Lim F, Hartley D, Starr P, Lang P, Song S, Yu L, Wang Y, Geller A I
Children's Hospital, Boston, MA, USA.
Biotechniques. 1996 Mar;20(3):460-9. doi: 10.2144/19962003460.
Vectors based on herpes simplex virus type 1 (HSV-1) show promise for gene transfer into mammalian cells because of their wide host range, efficient infection and ability to deliver genes to nondividing cells. Defective HSV-1 vectors, or amplicons, are plasmid vectors which are unable to propagate on their own but contain specific HSV-1 sequences that, in the presence of helper virus, support DNA replication and subsequent packaging into virus particles. We compared three replication-incompetent HSV-1 mutants (KOS strain 5dl1.2, strain 17 D30EBA, KOS strain d120) as the helper virus for packaging the prototype defective HSV-1 vector, pHSVlac, which uses the HSV-1 immediate-early (1E) 4/5 promoter to regulate expression of the Escherichia coli lacZ gene. Use of 5dl1.2, which contains a deletion in the IE 2 gene, consistently produced virus stocks that contained a high level of vector, undetectable levels of wild-type HSV-1 and a ratio of vector to helper greater than 1. Virus stocks prepared using 5dl1.2 were superior to those prepared using helper viruses that harbor a deletion in the IE 3 gene, either D30EBA or dl20, and supported more efficient gene transfer than possible with previously published procedures. Lactate dehydrogenase efflux assays in rat cortical cultures showed that 5dl1.2 was no more cytotoxic than either D30EBA or dl20, despite the expression of more viral genes. Rat cortical cultures infected with pHSVlac packaged with either 5dl1.2 or D30EBA were used to quantify the stability of vector expression. Our results show a decrease in the number of cells with detectable levels of beta-galactosidase to 30% of peak levels after one week, irrespective of the helper virus used. However, simultaneous superinfection with 5dl1.2, but not with either D30EBA or dl20, produced a transient increase in the number of cells expressing beta-galactosidase. Superinfection with 5dl1.2 at 9 days after gene transfer increased the number of cells expressing detectable beta-galactosidase back to peak levels, most probably because of reactivation of the IE 4/5 promoter in pHSVlac. These results thus provide the first quantitative demonstration of long-term persistence of defective HSV-1 vectors in neurons.
基于单纯疱疹病毒1型（HSV-1）的载体因宿主范围广、感染效率高以及能够将基因传递给非分裂细胞，在将基因导入哺乳动物细胞方面显示出前景。缺陷型HSV-1载体，即扩增子，是无法自行繁殖的质粒载体，但含有特定的HSV-1序列，在辅助病毒存在的情况下，这些序列支持DNA复制并随后包装到病毒颗粒中。我们比较了三种复制缺陷型HSV-1突变体（KOS株5dl1.2、17株D30EBA、KOS株d120）作为辅助病毒，用于包装原型缺陷型HSV-1载体pHSVlac，该载体使用HSV-1立即早期（IE）4/5启动子来调节大肠杆菌lacZ基因的表达。使用在IE 2基因中有缺失 的5dl1.2，始终能产生含有高水平载体、未检测到野生型HSV-1且载体与辅助病毒比例大于1的病毒储备液。使用5dl1.2制备的病毒储备液优于使用在IE 3基因中有缺失的辅助病毒（D30EBA或dl20）制备的病毒储备液，并且比以前发表的方法支持更有效的基因转移。大鼠皮质培养物中的乳酸脱氢酶外排试验表明，尽管表达了更多的病毒基因，但5dl1.2的细胞毒性并不比D30EBA或dl20更强。用5dl1.2或D30EBA包装的pHSVlac感染的大鼠皮质培养物用于量化载体表达的稳定性。我们的结果表明，无论使用何种辅助病毒，一周后β-半乳糖苷酶可检测水平的细胞数量降至峰值水平的30%。然而，同时用5dl1.2进行超感染，而不是用D30EBA或dl20，会使表达β-半乳糖苷酶的细胞数量短暂增加。在基因转移后9天用5dl1.2进行超感染，使表达可检测β-半乳糖苷酶的细胞数量恢复到峰值水平，这很可能是由于pHSVlac中IE 4/5启动子的重新激活。因此，这些结果首次定量证明了缺陷型HSV-1载体在神经元中的长期持久性。
