Tan P H, Manunta M, Ardjomand N, Xue S A, Larkin D F P, Haskard D O, Taylor K M, George A J T
Department of Immunology, Division of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK.
J Gene Med. 2003 Apr;5(4):311-23. doi: 10.1002/jgm.358.
One of the drawbacks of the currently available vectors for gene therapy is the lack of selectivity in gene delivery. We have therefore investigated a strategy to generate immunoliposomes to target non-viral vectors to cell surface receptors on endothelium.
We have developed a novel method of coupling antibodies (Abs) to liposomes complexed to DNA, using mild heat treatment to aggregate the immunoglobulin G (IgG). The interaction of plasmid DNA, liposomes and Abs was measured using a gel retardation assay and a resonant mirror biosensor. The size of the transfection complex was determined by light scattering, and the binding and internalization of the complex to cells was followed using flow cytometry. The transfection ability was tested on cell lines and primary cells in vitro and human corneal or vascular tissues ex vivo.
The interaction of antibodies with liposomes is relatively stable (t(1/2) congruent with 45 min). The size of the liposome, Ab and DNA complex was found to be around 500 nm in 4% BSA. The addition of anti-transferrin receptor Abs increased the internalization of the liposome-DNA complex into cells. Abs against both transferrin receptor and E-selectin were shown to augment transfection efficiency of liposomes to cell expressing the appropriate antigens. They are also shown to be efficient in mediating gene delivery to corneal and vascular tissues ex vivo.
We have shown that our novel vector is capable of in vitro and ex vivo gene delivery to cells and human tissues including cornea, artery and vein. In particular, an Ab against E-selectin was effective at selectively delivering genes to activated endothelial cells expressing the adhesion molecule. Such a strategy will have applications for targeting these tissues prior to transplantation or autologous grafting, and, in the longer term, may allow in vivo targeting of gene therapy to inflammatory sites.
目前用于基因治疗的载体的缺点之一是基因传递缺乏选择性。因此，我们研究了一种策略，以生成免疫脂质体，将非病毒载体靶向到内皮细胞表面受体。
我们开发了一种新方法，通过温和热处理使免疫球蛋白G（IgG）聚集，将抗体（Abs）偶联到与DNA复合的脂质体上。使用凝胶阻滞分析和共振镜生物传感器测量质粒DNA、脂质体和Abs之间的相互作用。通过光散射确定转染复合物的大小，并使用流式细胞术跟踪复合物与细胞的结合和内化。在体外细胞系和原代细胞以及离体人角膜或血管组织上测试转染能力。
抗体与脂质体的相互作用相对稳定（半衰期约为45分钟）。在4%牛血清白蛋白中，脂质体、Ab和DNA复合物的大小约为500nm。添加抗转铁蛋白受体Abs可增加脂质体-DNA复合物进入细胞的内化。针对转铁蛋白受体和E-选择素的Abs均显示可提高脂质体对表达相应抗原细胞的转染效率。它们还显示在介导基因传递到离体角膜和血管组织方面有效。
我们已表明，我们的新型载体能够在体外和离体条件下将基因传递到细胞和包括角膜、动脉和静脉在内的人体组织。特别是，针对E-选择素的Ab在将基因选择性传递到表达粘附分子的活化内皮细胞方面有效。这种策略将可应用于在移植或自体移植前靶向这些组织，并且从长远来看，可能允许在体内将基因治疗靶向炎症部位。
