‘Autoreplication’ of the vector genome in recombinant adenoviral vectors with different E1 region deletions and transgenes
‘Autoreplication’ of the vector genome in recombinant adenoviral vectors with different E1 region deletions and transgenes
复制标题
具有不同 E1 区域删除和转基因的重组腺病毒载体中载体基因组的“自动复制”
作者：
U. Marienfeld;A. Haack;P. Thalheimer;S. Schneider;H. Brackmann;W. Poller
据报道，腺病毒介导的基因转移后，在免疫缺陷宿主中转基因的稳定性可达1年或更长时间。这种持续时间的转基因持久性可能是由于附加型病毒载体DNA固有的高稳定性。另一种解释是转基因载体DNA的有限“自动复制”，仅足以抵消宿主细胞内缓慢但持续的降解。仅基于腺病毒DNA复制系统的残余活性，在不产生任何感染性病毒颗粒的情况下可发生自动复制。为了检验这一假设，在用不同载体转染的非允许细胞培养物中进行了一系列DNA代谢标记研究。由于广泛的E1区缺失，没有一种载体能够在非允许细胞中产生病毒子代。然而，就其自动复制潜力而言，载体分为两类。在非允许载体转染的细胞中，载体DNA的新合成在“A型”载体中容易检测到，但在“B型”载体中不能检测到。除了它们不同的转基因表达盒之外，载体DNA测序显示与B型载体（核苷酸325-3523）相比，A型载体（野生型病毒的核苷酸453-3333）中E1缺失的范围较小。与B型载体相比，自动复制还与几种病毒基因（E1 B-14 k，腺病毒DNA聚合酶，单链DNA结合蛋白，E4- 25 k）的高转录活性相关。除了这些“野生型”转录本，在含有转基因cDNA与腺病毒载体序列的自动复制载体中检测到“不规则”重组转录本。外源性或隐蔽性启动子可以（在某些条件下）以发生自动复制的方式增强载体的转录活性。决定转录增强水平的条件（E1缺失的程度、启动子和转基因的类型等）需要在具有高自动复制能力的腺病毒载体的合理设计成为可能之前进一步确定。总之，我们已经证明了自身复制是某些E1缺失腺病毒载体的一个新特征，可能与其体内稳定性相关，但也可能对靶细胞功能或载体免疫原性产生不利影响。因此，腺病毒载体系统的完整表征应包括其自身复制能力的描述。
High transgene stabilities of 1 year and more have been reported in immunodeficient hosts after adenovirus-mediated gene transfer. Transgene persistence of this duration could be due to inherently high stability of the episomal viral vector DNA. An alternative explanation would be limited ‘autoreplication’ of transgenic vector DNA, just sufficient to counteract slow but continuous degradation within the host cells. Autoreplication could occur in the absence of any production of infectious virus particles, based on residual activity of the adenoviral DNA replication system only. To test this hypothesis, a series of DNA metabolic labeling studies in non-permissive cells cultures transfected with different vectors was conducted. Due to extensive E1 region deletions none of the vectors was able to produce viral progeny in non-permissive cells. Vectors fell into two categories, however, with respect to their autoreplication potential. Neosynthesis of vector DNA in non-permissive vector-transfected cells was readily detectable in ‘type A’, but not in ‘type B’ vectors. In addition to their different transgene expression cassettes, vector DNA sequencing showed a less extensive E1 deletion in type A (nucleotides 453–3333 of wild-type virus) as compared to type B vectors (nucleotides 325–3523). Autoreplication was also associated with high transcriptional activity of several viral genes (E1B-14k, adenoviral DNA polymerase, single-strand DNA-binding protein, E4-25k), in contrast to type B vectors. In addition to these ‘wild-type’ transcripts, ‘irregular’ recombinant transcripts were detected in autoreplication vectors which contained the transgenic cDNA in conjunction with adenoviral vector sequences. Exogenous or cryptic promotors may (under certain conditions) enhance the transcriptional activity of a vector in such a way that autoreplication occurs. Conditions determining the level of transcriptional enhancement (extent of E1 deletion, type of promoter and transgene, etc) need to be further defined before rational design of adenovectors with high autoreplication capacity becomes possible. In summary, we have shown autoreplication to be a novel feature of certain E1-deleted adenovectors with likely relevance for their stability in vivo, but also with possibly adverse consequences for target cell function or vector immunogenicity. Full characterization of adenoviral vector systems should therefore include a description of their autoreplication capacity.
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DOI：
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发表时间：
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影响因子：
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DOI：
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