RECOMBINANT ADENO ASSOCIATED VIRUS

非致病性人类依赖病毒， 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被 如果以反式提供，则整个AAV基因组可以被替换为 感兴趣的基因然后，所得重组AAV（rAAV）可以 在细胞培养物中瞬时产生，并且rAAV颗粒 根据病毒粒子的物理性质进行浓缩和纯化。 AAV的原型是血清型2（AAV 2）。直到最近，所有 AAV遗传学、分子生物学和载体的表征 已经用AAV 2进行了开发。我们克隆了， 按顺序对4型和5型AAV进行测序和部分表征， 以确定非结构性Rep蛋白的活性结构域， 开发具有与AAV 2不同的组织特异性的载体。 AAV 4型- AAV 2和AAV 4 rep ORF序列在90%相同， 氨基酸水平。不同的序列出现在不同的区域 能够容忍突变的基因然而，AAV 4的序列 衣壳蛋白基因与AAV 2只有62%相同。的 衣壳蛋白的差异导致重叠但不同 向性为了确定不同的向性 由于利用不同的受体，细胞被 用报告病毒和非评分“冷AAV 2”共感染。 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。此外，胰蛋白酶 细胞处理降低了rAAV 2-lacZ的转导百分比 80%，而rAAV 4-lacZ转导几乎不受影响。的 竞争实验和对胰蛋白酶的敏感性强 表明AAV 2和AAV 4的摄取机制是 不同. AAV 5型-另一种血清型， 其特征在于AAV 5型。我们克隆了病毒基因组， 确定了序列。代表打开类型2的阅读帧 和5是67%相同的，并且衣壳开放阅读框是 60%相同因此，血清型2和5是最不同的 已知的AAVs序列比对显示，AAV 5是 与鹅或番鸭细小病毒近似同等相关 关于AAV 2 Rep多肽的功能似乎是 保守，但改变了核酸内切酶活性的特异性 相对于2型。AAV 5的摄取机制似乎是 基于与2型病毒的竞争而不同于AAV 2。AAV 2已被证明结合硫酸乙酰肝素蛋白聚糖和可溶性 肝素中和对细胞的吸附。AAV 4和 用可溶性肝素中和AAV 5。

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.