PACKAGING CAPACITY AND STABILITY OF HUMAN ADENOVIRUS TYPE-5 VECTORS
PACKAGING CAPACITY AND STABILITY OF HUMAN ADENOVIRUS TYPE-5 VECTORS
复制标题
DOI：
10.1128/jvi.67.10.5911-5921.1993
复制
发表时间：
1993-10-01
影响因子：
5.4
通讯作者：
GRAHAM, FL
中科院分区：
文献类型：
--
作者：
BETT, AJ;PREVEC, L;GRAHAM, FL
腺病毒载体广泛用于哺乳动物细胞中蛋白质的高水平表达，并且由于其作为活重组疫苗和作为用于基因治疗的转导病毒的潜在用途而受到越来越多的关注。尽管人们普遍认为腺病毒载体的主要优点之一是它们的相对稳定性，但这还没有得到彻底的研究。为了检测5型腺病毒载体的遗传稳定性，特别是检测遗传稳定性与基因组大小之间的关系，构建了具有4.88（单纯疱疹病毒1型gB），4.10（单纯疱疹病毒1型gB），或3.82（LacZ）kb与1.88-kb E3缺失或与新产生的2.69-kb E3缺失组合。DNA超过野生型（wt）基因组大小的净过量范围为1.13至3.00 kb或3.1至8.3%。在组织培养中连续传代期间对这些载体的分析揭示，当大小超过野生型基因组长度的105%约1.2kb（4.88-kb插入物与1.88-kb缺失物组合）时，所得载体生长非常差并经历快速重排，导致仅在几次传代后插入物丢失。相比之下，具有插入物的载体导致病毒DNA接近或小于wt的净基因组大小的105%，并且生长良好并且相对稳定。一般来说，基因组仅略高于野生型105%的病毒是不稳定的，重排发生的速度与插入片段的大小相关。这些发现表明，可以包装成病毒体的DNA的量有一个相对严格的限制，超过限制会导致病毒生长速率急剧下降。由此产生的对经历重排的变体的强选择，产生较小的基因组，表现为病毒群体的遗传不稳定性。
Adenovirus vectors are extensively used for high-level expression of proteins in mammalian cells and are receiving increasing attention for their potential use as live recombinant vaccines and as transducing viruses for use in gene therapy. Although it is commonly argued that one of the chief advantages of adenovirus vectors is their relative stability, this has not been thoroughly investigated. To examine the genetic stability of adenovirus type 5 vectors and in particular to examine the relationship between genetic stability and genome size, adenovirus vectors were constructed with inserts of 4.88 (herpes simplex virus type 1 gB), 4.10 (herpes simplex virus type 1 gB), or 3.82 (LacZ) kb combined with a 1.88-kb E3 deletion or with a newly generated 2.69-kb E3 deletion. The net excess of DNA over the wild-type (wt) genome size ranged from 1.13 to 3.00 kb or 3.1 to 8.3%. Analysis of these vectors during serial passage in tissue culture revealed that when the size exceeded 105% of the wt genome length by approximately 1.2 kb (4.88-kb insert combined with a 1.88-kb deletion), the resulting vector grew very poorly and underwent rapid rearrangement, resulting in loss of the insert after only a few passages. In contrast, vectors with inserts resulting in viral DNA close to or less than a net genome size of 105% of that of the wt grew well and were relatively stable. In general, viruses with genomes only slightly above 105% of that of the wt were unstable and the rapidity with which rearrangement occurred correlated with the size of the insert. These findings suggest that there is a relatively tight constraint on the amount of DNA which can be packaged into virions and that exceeding the limit results in a sharply decreased rate of virus growth. The resultant strong selection for variants which have undergone rearrangement, generating smaller genomes, is manifested as genetic instability of the virus population.