Efficient and Selective AAV2-Mediated Gene Transfer Directed to Human Vascular Endothelial Cells

Abstract

Gene therapy vectors based on adeno-associated virus-2 (AAV2) offer considerable promise for human gene therapy. Applications for AAV vectors are limited to tissues efficiently transduced by the vector due to its natural tropism, which is predominantly skeletal muscle, neurons, and hepatocytes. Tropism modification to elevate efficiency and/or selectivity to individual cell types would enhance the scope of AAV for disease therapies. The vascular endothelium is implicitly important in cardiovascular diseases and cancer, but is relatively poorly transduced by AAV vectors. We therefore genetically incorporated the peptide SIGYPLP, which targets endothelial cells (EC), into position I-587 of AAV capsids. SIGYPLP-modified AAV (AAVsig) showed enhanced transduction of human EC compared with AAV with a wild-type capsid (AAVwt), a phenotype independent of heparan sulphate proteoglycan (HSPG) binding. In contrast, AAVsig did not enhance transduction of primary human vascular smooth muscle cells or human hepatocytes, principal targets for AAV vectors in local or systemic gene delivery applications, respectively. Furthermore, infection of EC in the presence of bafilomycin A(2) indicated that intracellular trafficking of AAV particles was altered by targeting AAV by means of SIGYPLP. AAV vectors with enhanced tropism for EC will be useful for diverse gene therapeutics targeted at the vasculature.

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library

“…1 Among the tolerated insertion sites for small peptides, insertions in the AAV2 heparin binding domain 26,27 adjacent to amino acids 587 or 588 was most successful. 17,23,[28][29][30][31][32] An inherent problem of genetic modification of the AAV capsid is the design of the targeting peptide. Our knowledge about cell surface receptors and their ligands is incomplete, and the toleration and performance of ligands identified by phage display are so far not predictable within the constraints of the AAV capsid structure, although it was successful in several instances.…”

Adeno-associated virus (AAV)-7 and -8 poorly transduce vascular endothelial cells and are sensitive to proteasomal degradation

“…However, poor transduction of the vascular endothelium by AAV-2 both in vitro and in vivo has been noted. 9,11,12 This has been attributed to sequestration of AAV-2 in the extracellular matrix by heparan sulphate proteoglycans (HSPG), thus preventing cell entry 12 and sensitivity to the proteasome, 11 a mechanism previously established as an obstacle in the transduction of airway epithelial cells. 13,14 Implications for the proteasome-sensitivity in vascular cells is an important consideration for clinical applications since the ubiquitin-proteasome system remains active in diseased blood vessels.…”

“…[15][16][17] Alternate strategies to improve endothelial cell transduction by AAV have been actively sought. 18 These include AAV-2 retargeting using vascular cell-selective peptides 11,19,20 or the use of alternate serotypes. 21 We previously reported that for vascular cells (including endothelial and smooth muscle cells) AAV-2 produced the highest levels of transduction compared to AAV-3, -4, -5 or -6, although these levels were lower than those achieved in the more permissive cell type HeLa.…”

Post-intervention vessel remodeling

“…Accordingly several biologic targeting systems have recently been introduced. [4][5][6][7][8][9][10] In spite of relatively low transfection efficiency adenoviruses are still the most widely used viral vectors for vascular applications since they can transfect both proliferating and non-proliferating cells. 2,11 However, adenoviruses also transfect many unwanted organs and peripheral blood monocytes.…”

“…Thus, adeno-associated viruses (AAV) have been successfully used to transduce rabbit jugular veins with expression lasting beyond 30 days, 15 and a method to target AAV gene therapy to vascular endothelial cells has been described. 8 Furthermore, modified retrovirus, Semliki Forest virus, Baculovirus and herpes simplex virus vectors have been used for vascular gene transfer in vivo, but these vectors have not yet solved all problems related to vascular gene transfer. 7,[16][17][18] The most widely used non-viral vector for vascular gene therapy is plasmid DNA with or without carrier molecules.…”