848. Development of AAV Vectors Targeted to Vascular Endothelium

Abstract

Top of pageAbstract Many diseases, such as cancer and forms of cardiovascular disease, share a common process of vascular proliferation. It is thought that the ability to deliver and express genes in the vasculature will be valuable for the development of gene therapies aimed at these diseases. While adeno-associated virus (AAV) vectors can transduce a wide variety of cell types, certain cells (including cells of the vascular system) remain refractive to AAV transduction. We have pursued several different approaches to develop AAV vectors capable of gene delivery to the vasculature. Peptides, targeted towards vascular endothelial growth factor receptor 2 (VEGFR2) or integrins and peptides homologous to the N-terminal fragment of human high mobility group protein 2 (HMGN2), were incorporated into the capsids of AAV vectors. Two different sites were utilized for peptide insertion. The first site was designed to display inserted peptides on all viral capsid proteins (VP1, VP2, and VP3), while the second site was chosen to display peptides only on the VP2 capsid protein. Incorporating peptides into all capsid proteins allows for a greater number of the peptides to be displayed, but the size of the peptide was limited to |[le]| 24 amino acids. While the number of peptides displayed following VP2 insertion is limited (|[sim]|3 per viral particle), this site allows for the display of a much larger peptides while maintaining vector infectivity. Near wild type titers were obtained for all modified vectors. We also generated AAV2 vectors unable to bind their natural receptor, heparan sulfate proteoglycan (HSPG), by incorporating two point mutations into the VP3 coding region of the AAV2 genome (R585A/R588A). A solid-phase heparin sulfate binding assay was developed and used to show that these HS-ablated AAV2 vectors, as well as the HS-ablated AAV2 vectors containing targeting ligand insertions were unable to bind HS. Targeting insertions were made in AAV1, AAV2, and HS-ablated AAV2 backbones with the idea that HS-ablated AAV2 vectors might allow for the highest specificity, while targeted AAV1 vectors might allow for the highest level of in vivo vascular transduction due to the greater in vivo gene transfer observed with unmodified AAV1 vectors compared to AAV2 vectors. Along with the targeting peptide, a biotin acceptor peptide (BAP), used to purify modified AAV1 and HS-ablated AAV2 vectors, was incorporated into some modified AAV capsids. Vectors were used to infect four different human endothelial cell populations (saphenous vein endothelial cells (HSaVEC), umbilical vein endothelial cells (HUVEC), coronary artery endothelial cells (HCAEC), and coronary microvascular endothelial cells (HCMVEC)). Integrin targeted AAV1 and the integrin targeted/BAP chimeric AAV1 vectors displayed significantly higher levels of gene transfer to all endothelial cell types compared to unmodified AAV1 vector. Transduction of HSaVECs was greater than 20 fold (p<0.002) more efficient with the RGD/BAP chimeric AAV1 than with untargeted AAV1. Similarly, in HCAECs, AAV1-RGD/BAP was greater than 65 fold (p<0.0001) more efficient, and AAV1-VEGFR2E/BAP was 4 fold (p=0.044) more efficient than untargeted vectors.