Gene therapy for ornithine transcarbamylase deficiency by recombinant AAV vector

为实现对鸟氨酸转氨甲酰酶缺乏症（OTCD）的有效、持久的基因治疗，我们尝试制备重组腺相关病毒（rAAV）载体。首先，我们制备了两个载体质粒，它们分别含有GAG启动子和一个在ITR之间有TK neo（5.35kb，OTC 2）或无TK neo（4.4kb，OTC 3）的hOTC cDNA。为了评估这些质粒，我们用先前描述的每种质粒制备rAAV（人类遗传学中的最新方案）。以10^4cfu/ml制备具有OTC 2的rAAV载体，但没有获得酶活性。另一个含有OTC 3的载体以10^5个颗粒/ml获得，2 × 10^5个该载体颗粒在10^6个HepG 2细胞中表达1.265 μ mol/mg蛋白/hr的OTC活性（内源活性; 0.02 - 0.03 μ mol/mg蛋白/hr）。为了获得更高滴度的rAAV，我们尝试用载体质粒和辅助质粒以及载体质粒的新限制条件制备新的包装细胞系。

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.