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Updated: Feb 21, 2026
Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice
Published on: August 2, 2018
Ablation of PKCα Phosphorylation by CRISPR-Cas9 Base Editing Rescues Heart Failure
Tomonori Tadokoro1,2, Hui Li1,2, Peiheng Gan1,2,3
1Department of Molecular Biology (T.T., H.L., P.G., Z.X., W.T., D.A., E.S.-O., J.R.M., N.L., E.N.O.), University of Texas Southwestern Medical Center, Dallas.
Insights
Gene editing of protein kinase C alpha (PKCα) by targeting threonine 497 (T497) phosphorylation offers a novel therapeutic strategy for heart failure. This approach protects against cardiac dysfunction and remodeling, showing promise for treating human cardiac diseases.
Area of Science:
- Cardiovascular Research
- Molecular Cardiology
- Gene Therapy
Background:
- Increasing global prevalence of heart failure necessitates novel therapeutic strategies.
- Protein kinase C alpha (PKCα) is implicated in heart failure pathogenesis.
- Phosphorylation of PKCα at threonine 497 (T497) is crucial for its activation and stability.
Purpose of the Study:
- To investigate the therapeutic potential of ablating PKCα T497 phosphorylation in heart failure.
- To assess the efficacy of CRISPR-Cas9 adenine base editing for introducing the T497A mutation in vivo and in vitro.
Main Methods:
- Generation of germline phospho-resistant PKCα (T497A) mutant mice using CRISPR-Cas9.
- Postnatal genome editing in wild-type mice via adeno-associated virus 9-delivered base editors to introduce the T497A substitution.
- Modeling heart failure using transverse aortic constriction, followed by cardiac function, histological, and transcriptomic analyses.
- Editing of human induced pluripotent stem cells to introduce the PRKCA T497A mutation for in vitro studies on cardiomyocyte function and calcium homeostasis.
Main Results:
- The T497A mutation led to PKCα protein degradation and prevented its activation.
- PKCα phospho-resistant mice were protected against transverse aortic constriction-induced cardiac hypertrophy, fibrosis, and functional decline.
- In vivo base editing achieved similar cardioprotective effects, and in vitro studies demonstrated protection of edited cardiomyocytes from AngII-induced impairments.
Conclusions:
- Ablating PKCα T497 phosphorylation confers significant cardioprotection.
- CRISPR-Cas9 adenine base editing targeting PRKCA T497A is a viable strategy for treating heart failure.
- This gene editing approach holds potential as a therapeutic strategy for human cardiac diseases.
Background:
The prevalence of heart failure is increasing globally, with poor prognosis, highlighting the need for novel therapeutic strategies. PKCα (protein kinase C alpha), encoded by PRKCA, plays a central role in heart failure pathogenesis. Phosphorylation of PKCα at threonine 497 (T497) triggers a series of intramolecular phosphorylation events, leading to its activation. Ablation of T497 phosphorylation leads to reduced stability and activity of PKCα.
Methods:
We generated mice harboring a phospho-resistant PKCα (T497A) mutation in the germline using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated homology-directed repair. To assess the clinical feasibility of postnatal genome editing, we used CRISPR-Cas9 adenine base editing delivered by adeno-associated virus 9 to introduce the T497A substitution into the Prkca gene (PrkcaT497A) in wild-type mice. Mice underwent transverse aortic constriction to model heart failure. Cardiac function, hypertrophy, fibrosis, and transcriptional changes were evaluated by echocardiography, wheat germ agglutinin staining, Masson's trichrome staining, and RNA-sequencing. The editing efficiency of PrkcaT497A was assessed using Sanger sequencing and deep amplicon sequencing. To further explore its clinical potential, we introduced the PRKCAT497A mutation into human induced pluripotent stem cells by nucleofection-mediated adenine base editing. Ca2+ homeostasis was analyzed in Fura-2-loaded human induced pluripotent stem cell-derived cardiomyocytes with PRKCAT497A under chronic AngII (angiotensin II) stimulation.
Results:
The T497A mutation in PKCα prevented its subsequent phosphorylation and led to PKCα protein degradation. Four weeks after transverse aortic constriction surgery, wild-type mice showed impaired cardiac function, cardiac remodeling, and increased lung weight. In contrast, PKCα phospho-resistant mice showed protection against heart failure-related aberrant changes in cardiac hypertrophy, fibrosis, and cardiac gene expression. Mice administered with adeno-associated virus 9 base editors to prevent T497 phosphorylation exhibited similar cardioprotective effects. In vitro, PKCα-edited induced pluripotent stem cell-derived cardiomyocytes were protected from AngII-induced impairments in contractility and Ca2+ transients.
Conclusions:
The editing of PRKCAT497A through adenine base editing represents a potential therapeutic approach for human cardiac diseases.
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