RECOMBINANT ADENO ASSOCIATED VIRUS

基于腺相关病毒(AAV)的哺乳动物转导载体可能 保留了野生型病毒的特性。野生型AAV 在允许的细胞中复制到高滴度，具有物理稳定性 粒子，整合到细胞基因组中，是非细胞遗传学的，并且是 被认为具有广泛的寄主范围，似乎不受限制 在组织嗜性方面。此外，不考虑细胞分裂。 对于病毒感染是必不可少的。重组AAV(RAAV)的生产 需要在反式和存在的情况下表达AAV rep和capp基因 的AAV反向末端重复(ITR)在顺式。重组腺病毒的产生 可能受到病毒结构基因拷贝数的限制 而在野生型AAV感染中，复制 随着病毒基因组的复制，病毒数量呈几何级数增加。至 克服这一障碍，我们利用了SV40复制起点 在含有Rep和CAP的质粒上。该质粒在细胞中被扩增 表达SV40T-Ag。与高效率相结合 电穿孔，提高了重组病毒颗粒的产量 是非复制辅助质粒的60倍。遵循Cscl等密度线 梯度离心法，对含有rAAV的组份进行鉴定 通过瞬时感染试验和DNA斑点杂交。这个 RAAV颗粒密度为1.40~1.42g/ml。 梯度分级结果表明，重组AAV基本上是 无污染的腺病毒。此外，颗粒大小为 条带的电子显微照片显示约为20-25 nm 材料。浮力密度和物理外观都很相似 与为野生型粒子所确定的结果相一致。 这种新型的rAAV包装系统已被用于生产rAAV颗粒 它含有T细胞共刺激蛋白B7-2的基因。 人非贴壁淋巴样细胞系LP-1的转导 转导编码AAV的B7-2后78%的细胞表达 B7-2。B7-2在人淋巴样细胞系RPMI8226中的表达 也大幅增加了。

Mammalian transduction vectors based on adeno-associated virus (AAV) may retain properties attributed to the wild-type virus. Wild-type AAV replicates to high titer in permissive cells, has a physically stable particle, integrates into the cellular genome, is non-cytogenic, and is considered to have broad host range and does not appear to be restricted in tissue tropism. In addition, cell division is not considered essential for viral infection. Production of recombinant AAV (rAAV) requires expression of AAV rep and cap genes in trans and the presence of the AAV inverted terminal repeats (ITR) in cis. Generation of rAAV may be limited by the copy number of the viral structural genes transfected into cells, whereas in a wild-type AAV infection, the copy number increases geometrically as the viral genome is replicated. To overcome this obstruction, we have utilized an SV40 origin of replication on a rep and cap containing plasmid. This plasmid is amplified in cells expressiong SV40 T-Ag. In conjunction with high efficiency electroporation, the yield of recombinant viral particles was increased 60-fold over a non-replicating helper plasmid. Following CsCl isopycnic gradient centrifugation, the fractions containing rAAV were identified by transient infection assays and DNA dot blot hybridization. The density of the rAAV particles was 1.40 - 1.42 g/ml. Hybridization of the gradient fractions demonstrated that the recombinant AAV was essentially free of contaminating adenovirus. Furthermore the particle size was of approximately 20-25 nm was determined by electromicrographs of the banded material. Both the buoyant density and physical appearence are similar to those determined for wild type particles. This novel rAAV packaging system has been used to produce rAAV particles which contains the gene for the T-cell co-stimulatory protein, B7-2. Transduction of the human, non-adherent lymphoid cell line, LP-1 transduced with B7-2 encoding AAV resulted in 78% of cells expressing B7-2. Expression of B7-2 in the human lymphoid cell line RPMI 8226 was also substantially increased.