Retargeting of AAV9 to Cardiac and Skeletal Muscle Fibroblasts

Tarik Bozoglu (München)1, A. Hönig (München)1, S. Hanusch (München)1, T. Ziegler (München)1, C. Kupatt (München)1

1Klinikum rechts der Isar der Technischen Universität München Klinik und Poliklinik für Innere Medizin I München, Deutschland

 

Background:

Adeno-associated virus (AAV) is employed for in vivo gene transfer and gene editing applications, owing to its low integration rate and minimal immunogenicity. Additionally, various natural and engineered serotypes offer diverse tissue and cell type tropism. AAV serotype 9 exhibits strong tropism for cardiac and skeletal muscle. However, within these organs, the myocytes are the primary target of AAV9, with inefficient transduction observed in other constituent cells like endothelial cells and fibroblasts of the heart. This specific cell type targeting limits the potential applications of AAV-mediated gene transfer in vascular medicine and research. In a previous study, we successfully redirected AAV9 particles to microvascular endothelium (1). Our current objective is to employ a similar methodology to redirect AAV9 to cardiac fibroblasts.

Methods and results:

Bio-panning of a phage display library (Ph.D. CX7C) on primary rhesus left ventricular fibroblasts underwent two rounds. The resulting refined library was then administered to wild-type C57BL/6 mice following chloroquine injections. Cardiac and peripheral muscle fibroblasts were isolated through Liberase digestion and magnetic sorting for PDFGRa. NGS-amplicon sequencing was employed to acquire heptameric peptide sequences. Following normalization of sequence counts to address bacterial amplification bias, 20 peptides were chosen for synthesis and linked to PAMAM G2 dendrimers through a PEG linker. A pre-screening phase involved coating AAV9-eGFP with the modified PAMAMs and administering them via I.M. injections to wild-type C57BL/6 mice. The relative efficiencies in fibroblast transduction were evaluated through flow cytometric analysis of eGFP+/PDGFRa+ cells.

To further explore the most effective peptides, an AAV9 encoding Cre under PDGFRa promoter control (AAV9-proPDGFRa-Cre) was generated. The five most efficient peptide-PAMAM conjugates were utilized to coat AAV9-proPDGFRa-Cre, which was then administered to mTmG reporter mice through tail vein injections. The efficacy of fibroblast retargeting was assessed using flow cytometry and confocal microscopy.

Flow cytometric analysis of skeletal muscle cell suspensions from transduced mTmG mice indicated that up to 15% of PDGFRa+ cells were positive for eGFP. Confocal imaging of cardiac and skeletal muscle sections revealed transduced PDGFRa+ and Vimentin+ cells, with ratios consistent with the results obtained through flow cytometry.

Conclusion:

Attaining successful gene transfer to fibroblasts is crucial in clinical scenarios, especially when addressing cardiac conditions such as hypertension or aortic stenosis, which are associated with elevated fibrosis in the myocardium. Elevated fibrosis frequently leads to complications like arrhythmia. Therefore, the utilization of AAV9 specifically retargeted to fibroblasts emerges as an innovative solution for researchers to overcome this constraint in cardiovascular medicine and research.

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