Recombinant Adeno Associated Virus

重组腺相关病毒

• 批准号: 6546775
• 负责人: Robert Kotin
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6546775
• 关键词: DNA replication origin; adeno associated virus group; biological signal transduction; capsid; human genetic material tag; microorganism culture; open reading frames; plasmids; recombinant virus; transfection /expression vector; virus classification; virus genetics; virus replication; western blottings

The non-pathogenic human dependoviruses, adeno-associated viruses (AAV), infect either proliferating or post-mitotic cells. Since all the AAV and helper virus genes can be provided in trans, the entire AAV genome may be replaced with the gene of interest. The resulting recombinant AAV (rAAV) may then be produced transiently in cell culture and the rAAV particles concentrated and purified based on physical properties of the virion. The prototype for AAV is serotype 2 (AAV2). Until recently, all characterizations of AAV genetics, molecular biology, and vector development have been performed with AAV2. We have cloned, sequenced and partially characterized AAV types 4 and 5 in order to define activity domains of the non-structural Rep proteins and develop vectors with different tissue specificities than AAV2. AAV type 4 - AAV 2 and AAV4 rep ORF sequences are 90% identical at the amino acid level. The divergent sequences appear in regions that are tolerant of mutation. However, the sequence of the AAV4 capsid protein genes were only 62% identical to AAV2. The difference in capsid proteins resulted in overlapping but distinct tropism. In order to determine whether the different tropism resulted from utilization of different receptors, cells were co-infected with a reporter virus and a non-scoring "cold AAV2". A 72-fold excess of cold AAV2 was needed to inhibit AAV4-lacZ transduction 50%. Whereas rAAV2-lacZ was competed with approximately a 10-fold excess of cold AAV2. Furthermore, trypsin treatment of cells reduced the percent transduction of rAAV2-lacZ by 80%, yet rAAV4-lacZ transduction was virtually unaffected. The competition experiments and sensitivity to trypsin are strong indicators that the uptake mechanisms of AAV2 and AAV4 are different. AAV type 5 - Another serotype that had not been characterized is AAV type 5. We cloned the virus genome and determined the sequence. The Rep open reading frames of types 2 and 5 are 67% identical and the capsid open reading frames are 60% identical. Thus, serotypes 2 and 5 are the most divergent of the known AAVs. Sequence alignments revealed that AAV 5 is approximately equally related to goose or moscovy duck parvovirus as to AAV 2. Functions of the Rep polypeptides appear to be conserved, but specificity for endonuclease activity is altered relative to type 2. The uptake mechanism of AAV 5 seems to be distinct from AAV 2 based on competition with type 2 virus. AAV 2 has been shown to bind heparan sulfate proteoglycans and soluble heparin neutralizes adsorption to the cell. Neither AAV 4 nor AAV5 are neutralized with soluble heparin.

非致病性人依赖病毒，腺相关病毒（AAV），感染增殖或有丝分裂后的细胞。由于所有的AAV和辅助病毒基因都可以反式提供，因此整个AAV基因组可以用感兴趣的基因替换。然后可以在细胞培养物中瞬时产生所得重组AAV（rAAV），并基于病毒体的物理性质浓缩和纯化rAAV颗粒。AAV的原型是血清型2（AAV 2）。直到最近，AAV遗传学、分子生物学和载体开发的所有表征都用AAV 2进行。我们已经克隆，测序和部分特征的AAV 4型和5型，以确定非结构Rep蛋白的活性结构域和开发载体与不同的组织特异性比AAV 2。AAV 4型- AAV 2和AAV 4 rep ORF序列在氨基酸水平上90%相同。不同的序列出现在耐受突变的区域。然而，AAV 4衣壳蛋白基因的序列与AAV 2仅62%相同。衣壳蛋白的差异导致重叠但不同的向性。为了确定不同的向性是否由利用不同的受体引起，用报告病毒和非评分“冷AAV 2”共感染细胞。需要72倍过量的冷AAV 2来抑制AAV 4-lacZ转导50%。而rAAV 2-lacZ与约10倍过量的冷AAV 2竞争。此外，胰蛋白酶处理细胞使rAAV 2-lacZ的转导百分比降低80%，而rAAV 4-lacZ转导几乎不受影响。竞争实验和对胰蛋白酶的敏感性是AAV 2和AAV 4的摄取机制不同的有力指标。AAV 5型-另一种尚未表征的血清型是AAV 5型。我们克隆了病毒基因组并确定了序列。2型和5型的Rep开放阅读框为67%相同，衣壳开放阅读框为60%相同。因此，血清型2和5是已知AAV中最不同的。序列比对显示，AAV 5与鹅或番鸭细小病毒的相关性与AAV 2的相关性大致相等。Rep多肽的功能似乎是保守的，但相对于2型核酸内切酶活性的特异性改变。AAV 5的摄取机制似乎不同于基于与2型病毒竞争的AAV 2。已经显示AAV 2结合硫酸乙酰肝素蛋白聚糖，并且可溶性肝素中和对细胞的吸附。AAV 4和AAV 5都不被可溶性肝素中和。