Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted
Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted
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DOI：
10.1128/jvi.72.2.926-933.1998
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发表时间：
1998-02-01
影响因子：
5.4
通讯作者：
Chamberlain, JS
中科院分区：
文献类型：
--
作者：
Amalfitano, A;Hauser, MA;Chamberlain, JS
腺病毒载体在基因治疗遗传性和非遗传性疾病方面具有巨大的应用潜力。虽然能够有效地转导分裂和静止细胞，但Ad载体受到许多问题的限制。大多数Ad载体被工程化，使得转基因取代rid E1 a、E1 b和E3基因;随后，复制缺陷型载体只能在反式提供缺失的E1基因功能的人293细胞中增殖。不幸的是，已经反复证明使用高滴度的E1缺失载体导致仍然驻留在载体中的病毒基因的低水平表达。此外，通过与存在于293细胞中的E1序列的重组事件产生可复制的Ad（RCA）进一步限制了E1缺失的Ad载体的有用性。我们通过分离缺失E1、E3和E2 b基因功能的新Ad载体解决了这些问题。新载体可以容易地生长到高滴度并且具有几个改进，包括增加的携带能力和理论上降低的产生RCA的风险。我们还证明了与常规的E1-缺失的载体相比，通过E1和E2 b基因缺失提供的对Ad载体复制的进一步阻断显著降低了Ad晚期基因表达，这些结果表明，这些修饰的载体对于体外和体内基因治疗应用都非常有用。
Adeno virus (Ad)-based vectors have great potential for use in the gene therapy of multiple diseases, both genetic and nongenetic. While capable of transducing both dividing and quiescent cells efficiently, Ad vectors have been limited by a number of problems, Most Ad vectors are engineered such that a transgene replaces the rid E1a, E1b, and E3 genes; subsequently the replication-defective vector can be propagated only in human 293 cells that supply the deleted El gene functions in trans, Unfortunately, the use of high titers of El deleted vectors has been repeatedly demonstrated to result in low-level expression of viral genes still resident in the vector. In addition, the generation of replication-competent Ad (RCA) by recombination events with the E1 sequences residing in 293 cells further limits the usefulness of E1-deleted Ad vectors, We addressed these problems by isolating new Ad vectors deleted for the E1, E3, and the E2b gene functions, The new vectors can be readily grown to high titers and have several improvements, including an increased carrying capacity and a theoretically decreased risk for generating RCA, We have also demonstrated that the further block to Ad vector replication afforded by the deletion of both the E1 and E2b genes significantly diminished Ad late gene expression in comparison to a conventional E1-deleted vector, without destabilization of the modified vector genome, The results suggested that these modified vectors may be very useful both for in vitro and in vivo gene therapy applications.