A novel AAV9 random peptide library to select for endothelial cell – directed gene transfer vectors

Abstract

Endothelial cells play a central role in vascular diseases and represent therefore a clinically relevant cell type for gene therapeutic approaches. However, the endothelium is difficult to transduce with wtAAV vectors at feasible levels. The potential of random peptide libraries displayed on AAV2 to select for AAV2 vectors with improved efficiency of endothelial-directed gene transfer has been demonstrated. AAV9, however, may have advantages over AAV2 because of a lower prevalence of neutralizing antibodies in humans and more efficient gene transfer in vivo. The present study provides evidence that random peptide libraries can be displayed on AAV9 and can be utilized to select for AAV9 capsids redirected to human endothelium, as previously shown for AAV2. An AAV9 peptide display library which ensures that the displayed peptides correspond to the packaged genomes was generated. Four consecutive selection rounds on human coronary artery endothelial cells (HCAEC) performed in vitro yielded AAV9 library capsids with distinct peptides that strongly outperformed transduction of wild type AAV9. A central point of this study is posed by the finding that incorporation of sequences selected from AAV2 libraries into AAV9 capsids could not increase transduction as efficient as peptides selected in the AAV9 library context, justifying the generation and selection of an AAV9 library. Furthermore, AAV9 vectors with targeting sequences selected from AAV9 libraries revealed an increased transduction efficiency in presence of neutralising human intravenous immunoglobulins suggesting a reduced immunogenicity and a better suitability of AAV9 as a vector backbone. However, enriched peptides did not restrict AAV9 specificity towards HCAEC. The attempt to restrict transgene expression to this cell type by transcriptional targeting using a murine endothelium-specific promoter may be promising as an additional targeting level. To determine the potential of selected AAV on endothelial cells in the intact natural vascular context, murine mesenteric arteries and human umbilical veins were incubated in vivo or in situ, respectively, using the most efficient AAV9 vector. Analysis revealed a highly efficient transduction of human umbilical vein endothelial cells (HUVEC) by the vector mutant. Similar to the negligible transduction by wtAAV9 vectors, no transduction of murine mesenteric artery endothelial cell (MMAEC) could be detected. A closer comparative analysis of two selected vectors displaying peptides with divergent sequences revealed a HSPG-independent transduction of and the partial use of a common transduction. The results obtained in the present study permit the conclusion that the novel AAV9 peptide library is functional and can be used to select for vectors for future preclinical and clinical gene transfer applications.