Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but they are inefficiently transduced with adeno-associated viral (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new highthroughput selection and monitoring protocol, we identified a capsid variant, AAV-V EC , which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved, resulting in significantly higher transgene expression levels from singlestranded vector genomes detectable already few hours post-transduction. Notably, AAV-V EC transduced not only proliferating EC but also quiescent EC, although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-V EC transduction. Thus, overcoming barriers by capsid engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.

To ascertain the importance of amino-terminal proximal capsid protein (CP) sequences in cel-to-cell movement, virion formation, and stabilization, two CP mutants of cucumber mosaic virus (CMV) were generated by deletion of sequences encoding CP amino acids 15-40 (delta Sal-Nru) or 26-40 (delta Sac-Nru). Wildtype CMV and CMV containing delta Sac-Nru could infect systemically four host species, although symptoms induced by the two viruses usually were different CMV containing delta Sal-Nru could only infect Nicotiana benthamiana and N. clevelandii systemically, but only slowly, suggesting phloem-independent long-distance movement. A variant mutant designated delta Sal-Nru* could systemically infect N. tabacum as well as the above two Nicotiana species, rapidly, but could not systemically infect Cucurbita pepo. Virus particles could not be detected in plants infected by delta Sal-Nru, while delta Sal-Nru* and delta Sac-Nru formed particles of lower stabilities than for wildtype virus. The CPs of delta Sal-Nru and delta Sal-Nru* could bind RNA in vitro, although less strongly than delta Sac-Nru or wildtype CMV. These data indicate that amino-terminal proximal sequences of the CMV CP interact with viral RNA and are required for the formation of stable virions. Moreover, while the CP is necessary for cell-to-cell movement, the ability to form virions is not a prerequisite for cell-to-cell movement.