基于AS1411与NLS的视网膜级联靶向非病毒基因载体构建及其抗CNV机制研究

Choroidal neovascularization (CNV) is a vital reason which lead to the visual loss of AMD patients. And the formation of CNV is triggered by the significant increase in the VEGF level. Ocular gene therapy, as a hotspot of CNV relative diseases, could disrupt VEGF autocrine loops and fundamentally reduce the VEGF level. Gene delivery to the posterior segment of the eye is severely hindered by multiple barriers including blood-retinal, cytomembrane and nuclear membrane. Accordingly, it is quite important to enhance the transfection efficiency of gene delivery systems. In this study, nucleolin is selected as the therapeutic target because it is highly expressed on the surface of retinal cells. AS1411, an aptamer possessing inhibitory effect on CNV and showing specific binding to nucleolin was used as cytomembrane targeted ligand. N-terminal stearylated nuclear (NLS) was used as nuclear membrane targeted ligand. Consequently, a dual targeted ternary lipopolyplexes guided by aptamer AS1411 and NLS is designed for retinal delivery of Flt23k anti-VEGF intraceptor plasmid genes through intravitreal injection. Quantitative relation between preparation properties with celluar uptake or transfection efficiency will be studied systematically to screen the gene vector. The mechanisms of celluar uptake and intracellular transport of the non-viral gene delivery system will be illuminated. In vitro and in vivo pharmacodynamics will be used as evaluation indexes to explore the mechanisms that how the system inhibit intraocular CNV formation. The study would be hopeful to provide theoretical foundation for the gene therapy of wet AMD.

脉络膜新生血管（CNV）是导致湿性AMD患者失明的重要原因，而触发CNV形成的关键因素是VEGF水平的明显升高，因此以眼部基因治疗阻断VEGF自分泌环路、从根源上下调VEGF水平，是CNV相关疾病治疗的研究重点。眼后段基因递送面临着血-视网膜、细胞膜和核膜等多重屏障，切实提高载体的转染率亦非常关键。故本项目选择视网膜细胞表面过表达的核仁素作为治疗靶点，以兼具CNV抑制作用的AS1411适配体作为细胞膜靶向配体，以核定位信号（NLS）作为细胞核靶向配体，构建脂质体/NLS/DNA复合物级联靶向基因递送系统，经玻璃体注射将抗VEGF内感受器质粒（Flt23K）高效递送至眼后段视网膜，研究系统的制剂性质与细胞摄取、转染效率间的定量关系，筛选出高效转染的基因载体，阐明系统的细胞摄取和胞内转运机制，以体内外药效学为评价指标，探索制剂抑制眼内CNV形成的机制，为湿性AMD基因治疗载体研究提供理论支撑。

本课题组在依托单位沈阳药科大学的支持下，通过与辽宁科技学院的紧密合作，顺利完成了研究计划，成功制备了新型视网膜级联靶向的非病毒基因载体，并对其抗脉络膜新生血管（CNV）机制进行了研究。.本课题首先以精氨酸、组氨酸和苯丙氨酸修饰的树形高分子（PRHF）和核定位信号（NLS）对基因进行压缩，使构建的载体增强细胞摄取、提高溶酶体逃逸能力并将基因转运进细胞核，有效克服细胞内所遇到的屏障。通过对基因载体的最佳比例进行考察和研究，为后续扩展其应用奠定了基础。.随后，考虑到眼部独特的生理环境和视网膜细胞表面表达的CD44受体，我们将所构建的载体载入脂质体中并在最外层修饰HA后作为细胞膜靶向配体，成功构建了可靶向视网膜的新型非病毒基因载体。对所构建的非病毒基因载体进行细胞学评价，结果表明：制备的基因载体具有较高的转染效率，对眼部细胞基本安全，进入细胞后的载体还能有效的从溶酶体逃逸并转运进细胞核，并且包载治疗基因抗VEGF内感受器质粒（Flt23K）后能有效抑制缺氧条件下VEGF的表达。玻璃体注射后，可在小鼠视网膜处发现EGFP的表达，且具有良好的生物安全性。.本课题所构建的级联靶向载体利用透明质酸衍生物实现眼后段视网膜靶向，NLS实现细胞核靶向，从而达到级联靶向和高效转染等目的并能克服转染过程中遇到的各种屏障。为包括CNV在内的视网膜疾病基因治疗载体的研究提供了新的思路和途径。