Helper-free Fully-deleted Adenovirus Vector System

无辅助完全删除腺病毒载体系统

• 批准号: 6605636
• 负责人: VALERI A KROUGLIAK
• 金额: $ 4.54万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2003-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6605636
• 关键词: Adenoviridae; bioreactors; biotechnology; cell line; gene delivery system; recombinant virus; transfection /expression vector; virion; virus genetics; virus replication

DESCRIPTION (provided by applicant):Although first-generation adenovirus vectors have many attractive features for gene therapy applications, they do not persist in vivo due to the strong host immune response against adenovirus-encoded proteins. The development of so-called "gutless" or fully-deleted adenovirus vectors (FD-AdV) which do not encode any adenovirus genes, allowed this limitation to be at least partially overcome. However the inability of these vectors to replicate in the absence of a helper virus complicates the vector propagation and purification procedures and results in the contamination of all FD-AdV stocks by the helper virus, which is potentially as immunogenic as first-generation vectors. We propose FD-AdVs can be safely and efficiently generated and propagated through the substitution of the infectious adenovirus helper by a baculovirus/adenovirus (Bac/Ad) hybrid virus that carries a Cre recombinase-excisable copy of an adenovirus genome that is deleted for El and the packaging signal (helper genome). Additionally, the adenovirus ITRs in this construct will be joined to form an ITR junction known to be a functional adenovirus origin of replication, so that Cre-mediated excision will generate a replication-competent, packaging-deficient circular adenovirus genome. Such a hybrid virus will be propagated in insect cells and used to infect Cre-expressing, El-complementing cells in which FD-AdVs are produced. In these cells, the circular adenovirus genome will be excised from the hybrid. Adenovirus genes expressed from this helper genome will complement the replication of both the FD-AdV and the helper genome, while only the FD-AdV will be packaged into virions. Unlike the helper virus in the traditional helper-dependent system, the Bac/Ad hybrid helper cannot reproduce in mammalian cells and the excised helper genome cannot be incorporated into virus particles due to the lack of the adenovirus packaging signal. To prevent generation of packaging-competent adenovirus through homologous recombination, two approaches will be applied. In the first, the size of the helper genome will be increased to exceed the adenovirus packaging limit through the insertion of a staffer fragment into the E3 region of the helper genome. In the second approach, the FD-AdV will be propagated in the cell lines that are not prone to the generation of replication-competent adenovirus (RCA), This will allow the production of FD-AdVs that are free of contamination by both helper virus and RCk Additionally, a similar system will be developed to produce FD-AdVs based on unrelated adenovirus serotypes that potentially can overcome the problems associated with pre-existing immunity and allow repeat administration of FD-AdVs based on different serotypes. Once these systems are optimized, they will be used for production of safer, more persistent FD-AdVs for gene therapy applications.

描述（由申请人提供）：尽管第一代腺病毒载体对于基因治疗应用具有许多有吸引力的特征，但是由于针对腺病毒编码的蛋白质的强宿主免疫应答，它们不能在体内持续存在。不编码任何腺病毒基因的所谓的“无肠”或完全缺失的腺病毒载体（FD-AdV）的开发允许至少部分克服这种限制。然而，这些载体在没有辅助病毒的情况下不能复制，使得载体繁殖和纯化程序复杂化，并导致所有FD-AdV储备物被辅助病毒污染，辅助病毒可能与第一代载体一样具有免疫原性。我们提出FD-AdV可以通过杆状病毒/腺病毒（Bac/Ad）杂合病毒替换感染性腺病毒辅助病毒安全有效地产生和繁殖，所述杆状病毒/腺病毒（Bac/Ad）杂合病毒携带缺失El和包装信号（辅助基因组）的腺病毒基因组的Cre重组酶可切除拷贝。此外，该构建体中的腺病毒ITR将连接以形成已知为功能性腺病毒复制起点的ITR连接，使得Cre介导的切除将产生有复制能力的包装缺陷型环状腺病毒基因组。这样的杂合病毒将在昆虫细胞中繁殖，并用于感染表达Cre的El互补细胞，其中产生FD-AdV。在这些细胞中，环状腺病毒基因组将从杂交体中切除。从该辅助基因组表达的腺病毒基因将补充FD-AdV和辅助基因组的复制，而只有FD-AdV将被包装到病毒体中。与传统辅助病毒依赖性系统中的辅助病毒不同，Bac/Ad杂合辅助病毒不能在哺乳动物细胞中复制，并且由于缺乏腺病毒包装信号，切除的辅助病毒基因组不能掺入病毒颗粒中。为了防止通过同源重组产生有包装能力的腺病毒，将应用两种方法。在第一种方法中，通过将staffer片段插入辅助基因组的E3区，辅助基因组的大小将增加到超过腺病毒包装极限。在第二种方法中，FD-AdV将在不易于产生可复制腺病毒（RCA）的细胞系中繁殖。这将允许产生不受辅助病毒和RCK两者污染的FD-AdV。将开发类似的系统以基于不相关的腺病毒血清型生产FD-AdV，其潜在地可以克服与pre-AdV相关的问题。现有的免疫力，并允许基于不同血清型重复施用FD-AdV。一旦这些系统得到优化，它们将用于生产更安全，更持久的FD-AdV，用于基因治疗应用。