具有双重精准识别功能的单抗靶向型脱氧核酶传输体系的构建与评价

结题报告

项目介绍

AI项目解读

基本信息

批准号：
81373344
项目类别：
面上项目
资助金额：
65.0万
负责人：
李全顺
依托单位：
吉林大学
学科分类：
H3408.药剂学
结题年份：
2017
批准年份：
2013
项目状态：
已结题
起止时间：
2014-01-01 至2017-12-31

项目参与者：
孙严彤；孙凤英；滕乐生；王冠；相世栋；张剑旭；李春杰；孙玉波；
关键词：
肿瘤靶向 HLA-G单克隆抗体 PEI衍生物脱氧核酶 Michael加成

项目摘要

In this project, PEI25K is first modified by N-isopropyl acrylamide via Michael addition reaction to achieve PEI derivative (PEN) with high gene transfection efficiency and low toxicity. Through NHS-PEG5K-Mal strategy, PEN is chemically conjugated to an anti-HLA-G monoclonal antibody. As HLA-G is highly expressed in many tumor cells, the antibody-PEN conjugate can be a novel specific gene carrier with high tumor-targeting ability. Meanwhile, the involvement of hydrophilic PEG segment could efficiently facilitate the circulation time of carriers in the blood. Then, the electrostatic assemble strategy is employed to construct a nanocomplex of the tumor-targeting gene carrier and anti-IGF-II P3 mRNA DNAzyme with high sequence recognition specificity and cleavage ability. Finally, using the transfection assay of this nanocomplex, the ability to induce the apoptosis of tumor cells and detailed tumor-targeting mechanism are systematically evaluated on both cell and animal levels. In conclusion, the project constructs a novel gene delivery system with dual precise recognition, in which monoclonal antibody realizes the location of carrier in the tumor cells and DNAzyme achieves the specific recognition and cleavage towards the targeting mRNA sequence. Thus, the project provides a new insight into the development of tumor-targeting gene medicine and the clinic appliction of cancer gene therapy.

本项目首次通过Michael加成反应，实现N-异丙基丙烯酰胺对PEI25K的修饰，合成了高基因转染效率、低毒的基因传输载体PEN；通过NHS-PEG5K-Mal键合策略，将全新的、特性异针对恶性肿瘤细胞表面高表达的HLA-G的单克隆抗体与PEN偶联，并引入亲水性PEG分子，构建靶向型、体内长循环基因传输载体；通过静电组装的策略，将该肿瘤靶向型基因传输载体与对肝癌细胞中IGF-II P3 mRNA具有特异性识别与切割能力的脱氧核酶形成纳米复合物，并在细胞和动物水平上系统评估其诱导肿瘤细胞凋亡的能力与靶向作用机制，实现脱氧核酶的高效、稳定、靶向传输。本项目的实施，将构建一类单克隆抗体精确定位肿瘤细胞和脱氧核酶靶向切割癌基因序列的具有双重精准识别功能的基因药物传输体系，为靶向型基因治疗药物的研究开发及肿瘤基因治疗的临床应用提供新的思路。

结项摘要

本项目立足于脱氧核酶所介导的肿瘤基因治疗，构建了阳离子聚合物及外泌体型脱氧核酶传输体系，实现了脱氧核酶分子的精准、稳定、靶向递送，具体研究内容包括：(1) 以N-异丙基丙烯酰胺修饰PEI为载体，介导脱氧核酶Dz13的高效递送，实现了对肿瘤细胞增殖、迁移与浸润的抑制，为该具有临床应用前景的脱氧核酶分子高效应用提供了新的思路；(2) 以疏水性氨基酸修饰PEI衍生物为载体，介导以极光激酶A为靶点脱氧核酶的高效、稳定递送，实现了脱氧核酶分子的高效入胞及内涵体逃逸，同时脱氧核酶的传输实现了对肿瘤细胞增殖、迁移与浸润的抑制，为未来脱氧核酶基因药物的开发奠定了良好的基础；(3) 基于计算机辅助设计的策略，筛选获得了高效切割靶基因SALL4 mRNA的脱氧核酶分子，并以外泌体为传输载体，通过该脱氧核酶的高效递送达到了对肿瘤增殖与迁移的抑制，同时该传输体系能实现药物在肿瘤组织的深层渗透。本项目的实施，构建了一类脱氧核酶靶向切割癌基因序列的新型精准识别功能的基因药物传输体系，为靶向型基因治疗药物的研究开发及肿瘤基因治疗的临床应用提供新的思路。