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。在10^5颗粒/ml和2*10^5颗粒中获得了另一个具有OTC3的载体，该载体的颗粒表达了1.265mumo1/mg蛋白/mg蛋白/hr的OTC活性在HEPG2的10^6细胞中（内o.02--活性; O.02--; o.02-- O.03mumol/mg蛋白/hr）。但是，这种浓度不足以体内基因治疗。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

Nishiyori A.: "Y55D mutation in ornithine transcarbamylase associated with late-onest hyperammonemia in a male." Hum Mutant Suppl. 1. 131-133 (1998)

Nishiyori A.：“鸟氨酸转氨甲酰酶的 Y55D 突变与男性晚期高氨血症相关。”

Matsuda I.: "The ornithine transcarbamylase(OTC)gene : mutations in 50 Japanese families with OTC deficiency." Am J Med Genet. 71. 378-383 (1997)

Matsuda I.：“鸟氨酸转氨甲酰酶 (OTC) 基因：50 个日本 OTC 缺陷家庭的突变。”

Watanabe A.: "Prenatal diagnosis of ornithine transcarbamylase dificiency by using a single nucleated erythrocyte from maternal blood." Hum Genet. 102. 611-615 (1998)

Watanabe A.：“通过使用母血中的单核红细胞对鸟氨酸转氨甲酰酶缺陷进行产前诊断。”

Hisayasu S.: "In vivo targeted gene transfer into liver cells mediated by a novel galactosyl-D-lysine/D-seline copolymer." Gene Ther.(in press).

Hisayasu S.：“由新型半乳糖基-D-赖氨酸/D-丝氨酸共聚物介导的体内靶向基因转移到肝细胞中。”

Kubo S.: "Hepatocyte injury in tyrosinemia type 1 is induced by fumarylacetoacetate and is inhibited by caspase inhibitors." Proc Natl Acad Sci. 95. 9552-9557 (1998)

Kubo S.：“1 型酪氨酸血症的肝细胞损伤是由延胡索酰乙酰乙酸酯诱导的，并被半胱天冬酶抑制剂抑制。”