CRISPR-Cas9 base editing of pathogenic CaMKIIδ improves cardiac function in a humanized mouse model
Simon Lebek, … , Rhonda Bassel-Duby, Eric N. Olson
Simon Lebek, … , Rhonda Bassel-Duby, Eric N. Olson
Published October 19, 2023
Citation Information: J Clin Invest. 2024;134(1):e175164. https://doi.org/10.1172/JCI175164.
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CRISPR-Cas9 base editing of pathogenic CaMKIIδ improves cardiac function in a humanized mouse model

Abstract

Cardiovascular diseases are the most common cause of worldwide morbidity and mortality, highlighting the necessity for advanced therapeutic strategies. Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is a prominent inducer of various cardiac disorders, which is mediated by 2 oxidation-sensitive methionine residues within the regulatory domain. We have previously shown that ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing enables the heart to recover function from otherwise severe damage following ischemia/reperfusion (IR) injury. Here, we extended this therapeutic concept toward potential clinical translation. We generated a humanized CAMK2D knockin mouse model in which the genomic sequence encoding the entire regulatory domain was replaced with the human sequence. This enabled comparison and optimization of two different editing strategies for the human genome in mice. To edit CAMK2D in vivo, we packaged the optimized editing components into an engineered myotropic adeno-associated virus (MyoAAV 2A), which enabled efficient delivery at a very low AAV dose into the humanized mice at the time of IR injury. CAMK2D-edited mice recovered cardiac function, showed improved exercise performance, and were protected from myocardial fibrosis, which was otherwise observed in injured control mice after IR. Our findings identify a potentially effective strategy for cardioprotection in response to oxidative damage.

Authors

Simon Lebek, Xurde M. Caravia, Leon G. Straub, Damir Alzhanov, Wei Tan, Hui Li, John R. McAnally, Kenian Chen, Lin Xu, Philipp E. Scherer, Ning Liu, Rhonda Bassel-Duby, Eric N. Olson

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Figure 1

Analysis of on- and off-target editing in human iPSCs.

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Analysis of on- and off-target editing in human iPSCs. (A) Schematic sho...
(A) Schematic showing the structure of CaMKIIδ with its 3 domains (blue, catalytic domain; green, regulatory domain; brown, association domain). Upon oxidative stress, 2 critical methionine residues at position 281 and 282 become oxidized, thereby preventing association of the regulatory with the catalytic domain, and resulting in cardiac disease. We deployed 2 ABE strategies to ablate either 1 (sgRNA1) or both (sgRNA2) oxidative activation sites of CaMKIIδ. (B) Human iPSCs were nucleofected with ABE8e(V106W) and either sgRNA1 or sgRNA2. (C) Sequence of sgRNA1 (CAMK2D on-target, ON) and the corresponding DNA and PAM sequences of the top 8 predicted potential off-target sites (OT), as predicted by CRISPOR. Nucleotides highlighted in yellow are different from sgRNA1. (D) Percentage of adenine (A) to guanine (G) editing for all adenines within the on- and off-target sites (ordered from 5′ to 3′) following adenine base editing with ABE8e(V106W)-SpCas9 + sgRNA1 (n = 3). (E) Sequence of sgRNA2 (CAMK2D on-target, ON) and the corresponding DNA and PAM sequences of the top 8 predicted potential off-target sites, as predicted by CRISPOR. Nucleotides highlighted in yellow are different from sgRNA2. (F) Percentage of adenine (A) to guanine (G) editing for all adenines within the on- and off-target sites (ordered from 5′ to 3′) following adenine base editing with ABE8e(V106W)-SpRY + sgRNA2 (n = 3). All data are individual data points with mean ± SEM. Replicates are human iPSCs following 3 independent nucleofections.