AAV-mediated gene expression and deletion in cardiac fibroblasts in vivo

Introduction: Mechanical stress (e.g., pressure overload), cytokines, growth factors, and neurohumoral mediators stimulate cardiac fibroblast (CF) activation, which promotes extracellular matrix (ECM) protein synthesis leading to cardiac fibrosis and diastolic dysfunction. Therefore, CFs are an attractive target for reducing pathological cardiac remodeling, and understanding the underlying mechanisms of these processes is key to developing successful therapies for treating the pressure-overloaded heart. CF-specific knockout (KO) mouse lines with a Cre recombinase under the control of Human TCF21 (hTCF21) promoter provide a powerful tool for understanding CF biology in vivo in addition to the periostin promoter-driven myofibroblast-specific KO system. In addition to these transgenic mouse lines, one group recently reported the overexpression of Yes-associated protein (YAP) in CFs using an adeno-associated virus (AAV)-based system (Francisco J et al., Sci Rep. 2021). Aim: To test the feasibility of CF-selective gene expression and deletion in vivo using an AAV-based approach. Methods: The pAAV-hTCF21 promoter vector was generated by cloning the hTCF21 promoter fragment into a mammalian gene expression AAV vector (Vectorbuilder). AAV vectors were packaged into AAV serotype 9 (AAV9) and the viral particles were introduced into adult rats and mice via tail vein and retro-orbital injections, respectively. Gene expression or deletion in adult rats and mice were assessed 5-6-weeks after AAV9 injection. Results: First, tissue-specific gene delivery was evaluated in rats by injecting AAV9-hTCF21-GFP. GFP mRNA expression was highest in liver and heart tissues compared to other organs assessed by real-time qPCR. CF-specific GFP expression was confirmed by co-immunostaining ventricular tissue sections with antibodies against GFP and the CF marker vimentin. GFP was not detectable in cardiomyocytes, another major cell type in the heart. Next, we injected AAV9-hTCF21-iCre and -luciferase (as a control) into the mice in which critical exons of the protein kinase D1 (PKD1) gene are flanked with loxP sites (PKD1 fl/fl mice). PKD expression levels in CFs were quantified by co-immunostaining ventricular sections with PKD and vimentin antibodies. PKD was detectable in both CFs and cardiomyocytes in control hearts from AAV9-hTCF21-luciferase-injected PKD1 fl/fl mice. In contrast, significant decrease in PKD expression was observed only in CFs, but not in the cardiomyocyte area after AAV9-hTCF21-iCre injection. Conclusion: AAV9-mediated hTCF21-driven gene expression and deletion in CFs in vivo may be a powerful experimental platform in addition to the generation of CF-specific conditional KO mouse lines. R01HL136757 (to J.O.-U), AHA 18CDA34110091 (to B.S.J) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.