生物
辅助病毒
质粒
病毒学
腺相关病毒
开放式参考框架
衣壳
腺病毒基因组
重组DNA
基因
病毒
HEK 293细胞
腺病毒科
分子生物学
遗传学
载体(分子生物学)
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肽序列
作者
Angela Adsero,Brendan Chestnut,Sara Shahnejat-Bushehri,Lalita M Sasnoor,Travis McMurphy,Michael Swenor,Ryan Pasquino,Arun Pradhan,Víctor Agmo Hernández,Linas Padegimas,David Dismuke
出处
期刊:Human Gene Therapy
[Mary Ann Liebert]
日期:2023-12-07
摘要
Despite decades of research in adeno-associated virus (AAV) and the role of adenovirus in production, the interplay of AAV and adenovirus is not fully understood. Specific regions of the adenoviral genome containing E1, E2a, E4 ORFs, and VA RNA have been demonstrated as necessary for AAV production; however, incorporating these regions into either a producer cell line or subcloning into an Ad helper plasmid may lead to inclusion of neighboring adenoviral sequence or ORFs with unknown function. Because AAV is frequently used in gene therapies, removing excessive adenovirus sequences improves the Ad helper plasmid size and manufacturability, and may lead to safer vectors for patients. Furthermore, deepening our understanding of the helper virus genes required for rAAV production has the potential to increase yields and manufacturability of rAAV for clinical and commercial applications. One region continuously included in various Ad helper plasmid iterations is the adenoviral E2a promoter region that appears to be necessary for E2a expression. Due to the compact nature of viral genomes, the E2a promoter region overlaps with the Hexon Assembly/100K protein and the L4 region. The L4 region, which contains the coding sequences for 22K and 33K proteins, had not been thought to be necessary for AAV production. Through molecular techniques, this study demonstrates that the adenoviral 22K protein is essential for recombinant AAV (rAAV) production in HEK293 cells by triple transfection and that the 33K protein synergistically increases rAAV yield.
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