Gene therapy for ornithine transcarbamylase deficiency by recombinant AAV vector

重组AAV载体治疗鸟氨酸转氨甲酰酶缺乏症的基因治疗

• 批准号: 09470520
• 负责人: MATSUDA Ichiro
• 金额: $ 2.94万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470520/
• 关键词: gene therapy; AAV vector; vector plasmid; rAAV vector; OTC deficiency; packaging plasmid; packaging cell line; ベクター

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.

为了实现鸟氨酸经钙化酶缺乏症（OTCD）的有效和持续的基因治疗，我们试图制备重组腺体相关病毒（RAAV）载体。最初，我们制备了两个载体质粒，它们具有一个具有TK NEO（5.35kb，OTC2）的HotccDNA，或者在ITRS之间（4.4KB，OTC3）。为了评估这些质粒，我们按照先前所述的每种质粒制备了RAAV（人类遗传学中的当前方案）。在LO^4CFU/ML时制备了用OTC2的Raav载体，但没有获得酶活性。 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.为了获得更高的滴定拉文，我们试图制备具有载体质粒和辅助质粒和载体质粒的新约束的新包装细胞系。

项目成果

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