HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells.
HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells.
复制标题
DOI：
10.1089/hum.1997.8.3-359
复制
发表时间：
1997-02
影响因子：
4.2
通讯作者：
Karen M. Johnston;David R. Jacoby;Peter A. Pechan;C. Fraefel;Paul R. Borghesani;D. Schuback;Robert J. Dunn;Frances I. Smith;X. Breakefield
中科院分区：
文献类型：
--
作者：
Karen M. Johnston;David R. Jacoby;Peter A. Pechan;C. Fraefel;Paul R. Borghesani;D. Schuback;Robert J. Dunn;Frances I. Smith;X. Breakefield
新型杂交载体包含单纯疱疹病毒1型（HSV-1）扩增子载体和腺相关病毒（AAV）载体的关键元素，能够在分裂的胶质瘤细胞中维持转基因表达超过2周。这些载体结合了HSV-1载体的高传染性和大的转基因能力，以及AAV载体的表观扩增和染色体整合的潜力。杂交载体包含HSV-1的DNA复制源oriS和DNA切割/包装信号pac，它们允许在HSV-1病毒粒子中扩增子复制和包装。受CMV IE1启动子控制的lacZ报告基因两侧有AAV反向末端重复序列（ITR），这有利于该卡带在宿主细胞核中的复制和基因组整合。构建带或不带AAV rep基因（rep+和rep-），以评估其在延长转基因表达中的重要性。Western blot分析证实Rep蛋白的表达。用含有报告基因但不含AAV序列的HSV-1扩增子构建物作为对照。构建物被包装到有或没有辅助病毒的HSV-1病毒体中，这些载体库用于感染培养的人U87胶质瘤细胞。杂交载体支持转基因保留和表达超过2周，而对照扩增子载体在10天后失去转基因。与rep-杂交载体相比，rep+的表达时间稍长。使用无辅助病毒扩增子载体消除HSV-1辅助病毒引起的毒性。利用AAV和腺病毒或HSV-1辅助功能，转基因构建物也可以包装在AAV病毒粒子中。这些HSV/AAV杂交载体应该允许长期、无毒的DNA结构基因传递到分裂和非分裂细胞。
Novel hybrid vectors, which incorporate critical elements of both herpes simplex virus type 1 (HSV-1) amplicon vectors and adeno-associated virus (AAV) vectors, are able to sustain transgene expression in dividing glioma cells for over 2 weeks. These vectors combine the high infectibility and large transgene capacity of HSV-1 vectors with the potential for episomal amplification and chromosomal integration of AAV vectors. The hybrid vectors contain the HSV-1 origin of DNA replication, oriS, and the DNA cleavage/packaging signal, pac, which allow amplicon replication and packaging in HSV-1 virions. The lacZ reporter gene under control of the CMV IE1 promoter is flanked by AAV inverted terminal repeat (ITR) sequences, which facilitate replication and genomic integration of this cassette in the host cell nucleus. Constructs were generated with or without the AAV rep gene (rep+ and rep-) to assess its importance in extending transgene expression. Expression of Rep proteins was confirmed by Western blot analysis. An HSV-1 amplicon construct containing the reporter gene, but no AAV sequences, was used as a control. Constructs were packaged into HSV-1 virions with or without helper virus and these vector stocks were used to infect human U87 glioma cells in culture. The hybrid vectors supported transgene retention and expression for over 2 weeks, whereas the control amplicon vector lost the transgene after 10 days. Expression was somewhat longer for the rep+ as compared to the rep- hybrid vectors. Toxicity due to the HSV-1 helper virus was eliminated using helper virus-free amplicon vector stocks. Transgene constructs could also be packaged in AAV virions, using AAV and adenovirus or HSV-1 helper functions. These HSV/AAV hybrid vectors should allow long-term, nontoxic gene delivery of DNA constructs to both dividing and nondividing cells.