Barrett L B, Berry M, Ying W-B, Hodgkin M N, Seymour L W, Gonzalez A-M, Read M L, Baird A, Logan A
Molecular Neuroscience Group, Department of Medicine, University of Birmingham, Birmingham B15 2TH, UK.
J Gene Med. 2004 Apr;6(4):429-38. doi: 10.1002/jgm.524.
Efficient neuronal gene therapy is a goal for the long-term repair and regeneration of the injured central nervous system (CNS). We investigated whether targeting cDNA to neurons with cholera toxin b chain conjugated non-viral polyplexes led to increased efficiency of non-viral gene transfer in the CNS. Here, we illustrate the potential for this strategy by demonstrating enhanced transfection of a differentiated neuronal cell type, PC12.
In vitro transfection efficiency of a cholera toxin b chain-poly(D-lysine) molecular conjugate (CTb-K(100)) was compared by fluorescence-activated cell sorting (FACS) analysis of green fluorescent protein (GFP) expression and luminometric measurement of beta-galactosidase (beta-gal) expression, to untargeted poly(D-lysine) (K(100)) in undifferentiated and NGF-differentiated PC12 cells.
Transfection of undifferentiated PC12 cells with CTb-K(100) polyplexes resulted in a 36-fold increase in levels of pCMV-DNA(LacZ) expression and a 20-fold increase in the frequency of transduction with pCMV-DNA(GFP), compared with untargeted K(100) polyplexes. Treatment of PC12 cells with 50 ng/ml/day of NGF for 14 days led to differentiation to a neuronal phenotype. Transfection of NGF-differentiated cells with CTb-K(100) polyplexes resulted in a 133-fold increase in levels of pCMV-DNA(LacZ) expression and a 11-fold increase in the percentage of cells transduced with pCMV-DNA(GFP), compared with untargeted K(100) polyplexes. Transfection was dependent on CTb, with CTb-K(100)-mediated transfections competitively inhibited with free CTb in both PC12 phenotypes.
Non-viral systems for gene transfer in damaged CNS show superior toxicological profiles to most viruses but are limited by inefficient and non-selective gene expression in target tissue. Cholera toxin is known to interact preferentially with neuronal cells of the central and peripheral nervous systems, mediating binding through the b subunit, CTb, and the pentasaccharide moiety of the gangliosaccharide, GM1, which is present at high levels on the neuronal cell surface. Here, we show that a molecular conjugate of the CTb subunit, covalently linked to poly(D-lysine), is able to successfully target and significantly enhance transfection of a neuronal cell type, NGF-differentiated rat PC12 pheochromocytoma cells. This observation encourages the further development of non-viral strategies for the delivery of therapeutic genes to neurons.
高效的神经元基因治疗是受损中枢神经系统（CNS）长期修复和再生的目标。我们研究了用霍乱毒素b链偶联的非病毒多聚体将互补DNA（cDNA）靶向神经元是否能提高中枢神经系统中非病毒基因转移的效率。在此，我们通过证明分化的神经元细胞类型PC12的转染增强，阐述了该策略的潜力。
通过对绿色荧光蛋白（GFP）表达的荧光激活细胞分选（FACS）分析和β-半乳糖苷酶（β-gal）表达的发光测量，比较霍乱毒素b链-聚（D-赖氨酸）分子偶联物（CTb-K(100)）与未靶向的聚（D-赖氨酸）（K(100)）在未分化和神经生长因子（NGF）分化的PC12细胞中的体外转染效率。
与未靶向的K(100)多聚体相比，用CTb-K(100)多聚体转染未分化的PC12细胞导致pCMV-DNA(LacZ)表达水平增加36倍，pCMV-DNA(GFP)转导频率增加20倍。用50 ng/ml/天的NGF处理PC12细胞14天导致分化为神经元表型。与未靶向的K(100)多聚体相比，用CTb-K(100)多聚体转染NGF分化的细胞导致pCMV-DNA(LacZ)表达水平增加133倍，pCMV-DNA(GFP)转导的细胞百分比增加11倍。转染依赖于CTb，在两种PC12表型中，CTb-K(100)介导的转染均被游离CTb竞争性抑制。
用于受损中枢神经系统基因转移的非病毒系统在毒理学方面比大多数病毒更具优势，但受限于靶组织中低效和非选择性的基因表达。已知霍乱毒素优先与中枢和外周神经系统的神经元细胞相互作用，通过b亚基CTb和神经节苷脂GM1的五糖部分介导结合，GM1在神经元细胞表面高水平存在。在此，我们表明CTb亚基与聚（D-赖氨酸）共价连接的分子偶联物能够成功靶向并显著增强神经元细胞类型（NGF分化的大鼠PC12嗜铬细胞瘤细胞）的转染。这一观察结果鼓励进一步开发用于向神经元递送治疗性基因的非病毒策略。
