Site-Specific Glycation and Chemo-enzymatic Antibody Sortagging for the Retargeting of rAAV6 to Inflamed Endothelium

Hannah A Pearce; Hongwei Qian; Timothy U Connell; Dexing Huang; Claudia Gottstein; Paul S Donnelly; Karlheinz Peter; Paul Gregorevic; Christoph E Hagemeyer

doi:10.1016/j.omtm.2019.07.003

Site-Specific Glycation and Chemo-enzymatic Antibody Sortagging for the Retargeting of rAAV6 to Inflamed Endothelium

Mol Ther Methods Clin Dev. 2019 Jul 23:14:261-269. doi: 10.1016/j.omtm.2019.07.003. eCollection 2019 Sep 13.

Authors

Hannah A Pearce^{1

2}, Hongwei Qian^{3

4}, Timothy U Connell⁵, Dexing Huang², Claudia Gottstein⁶, Paul S Donnelly⁵, Karlheinz Peter², Paul Gregorevic^{3

4

7

8}, Christoph E Hagemeyer¹

Affiliations

¹ NanoBiotechnology Laboratory, Monash University, Melbourne, VIC, Australia.
² Atherothrombosis and Vascular Biology Laboratory, The Baker Heart and Diabetes Institute, Melbourne, VIC, Australia.
³ Muscle Research and Therapeutics Laboratory, The Baker Heart and Diabetes Institute, Melbourne, VIC, Australia.
⁴ Centre for Muscle Research, Department of Physiology, University of Melbourne, Melbourne, VIC, Australia.
⁵ School of Chemistry, Bio21 Molecular Science Institute, University of Melbourne, Melbourne, VIC, Australia.
⁶ Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA, USA.
⁷ Department of Biochemistry and Molecular Biology, Monash University, Australia.
⁸ Department of Neurology, University of Washington School of Medicine, Seattle, WA, USA.

Abstract

Gene therapy holds great potential for conditions such as cardiovascular disease, including atherosclerosis and also vascular cancers, yet available vectors such as the adeno-associated virus (rAAV) transduce the vasculature poorly. To enable retargeting, a single-chain antibody (scFv) that binds to the vascular cell-adhesion molecule (VCAM-1) overexpressed at areas of endothelial inflammation was site specifically and covalently conjugated to the exterior of rAAV6. To achieve conjugation, the scFv was functionalized with an orthogonal click chemistry group. This conjugation utilized site-specific sortase A methodology, thus preserving scFv binding capacity to VCAM-1. The AAV6 was separately functionalized with 4-azidophenyl glyoxal (APGO) via covalent adducts to arginine residues in the capsid's heparin co-receptor binding region. APGO functionalization removed native tropism, greatly reducing rAAV6-GFP transduction into all cells tested, and the effect was similar to the inhibition seen in the presence of heparin. Utilizing the incorporated functionalizations, the scFv was then covalently conjugated to the exterior of rAAV6 via strain-promoted azide-alkyne cycloaddition (SPAAC). With both the removal of native heparin tropism and the addition of VCAM-1 targeting, rAAV6 transduction of endothelial cells was greatly enhanced compared to control cells. Thus, this novel and modular targeting system could have further application in re-directing AAV6 toward inflamed endothelium for therapeutic use.

Keywords: AAV; VCAM-1; antibody; click chemistry; glyoxal; sortase.