Pearce Hannah A, Qian Hongwei, Connell Timothy U, Huang Dexing, Gottstein Claudia, Donnelly Paul S, Peter Karlheinz, Gregorevic Paul, Hagemeyer Christoph E
NanoBiotechnology Laboratory, Monash University, Melbourne, VIC, Australia.
Atherothrombosis and Vascular Biology Laboratory, The Baker Heart and Diabetes Institute, Melbourne, VIC, Australia.
Mol Ther Methods Clin Dev. 2019 Jul 23;14:261-269. doi: 10.1016/j.omtm.2019.07.003. eCollection 2019 Sep 13.
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.
基因治疗对于心血管疾病（如动脉粥样硬化以及血管癌）具有巨大潜力，然而，诸如腺相关病毒（rAAV）等现有载体对脉管系统的转导效果不佳。为实现重新靶向，一种与在内皮炎症区域过表达的血管细胞黏附分子（VCAM-1）结合的单链抗体（scFv）被位点特异性且共价地偶联到rAAV6的外部。为实现偶联，scFv用一种正交点击化学基团进行功能化修饰。这种偶联利用了位点特异性分选酶A方法，从而保留了scFv与VCAM-1的结合能力。AAV6通过共价加合物与衣壳肝素共受体结合区域中的精氨酸残基用4-叠氮基苯基乙二醛（APGO）进行单独的功能化修饰。APGO功能化消除了天然嗜性，极大地降低了rAAV6-GFP对所有测试细胞的转导，其效果类似于在肝素存在下所观察到的抑制作用。利用引入的功能化修饰，scFv随后通过应变促进的叠氮-炔环加成反应（SPAAC）共价偶联到rAAV6的外部。随着天然肝素嗜性的消除以及VCAM-1靶向作用的添加，与对照细胞相比，内皮细胞的rAAV6转导显著增强。因此，这种新型的模块化靶向系统在将AAV6重新导向炎症内皮用于治疗方面可能具有进一步的应用。
