DEVELOPMENT OF ADENO ASSOCIATED VIRUS/ADENOVIRUS HYBRID

腺相关病毒/腺病毒杂交体的开发

• 批准号: 6501117
• 负责人: KENNETH I. BERNS
• 金额: $ 12.63万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6501117
• 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，成功的基因治疗还需要 用于引入转基因的合适的媒介物或载体 进入细胞。对于宿主细胞的长期转化， 生物体，将转基因置于其被 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），一直被用作矢量，因为它从来没有 被确定为人类病原体，尽管大约90%的成年人 病毒的抗体腺相关病毒（AAV）整合 在19号染色体上的一个特定位点插入人类基因组， 甚至在非分裂细胞中整合其DNA（尽管在较低的细胞中）。 频率）。目前的AAV载体具有某些局限性：1）它们 难以高浓度生产; 2）它们具有 转基因大小的能力有限; 3）目前 它们不会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 在细胞内自主整合或复制， 只在短暂的基础上转变。然而，相对而言， 容易产生大量的广告载体， 转基因的能力几乎是目前AAV的两倍 向量。这个项目的目标是构建一个杂交病毒 AAV和Ad之间的载体，其将具有期望的性质 两者都是可以产生大量的载体， 转基因可以整合到19号染色体上的特定位点 永久表达。为了产生杂交载体转基因， 侧翼为关键的AAV末端序列，并且AAV rep基因 具体地点整合所需的 将不同的位点分离成Ad DNA的骨架。载体病毒会 在细胞培养中进行测试，以表征其功能 在分子水平和动物模型中评估宿主反应， 清除和长期转化的能力。