. Despite extensive efforts, it remains incurable, with only moderately effective treatments available for some patients. Gene therapy, particularly using CRISPR-Cas, holds promise as an advanced alternative. The ability to reprogram cell phenotypes by regulating gene expression via epigenetic reprogramming using the CRISPR-dCas9 variant has widened our options for complex disease therapies, like AD. It’s known that exercising modulates genes encoding for myokines that regulate other factors in the brain, leading to neurogenesis, reduced neuropathology, and fewer cognitive deficits in AD. e hypothesized that using CRISPR-dCas epigenetic reprogramming to upregulate exercise-responsive genes could mimic the beneficial effects of exercise in the brain, potentially slowing AD progression. As CRISPR delivery methods advance, there's optimism that this strategy could not only delay the onset of AD and its progression but also reduce cognitive decline in patients. Experiments. We used the CRISPR-dCas9-VPR-WPRE/BGH containing both viral (VP64, Rta) and human transactivation domains (RELA, p65), known to recruit epigenetic effector proteins. We tested six single-guide RNAs (sgRNAs) designed around 200 bases upstream of our top myokine gene transcription start site (Fig. 1A). We also tested two 3’ untranslated sequences (WPRE or BGH) to see if they affect mRNA stability and modulation efficiency. The cells used were iPS-derived neural progenitors. sgRNAs and mRNAs were transfected with cationic lipids. Our in vitro transcriptions incorporated pseudouridine instead of uridine into the mRNAs. Experiments have as readout the mRNA expression measured by TaqMan RT-qPCR (Fig. 1B). Results. For the six tested sgRNAs and 3’-UTRs, we obtained at least a 3-fold increase in mRNA expression of the target gene. Two-way ANOVA showed no interaction between sgRNA and 3’-UTR, but a slightly higher upregulation with the WPRE (F = 7.37_{df = 39}, P = 0.011, Fig. 1C). The fold-change among the six sgRNAs for the WPRE construct ranged from 3.3× up to 31× over the non-targeting control (Fig.1D). As a result, the top three sgRNAs with the largest fold-changes were W0-CB3 (31.2×), C1 (19.1×), and WE3 (10.8×). In summary, the sgRNAs located around -292 bases upstream of the transcription start site showed improved upregulation of the target transcript. Conclusions. We have shown that all sgRNAs can effectively upregulate the target gene mRNA up to 30-fold. Next experiments will evaluate if protein levels correlate with the mRNA’s. We will also try the transcriptional activation of selected sgRNAs on cortical neurons. In the future, in vivo experiments on Alzheimer’s disease mouse models will measure the effect of this approach on disease progression, memory, and neuropathology.

Thyago Leal Calvo^1,2, Viviana Salinas-Rios¹, Ilana Burg¹, Fernanda G. De Felice³, Fyodor D. Urnov<sup>1

1</sup>Innovative Genomics Institute, University of California Berkeley, Berkeley, CA,²Pioneer Science Initiative, D'Or Institute for Research and Education, Rio de Janeiro, Brazil,³Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON, Canada"