Parker Alan L, Collins Louise, Zhang Xiaohong, Fabre John W
Department of Clinical Sciences, Guys, Kings and St Thomas School of Medicine, The Rayne Institute, London SE5 9NU, UK.
J Gene Med. 2005 Dec;7(12):1545-54. doi: 10.1002/jgm.809.
The immunogenicity of viral DNA vectors is an important problem for gene therapy. The use of peptide motifs for gene delivery would largely overcome this problem, and provide a simple, safe and powerful approach for non-viral gene therapy.
We explored the functional properties of two motifs: the (Lys)(16) motif (for binding and condensing DNA, and probably also nuclear translocation of plasmids) and the fusogenic peptide motif of influenza virus (for acid-dependent endocytic escape of peptide/DNA particles). The physical properties and gene delivery efficiencies of (Lys)(16)-containing peptides in combination with free fusogenic peptide were evaluated, and compared with a single composite peptide incorporating both moieties. Post-mitotic corneal endothelial cells and growth-arrested HeLa were included, so as not to neglect the question of nuclear translocation of plasmids.
The fusogenic moiety in the composite peptide was able to adopt an alpha-helical configuration unhindered by the (Lys)(16) moiety, and retained acid-dependent fusogenic properties. The composite peptide gave remarkably high levels of gene delivery to dividing cell lines. However, in marked contrast to (Lys)(16)/DNA complexes plus free fusogenic peptide, the composite peptide was completely ineffective for gene delivery to post-mitotic and growth-arrested cells.
Attachment of the fusogenic peptide to (Lys)(16) appears to block (Lys)(16)-mediated nuclear translocation of plasmid, but not fusogenic peptide mediated endocytic escape. This strengthens the experimental basis for (Lys)(16)-mediated nuclear translocation of plasmids, and provides a single peptide with potent gene delivery properties, restricted to dividing cells. This property is potentially useful in experimental biology and clinical medicine.
病毒DNA载体的免疫原性是基因治疗中的一个重要问题。使用肽基序进行基因递送将在很大程度上克服这一问题，并为非病毒基因治疗提供一种简单、安全且有效的方法。
我们探究了两种基序的功能特性：（赖氨酸）16基序（用于结合和凝聚DNA，可能还参与质粒的核转运）和流感病毒的融合肽基序（用于肽/DNA颗粒的酸依赖性胞吞逃逸）。评估了含（赖氨酸）16的肽与游离融合肽结合后的物理性质和基因递送效率，并与包含这两种部分的单一复合肽进行比较。纳入了有丝分裂后角膜内皮细胞和生长停滞的HeLa细胞，以免忽略质粒核转运的问题。
复合肽中的融合部分能够不受（赖氨酸）16部分的阻碍而形成α螺旋结构，并保留酸依赖性融合特性。复合肽对分裂细胞系的基因递送水平显著较高。然而，与（赖氨酸）16/DNA复合物加游离融合肽形成鲜明对比的是，复合肽对有丝分裂后和生长停滞细胞的基因递送完全无效。
将融合肽连接到（赖氨酸）16上似乎会阻断（赖氨酸）16介导的质粒核转运，但不会阻断融合肽介导的胞吞逃逸。这加强了（赖氨酸）16介导质粒核转运的实验基础，并提供了一种仅对分裂细胞具有高效基因递送特性的单一肽。这一特性在实验生物学和临床医学中可能具有实用性。
