Krusch Stefan, Domann Eugen, Frings Monika, Zelmer Andrea, Diener Martin, Chakraborty Trinad, Weiss Siegfried
Division of Cell Biology and Immunology, GBF, German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany.
J Gene Med. 2002 Nov-Dec;4(6):655-67. doi: 10.1002/jgm.313.
Several approaches for gene therapy of cystic fibrosis using viral and non-viral vectors are currently being undertaken. Nevertheless, the present data suggest that vectors currently being used will either have to be further modified or, alternatively, novel vector systems need to be developed. Recently, bacteria have been proven as suitable vehicles for DNA transfer to a wide variety of eukaryotic cells. In this study, we assessed the ability of the facultative intracellular pathogen Listeria monocytogenes to deliver a cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR) to CHO-K1 cells, since these cells have been extensively used for heterologous CFTR expression.
An established in vitro gene transfer system based on antibiotic-mediated lysis of intracellular L. monocytogenes was exploited to transfer eukaryotic expression plasmids. Transient as well as stable CFTR transgene expression was analyzed by microscopical and biochemical methods; functionality was tested by whole-cell patch-clamp recordings.
L. monocytogenes mediated gene transfer to CHO-K1 cells was facilitated by an improved transfection protocol. In addition, the use of the isogenic mutant L. monocytogenes hlyW491A, engineered to produce a hemolysin variant with low toxigenic activity, greatly enhanced the efficiency of gene transfer. This strain allowed the transfer of functional CFTR to CHO-K1 cells.
This is the first demonstration of L. monoyctogenes mediated CFTR transgene transfer. The successful in vitro transfer suggests that L. monocytogenes might be a potential vector for cystic fibrosis gene therapy or alternative applications and deserves further investigation in vitro as well as in vivo.
目前正在采用多种方法，利用病毒和非病毒载体对囊性纤维化进行基因治疗。然而，目前的数据表明，目前使用的载体要么需要进一步修饰，要么需要开发新的载体系统。最近，细菌已被证明是将DNA转移到多种真核细胞的合适载体。在本研究中，我们评估了兼性细胞内病原体单核细胞增生李斯特菌将编码人囊性纤维化跨膜传导调节因子（CFTR）的cDNA传递至CHO-K1细胞的能力，因为这些细胞已被广泛用于异源CFTR表达。
利用基于抗生素介导的细胞内单核细胞增生李斯特菌裂解的成熟体外基因转移系统来转移真核表达质粒。通过显微镜和生化方法分析瞬时和稳定的CFTR转基因表达；通过全细胞膜片钳记录测试其功能。
改进的转染方案促进了单核细胞增生李斯特菌介导的基因向CHO-K1细胞的转移。此外，使用经过基因工程改造以产生低毒力溶血素变体的同基因突变体单核细胞增生李斯特菌hlyW491A，大大提高了基因转移效率。该菌株能够将功能性CFTR转移至CHO-K1细胞。
这是首次证明单核细胞增生李斯特菌介导CFTR转基因转移。体外转移成功表明，单核细胞增生李斯特菌可能是囊性纤维化基因治疗或其他应用的潜在载体，值得在体外和体内进行进一步研究。
