Introduction: Recombinant adeno-associated viral vector are valuable tools in basic research facilitating overexpression of genes of interest. Depending on the serotype used, rAAVs display a distinct tropism. The widely used serotype AAV9 shows high transductionrates in cardiomyocytes, muscle cells and neurons. However, a rAAV system enabling endothelial targeting of rAAVs remains elusive. We propose modifications to the rAAV platform to retarget the rAAV 2/9 to endothelial cells. To this end, rAAV virions are coated with second generation Polyamidoamins (G2 PAMAM). Via an PEG-OSS linker cell type specific targeting peptides are attached to the PAMAM nanoparticles (G2^CNN). Lastly, the endothelial cell specific Endoglin-promoter is used to drive transgene expression.

Here we seek out to modify eNOS activity, a crucial endothelial cell specific protein, which regulates endothelial NO-production, thus regulating systemic blood pressure. Packaging a Cre-recombinase as well as eNOS specific guide RNAs into the G2^CNN.pEndo.AAV we aim to knockout eNOS in endothelial cells of the transgenic mouse line LSL-Cas9, which expresses Cas9 after Cre-mediated recombination.

Methods: Transgenic C57Bl/6 mice expressing the Cas9 gene after Cre-mediated recombination (LSL-Cas9) were transduced with a recombinant adeno-associated viral vector carrying a Cre-recombinase together with guide RNAs targeting eNOS. The rAAVs were coated with PAMAM nanoparticles linked to an endothelial targeting peptide. Blood pressure was recorder before injection and 4 and 8 weeks after rAAV-transduction. After 8 weeks, invasive hemodynamic measurements were performed using a PV-loop catheter (Scisense catheter system, Transonic). Organs were harvested and MACS-sorted for editing efficiency and histological analysis.

Results: After G2^CNN.pEndo.AAV transduction lead to a robust Cas9 expression in 30% of endothelial cells with successful editing in 20% of endothelial cells. Compared to control mice, G2^CNN.pEndo.AAV treated mice displayed a drastic increase in systolic, diastolic and mean arterial pressure (systolic blood pressure 163 ± 3 vs. Ctrl. 144 ± 5 mmHg; diastolic blood pressure 133 ± 3 vs. Ctrl. 113 ± 4 mmHg; MAP 143 ± 3 vs. Ctrl. 123 ± 4 mmHg) as well as an increase in developed pressure (dP 116 ± 2 vs. Ctrl. 106 ± 3 mmHg). These results demonstrate the feasibility of rAAV retargeting to the endothelial compartment via nano-particle coating, targeting peptides and cell-line specific promoters.