N Deyerling (1,2), L Hüttermann (1,2), S S Hille (1,2), T Jonker (3), D Grimm (4), D Frank (2,5), G J J Boink (3), O J Müller (1,2)

1: Department of Internal Medicine V, University Hospital Schleswig-Holstein and University of Kiel, Kiel, Germany; 2: German Centre for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, Kiel, Germany; 3: Department of Medical Biology and Department of Cardiology, Academic Medical Centre, University of Amsterdam, Amsterdam, Netherlands; 4: Department of Infectious Diseases/Virology, Section Viral Vector Technologies, Medical Faculty of Engineering Sciences, BioQuant, German Centre for Infection Research (DZIF) and German Centre for Cardiovascular Research (DZHK), Heidelberg University, Heidelberg, Germany; 5: Department of Internal Medicine III, University Hospital Schleswig-Holstein and University of Kiel, Kiel, Germany

This study reports the development and initial evaluation of an AAV9-loxP 7-mer peptide library engineered for Cre recombinase-dependent targeting in the cardiac conduction system. To assess its in vivo utility, an AAV9-loxP peptide insertion library was intravenously administered into five HCN4-Cre-ERT2 x ROSA [mT/mG] transgenic mice (2 x 10¹² vg per mouse) at the Amsterdam University Medical Center. Four weeks post-injection, tissues were harvested, and both genomic DNA (gDNA) and complementary DNA (cDNA) from the sinoatrial node (SAN) and atrioventricular node (AVN) cells were isolated and analysed to evaluate peptide motif enrichment.

Despite representing only the first round of selection, the data revealed distinct and detectable enrichment of both gDNA and more notably, cDNA within SAN and AVN cells. This early enrichment suggests preliminary target-specific accumulation and supports the feasibility of Cre-dependent selection. Strikingly, the top five enriched peptide motifs differed entirely between SAN and AVN cells as well as between their respective gDNA and cDNA populations. This divergence emphasizes the potential for highly cell-type-specific AAV targeting.

These findings lay the groundwork for further rounds of in vivo selection and functional validation with the promise of generating novel AAV variants with improved gene expression profiles and enhanced cellular specificity, particularly within the cardiac conduction system.