单纯疱疹病毒1型重组载体与扩增子载体联用：复制能力完整型与缺陷型及其对实验性胶质瘤的治疗效果

Combined HSV-1 recombinant and amplicon piggyback vectors: replication-competent and defective forms, and therapeutic efficacy for experimental gliomas.

作者信息

Pechan P A, Herrlinger U, Aghi M, Jacobs A, Breakefield X O

机构信息

Department of Neurology, Massachusetts General Hospital East, Harvard Medical School Neuroscience Program, Boston 02129, USA.

出版信息

J Gene Med. 1999 May-Jun;1(3):176-85. doi: 10.1002/(SICI)1521-2254(199905/06)1:3<176::AID-JGM35>3.0.CO;2-T.

DOI:10.1002/(SICI)1521-2254(199905/06)1:3<176::AID-JGM35>3.0.CO;2-T

PMID:10738566

Abstract

BACKGROUND

The versatility of HSV-1 vectors includes large transgene capacity, selective replication of mutants in dividing cells, and availability of recombinant virus (RV) and plasmid-derived (amplicon) vectors, which can be propagated in a co-dependent, 'piggyback', manner.

METHODS

A replication-defective piggyback vector system was generated in which the amplicon carries either of two genes essential for virus replication, IE2 (ICP27) or IE3 (ICP4), as well as lacZ; the RV is deleted in both these genes, and vector stocks are propagated in cells transfected with one of the complementary genes. In the replication-competent system, the amplicon carries the IE2 and lacZ; the RV had a large deletion in the IE2; and stocks are propagated in untransfected cells. Titers over successive passages, recombination between amplicon and RV, and the structural integrity of vector genomes were evaluated. The replication-competent system was tested for therapeutic efficacy in subcutaneous 9L gliosarcoma tumors in nude mice with activation of ganciclovir via the viral HSV-thymidine kinase gene.

RESULTS

Both systems generated high titer amplicon vectors (about 10(7) tu/ml) and amplicon:RV ratios (0.6-3.0). No replication-competent RV was generated in either system. The replication-defective system showed low toxicity and increased packaging efficiency of amplicon vectors, as compared to single mutant RV helper virus. The replication-competent system allowed co-propagation of amplicon and RV; injection into tumors followed by ganciclovir treatment inhibited tumor growth without systemic toxicity.

CONCLUSION

New replication-defective and replication-competent piggyback HSV, vector systems allow gene delivery via amplicon vectors with reduced toxicity and co-propagation of both RV and amplicon vectors in target cells, with effective tumor therapy via focal virus replication and pro-drug activation.

摘要

背景

单纯疱疹病毒1型（HSV-1）载体具有多种特性，包括转基因容量大、突变体在分裂细胞中选择性复制，以及存在重组病毒（RV）和质粒衍生（扩增子）载体，这些载体能够以共依赖的“搭载”方式进行增殖。

方法

构建了一种复制缺陷型搭载载体系统，其中扩增子携带病毒复制所需的两个基因之一，即IE2（ICP27）或IE3（ICP4），以及lacZ；RV在这两个基因中均缺失，载体原液在转染了其中一个互补基因的细胞中进行增殖。在复制 competent 系统中，扩增子携带IE2和lacZ；RV在IE2中有大片段缺失；原液在未转染的细胞中进行增殖。评估了连续传代后的滴度、扩增子与RV之间的重组以及载体基因组的结构完整性。在通过病毒HSV - 胸苷激酶基因激活更昔洛韦的情况下，对复制 competent 系统在裸鼠皮下9L胶质肉瘤肿瘤中的治疗效果进行了测试。

结果

两个系统均产生了高滴度的扩增子载体（约10⁷tu/ml）和扩增子:RV比率（0.6 - 3.0）。两个系统均未产生具有复制能力的RV。与单突变RV辅助病毒相比，复制缺陷型系统显示出低毒性且扩增子载体的包装效率提高。复制 competent 系统允许扩增子和RV共同增殖；注入肿瘤后进行更昔洛韦治疗可抑制肿瘤生长且无全身毒性。