Targeted gene delivery of cardiac interstitial cells using AAV
A Shakouri, T Bozoglu, V Rajendran, A Dastmaltschi, N Raad, T Ziegler, C KupattAbstract
Background
Adeno-associated viruses (AAV) are currently the gold standard for gene transfer and gene editing applications in vivo, due to their low integration rate and low immunogenicity, as well as a variety of natural and engineered serotypes providing different tissue and cell type tropism. In this study, our goal is to develop a vector system that can regulate SRF gene expression in specific cardiac cell types. The approach we take is a combinatorial system with cell-type-specific promoters, miRNA seed sequence, and peptide coating. This would allow us to fine tune delivery in applications such as gene transfer and (epi-)genome editing, helping to dissect the modulation of MRTFA/SRF pathway in individual cell types.
Methods
We previously demonstrated efficient PAMAM-mediated retargeting of AAV9 from cardiomyocytes to microvascular endothelial cells, a major interstitial component of the myocardium. To enhance specificity, miR seed sequences were cloned into AAV reporter gene plasmids (eGFP) and validated in HEK293 cells co-transfected with miRNA mimics, confirming effective transgene downregulation. Subsequently, cell-specific promoters were introduced to achieve selective expression in endothelial cells, fibroblasts, and cardiomyocytes. Furthermore, PAMAM dendrimers conjugated to targeting peptides were utilized to increase AAV affinity for fibroblasts and endothelial cells. These modified viruses were administered to wild-type C57BL/6 mice, followed by the isolation of primary cardiac cells and downstream analysis.
Results
The inclusion of specific miRNA seed sequences significantly reduced GFP expression in HEK293 cells when co-delivered with the corresponding mimics, while scrambled controls showed no inhibition, confirming targeting specificity. Evaluation of cell-specific promoters (Endoglin, Periostin, and Troponin) demonstrated that miRNA seed sequences successfully suppressed off-target transgene expression in vitro. In vivo analysis following FACS-mediated cell isolation yielded high-purity populations of target cell types. qPCR analysis confirmed that the engineered vectors specifically targeted cardiomyocytes, fibroblasts, and endothelial cells. Consequently, the utilization of AAV9 specifically retargeted to fibroblasts emerges as an innovative solution to overcome current delivery constraints in the myocardium.
Conclusion
The gene delivery toolkit we are developing in this study, comprising retargeting capabilities of promoter–mir seed sequence–PAMAM peptide formulations, would enable the spatial and temporal resolution required to dissect a range of clinically relevant cardiovascular processes, from fibrosis to angiogenesis.