DEVELOPMENT OF ADENO ASSOCIATED VIRUS/ADENOVIRUS HYBRID

腺相关病毒/腺病毒杂交体的开发

• 批准号: 6110879
• 负责人: KENNETH I. BERNS
• 金额: $ 12.87万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-28 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6110879
• 关键词: Adenoviridae; adeno associated virus group; cell transformation; method development; recombinant virus; tissue /cell culture; transfection /expression vector; virus genetics; virus integration

In addition to the desired gene, successful gene therapy requires an appropriate vehicle or vector for introduction of the transgene into the cell. For long term transformation of the host cell or organism, placing the transgene into a context in which it is replicated in synchrony with the cell genome is necessary. The human parvovirus, adeno-associated virus. (which requires adenovirus to multiply), has been used as a vector, because it has never been identified as a human pathogen, even though about 90% of adults have antibodies to the virus. Adeno-associated virus (AAV) integrates into the human genome at a specific site on chromosome 19 and can integrate its DNA even in non dividing cells (albeit at a lower frequency). Current AAV vectors have certain limitations: 1) they are difficult to produce in high concentration; 2) they have a limited capacity for the size of the transgene; 3) as currently made, they do not integrate in a site specific manner. Adenovirus (Ad) has also been used as a vector. Because Ad vectors do not integrate or replicate autonomously within the cell, they are able to transform only on a transient basis. However, it is relatively easy to produce large amounts of the Ad vector and the carrying capacity for the transgene is almost twice that of current AAV vectors. The goal of this project is to construct a hybrid virus vector between AAV and Ad which would have the desirable properties of both. Large amounts of the vector could be produced and the transgene could be integrated into a specific site in chromosome 19 for permanent expression. To create the hybrid vector the transgene, flanked by critical AAV terminal sequences, and the AAV rep gene required for site specific integration would be incorporated at separate sites into a backbone of Ad DNA. The vector virus will be tested both in cell culture in order to characterize its function at the molecular level and in animal models to assess host response, clearance and ability to transform in a long term manner.

除了所需的基因外，成功的基因治疗还需要 用于引入转基因的适当载体或载体 进入细胞。用于宿主细胞的长期转化或 有机体，将转基因置于其存在的环境中 与细胞基因组同步复制是必要的。人类 细小病毒、腺相关病毒。 （这需要腺病毒 乘法），已被用作向量，因为它从未被 被确定为人类病原体，尽管约 90% 的成年人患有 病毒的抗体。腺相关病毒（AAV）整合 进入人类基因组 19 号染色体上的特定位点，并且可以 即使在非分裂细胞中也能整合其 DNA（尽管速度较低） 频率）。目前的 AAV 载体有一定的局限性：1）它们 难以高浓度生产； 2）他们有一个 转基因大小的能力有限； 3) 就目前而言 制作后，它们不以特定于站点的方式集成。腺病毒 (Ad)也被用作载体。因为广告载体不 它们能够在细胞内自主整合或复制 仅在短暂的基础上进行转变。不过，相对而言 易于产生大量的Ad载体并携带 转基因的能力几乎是现有 AAV 的两倍 向量。该项目的目标是构建一种混合病毒 AAV 和 Ad 之间的向量将具有所需的属性 两者都有。可以产生大量的载体并且 转基因可以整合到 19 号染色体的特定位点 用于永久表达。为了创建转基因杂交载体， 两侧是关键的 AAV 末端序列和 AAV 代表基因 站点特定集成所需的内容将被纳入 将位点分离成 Ad DNA 的主干。载体病毒将是 在细胞培养物中进行测试以表征其功能 在分子水平和动物模型中评估宿主反应， 清除和长期转型的能力。