RECOMBINANT ADENO-ASSOCIATED VIRUS

重组腺相关病毒

• 批准号: 5203536
• 负责人: R 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/5203536
• 关键词: DNA replication origin; adeno associated virus group; density gradient ultracentrifugation; electron microscopy; electroporation; microorganism culture; nucleic acid repetitive sequence; plasmids; recombinant virus; simian virus 40; structural genes; transfection /expression vector; virus genetics; virus replication

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.

基于腺相关病毒（AAV）的哺乳动物转导载体可 保留了野生型病毒的特性。 野生型AAV 在允许细胞中复制到高滴度，具有物理稳定性 颗粒，整合到细胞基因组中，是非细胞遗传性的， 被认为具有广泛的宿主范围，并且似乎不受限制 在组织向性上。 此外，细胞分裂也不被考虑 对病毒感染至关重要。 重组AAV（rAAV）的生产 需要反式表达AAV rep和cap基因， AAV反向末端重复序列（ITR）的顺式。 rAAV的产生 可能受到病毒结构基因拷贝数的限制 转染到细胞中，而在野生型AAV感染中， 随着病毒基因组的复制，数量呈几何级数增长。 到 克服这个障碍，我们已经利用了SV 40的起源， 在含有rep和cap的质粒上复制。 该质粒 在表达SV 40 T-Ag的细胞中扩增。 结合高 高效电穿孔，重组病毒颗粒的产量为 比非复制型辅助质粒增加60倍。 CsCl后 等密度梯度离心后，将含有rAAV的级分离心。 通过瞬时感染试验和DNA斑点杂交鉴定。 rAAV颗粒的密度为1.40 - 1.42 g/ml。 杂交 的梯度洗脱证明重组AAV是 基本上不含污染的腺病毒。 此外，粒子 通过电子显微镜测定，其尺寸约为20-25 nm 的带状材料。 浮力密度和物理密度 表观与野生型颗粒的表观相似。 这种新的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。 人非粘附淋巴样细胞系LP-1的转导 用编码B7-2的AAV转导导致78%的细胞表达 B7-2 B7-2在人淋巴样细胞系RPMI 8226中的表达， 也大幅增加。