Gene therapy for ornithine transcarbamylase deficiency by recombinant AAV vector

为了实现对鸟氨酸氨基转移酶缺乏症(OTCD)的有效和持久的基因治疗，我们试图制备重组腺相关病毒(RAAV)载体。首先，我们制备了两个含有Gag启动子和hOTCcDNA的载体，在ITRs之间有Tk neo(5.35kb，OTC2)或没有(4.4kb，OTC3)。为了鉴定这些质粒，我们按照前面描述的方法(目前人类遗传学中的方法)分别制备了rAAV。当OTC2的浓度为104cfu/ml时，我们制备了含有OTC2的rAAV载体，但没有获得任何酶活性。另一种含OTC3的载体在10^6细胞中的内源性活性为0.02~0.03mumol/mg-蛋白/小时，表达的OTC活性为1.265 mumo1/mg-蛋白/小时，但这一浓度不足以用于体内基因治疗。为了获得滴度更高的rAAV，我们尝试用载体载体和辅助载体制备新的包装细胞系，并尝试用载体载体的新约束来制备新的包装细胞。

To achieve the efficient and persistent gene therapy for ornithine transcarbamylase deficiency (OTCD), we have tried to prepare recombinant adenoassociated virus (rAAV) vector. At first, we prepared two vector plasmids which had a GAG promotor and a hOTCcDNA with a TK neo (5.35kb, OTC2) or not (4.4kb, OTC3) between ITRs. To evaluate these plasmids, we prepared rAAV with each plasmid as previously described (Current protocols in human genetics).rAAV vector with OTC2 was prepared at lO^4cfu/ml, but no enzymal activity was obtained. Another vector with OTC3 was obtained at 10^5 particles/ml and 2*10^5 particles of this vector expressed 1.265mumo1/mg-protein/hr of OTC activity in 10^6 cells of HepG2 (endogeneous activity ; O.02-O.03mumol/mg-protein/hr).However, this concentration was insufficient for in vivo gene therapy. To obtain higher titered rAAV, we tried to prepare new packaging cell lines with vector plasmid and helper plasmid and new constraucts of vector plasmid.