Konopka K, Harrison G S, Felgner P L, Düzgüneş N
Department of Microbiology, School of Dentistry, University of the Pacific, San Francisco, CA 94115, USA.
Biochim Biophys Acta. 1997 Apr 24;1356(2):185-97. doi: 10.1016/s0167-4889(96)00176-0.
HIV-regulated expression of the diphtheria toxin A fragment gene (HIV-DT-A) is a potential gene therapy approach to AIDS. Since cationic liposomes are safe and non-immunogenic for in vivo gene delivery, we examined whether LipofectAMINE or DMRIE reagent could mediate the transfection of HIV-DT-A (pTHA43) or the HIV-regulated luciferase gene (pLUCA43) into HIV-infected or uninfected HeLa cells. pLUCA43 was expressed at a 10(3)-fold higher level in HeLa/LAV cells than in uninfected HeLa cells, while the extent of expression of RSV-regulated luciferase was the same in both cell lines. Co-transfection of HeLa cells with pTHA43 and the proviral HIV clone, HXB deltaBgl, resulted in complete inhibition of virus production. In contrast, the delivery of HIV-DT-A to chronically infected HeLa/LAV or HeLa/IIIB cells, or to HeLa CD4+ cells before infection, did not have a specific effect on virus production, since treatment of cells with control plasmids also reduced virus production. This reduction could be ascribed to cytotoxicity of the reagents. The efficiency of transfection, as measured by the percentage of cells expressing beta-gal, was approximately 5%. Thus, cationic liposome-mediated transfection was too inefficient to inhibit virus production when the DT-A was delivered by cationic liposomes to chronically- or de novo- infected cells. However, when both the virus and DT-A genes were delivered into the same cells by cationic liposomes, DT-A was very effective at inhibiting virus production. Our results indicate that the successful use of cationic liposomes for gene therapy will require the improvement of their transfection efficiency.
HIV调控的白喉毒素A片段基因（HIV-DT-A）表达是一种针对艾滋病的潜在基因治疗方法。由于阳离子脂质体对于体内基因传递是安全且无免疫原性的，我们研究了LipofectAMINE或DMRIE试剂是否能介导HIV-DT-A（pTHA43）或HIV调控的荧光素酶基因（pLUCA43）转染到HIV感染或未感染的HeLa细胞中。pLUCA43在HeLa/LAV细胞中的表达水平比未感染的HeLa细胞高10³倍，而呼吸道合胞病毒（RSV）调控的荧光素酶在两种细胞系中的表达程度相同。用pTHA43和前病毒HIV克隆HXB deltaBgl共转染HeLa细胞，导致病毒产生完全受到抑制。相比之下，将HIV-DT-A导入慢性感染的HeLa/LAV或HeLa/IIIB细胞，或在感染前导入HeLa CD4⁺细胞，对病毒产生没有特异性影响，因为用对照质粒处理细胞也会降低病毒产生。这种降低可能归因于试剂的细胞毒性。通过表达β-半乳糖苷的细胞百分比衡量的转染效率约为5%。因此，当通过阳离子脂质体将DT-A递送至慢性或初发感染细胞时，阳离子脂质体介导的转染效率太低，无法抑制病毒产生。然而，当通过阳离子脂质体将病毒和DT-A基因都递送至同一细胞时，DT-A在抑制病毒产生方面非常有效。我们的结果表明，要成功将阳离子脂质体用于基因治疗，需要提高其转染效率。
