Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector
Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector
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DOI：
10.1089/hum.1998.9.4-575
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发表时间：
1998-03-01
影响因子：
4.2
通讯作者：
Kinnon, C
中科院分区：
文献类型：
--
作者：
Hart, SL;Arancibia-Cárcamo, CV;Kinnon, C
由整合素靶向肽/DNA（ID）复合物组成的非病毒载体具有广泛应用于基因治疗的潜力，然而，这种载体的转染效率受到内体降解的限制。我们现在报道，将lipofectin（L）掺入ID复合物中增强整合素介导的转染，使荧光素酶表达增加100倍以上。通过β-半乳糖苷酶报告基因表达和X-gal染色评估的Lipofectin/整合素结合肽/DNA（LID）复合物的转染效率对于三种不同的细胞系从1%提高到10%到超过50%，并且在体外角膜内皮中从0%提高到约25%，转染复合物的转染效率已被优化，我们已经研究了它们的结构，功能和转染模式，ID和LID复合物均形成颗粒，与脂质体/DNA复合物（LD）形成的纤维状网络不同，LID复合物介导的整合素转染通过LKD复合物的显著较低的转染效率来证明，其中整合素结合肽取代K（16）（K）。此外，复合物的转染效率显示依赖于复合物中整联蛋白靶向配体的量。最后，用竞争性单克隆抗体实现了LID复合物的整联蛋白介导的转染效率降低34%。增强的机制可能涉及DNA的内体降解程度的降低。
Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy, The transfection efficiency of this vector, however, has been limited by endosomal degradation, We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing luciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by beta-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro, Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection, Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD), Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K(16) (K) Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex, Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody, The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection, The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA.