Development of the gene therapy technologies using adeno-associated virus (AAV)

我们研究了腺相关病毒（AAV）介导的基因转移方法. AAV载体生产方法的开发：我们通过Cre/loxP系统调节细胞毒性AAV蛋白的表达，开发了用于AAV载体生产的新型包装细胞系。首先，我们将使用变体loxP和野生型loxP的组合Cre/loxP系统应用于Rep和Cap表达的同时调节。其次，我们开发了一种新的293衍生的预包装细胞系，其组成型表达由loxP侧翼CMV启动子驱动的反义rep/cap。通过导入Cre重组酶基因将该细胞系转化为表达Rep/Cap的包装细胞系。AAV载体介导的转基因高灵敏检测方法的建立与应用：采用位于ITR D区的合适引物对进行长链PCR（有时与巢式PCR结合）。我们还从肌肉注射AAV载体的实验动物中收集了许多样品.开发使用AAV衍生组分（ITR和Rep基因）将靶向载体整合（TVI）到19号染色体上的确定基因座中的方法：使用TVI方法用neoγ基因转染293和K562细胞。我们通过RT-PCR扩增细胞和转基因序列之间的连接区。因此，没有细胞序列被认为是一个共同的识别基序的Rep蛋白被发现。我们还开发了突变的Rep表达载体与降低细胞毒性。

We studied the gene transfer methods using adeno-associated virus (AAV).1. Development of the method for AAV vector production: We developed novel packaging cell lines for AAV vector production by regulating the expression of cytotoxic AAV proteins through Cre/loxP system. First, we applied combined Cre/loxP system using variant loxP and wild-type loxP to the simultaneous regulation of Rep and Cap expressions. Second, we developed a novel 293-derived prepackaging cell line which constitutively expresses the antisense rep/cap driven by a loxP-flanked CMV promoter. This cell line was converted into a packaging cell line expressing Rep/Cap through the introduction of a Cre recombinase gene.2. Establishment and application of highly sensitive detection method for AAV vector-mediated transgenes : Long PCR (sometimes combined with nested PCR) was conducted with appropriate primer sets located on the D region of ITR. We also collected many samples from the experimental animals which received intramuscular injections of AAV vectors.3. Development of the method for targeted vector integration (TVI) into a defined locus on chromosome 19 using AAV-derived components (ITR and Rep gene) : 293 and K562 cells were transfected with the neoγ gene using the TVI method. We amplified junctional regions between cellular and transgene sequences by Alu-PCR. As a result, no cellular sequences regarded as a common recognition motif of the Rep proteins was found. We also developed mutant Rep-expression vectors with reduced cytotoxicity.