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Gene therapy for hemophilia A with simian immunodeficiency virus agmTYO1-based vectors carrying human factor VIII gene.

使用携带人类因子 VIII 基因的基于猿猴免疫缺陷病毒 agmTYO1 的载体对 A 型血友病进行基因治疗。

基本信息

批准号：
13671078
负责人：
MADOIWA Seiji
金额：
$ 1.86万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671078/
关键词：
Hemophilia A Factor VIII Gene therapy SIV virus vector Hematopoietic stem cell Adipocyte NOD SCID mice db db mice 肝細胞

项目摘要

(l)We efficiently transduced human cord bloodderived (CB)-CD34+ cells using a simian immunodeficiency virus agmTYO1 (SIVagm) vector carrying the enhanced green fluorescent protein gene in a dose-dependent manner, reaching a maximum (99.6±0.1%) at MOI 5 X 10(3) vg/cell. (2) After transducing CB-CD34+ cells with SIVagm-based lentiviral vector carrying the human coagulation factor VIII gene (SIVhFVIII), hFVIII was produced (274.3±20.1 ng) from 10(6) CB-CD34+ cells during 24hr in vitro incubation. (3) Transplantation of SIVhFVIII-transduced CB-CD34+ cells (5-10 X 10(5)) into NOD/SCID mice resulted in successful engraftment of CD34+ cells and production of hFVIII (minimun 1.2±0.9 ng/mL, maximum 3.6±0.8 ng/mL) for at least 60 days in vivo. Trancripts of hFVIII gene and antigen were also detected in the murine bone marrow cells. (4) There was no significant difference in lineage marker expression of human hematopoiteic cells in the bone marrow and the spleen between NOD/SCID mice receiving th … More e SIVhFVIII-transduced cells and those who received mock-transduced cells, suggesting that stable human FVIII gene tranduction with the SIV vector does not alter capability of CB-CD34+ cells to re-populate and expand in the mouse hematopoietic microenvironment. (5) Cultured human adipocytes were transduced with the SlVagm vector carrying the GFP gene in a dose-dependent manner and transduction of adipocytes with SIVhFVIII resulted in efficient expression of human FVIII (320±39.8 ng/10(6) adipocytes/24 hours) in vitro. (6) Based upon successful transduction of adipocytes by SIV vectors carrying the lacZ gene in vivo in mice, the adipose tissue of db/db mice was transduced with SIVhFVIII. There was a trasient appearance of human FVIII in mouse plasma (maximun 1.8 ng/mL) on day 11 after the injection. Transcripts of human FVIII transgene and antigen also were detected in the adipose tissue by RT-PCR and immunofluorescence on day 14, respectively. These data suggest that transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vector is safe and potentially applicable for gene therapy of hemophilia A patients, and that transduction of the adipocytes with vectors carrying the human FVIII gene may be also applicable for hemophilia A gene therapy. Less

(l)利用携带增强绿色荧光蛋白基因的猴免疫缺陷病毒agmTYO1 （SIVagm）载体，以剂量依赖的方式高效转导人脐带血来源(CB)-CD34+细胞，在MOI为5 X 10(3) vg/细胞时达到最高（99.6±0.1%）。(2)以sivagm为载体，携带人凝血因子VIII基因（SIVhFVIII）的慢病毒载体转导CB-CD34+细胞后，10(6)个CB-CD34+细胞体外培养24h，产生hFVIII（274.3±20.1 ng）。(3)将sivhfviii转导的CB-CD34+细胞（5-10 × 10(5)）移植到NOD/SCID小鼠体内，CD34+细胞成功植入，体内产生hFVIII（最小1.2±0.9 ng/mL，最大3.6±0.8 ng/mL）至少60天。在小鼠骨髓细胞中也检测到hFVIII基因和抗原的转录本。(4)接受sivhfviii转导细胞的NOD/SCID小鼠骨髓和脾脏中人造血细胞的谱系标记表达与接受模拟转导细胞的NOD/SCID小鼠之间没有显著差异，这表明SIV载体稳定的人FVIII基因转导不会改变CB-CD34+细胞在小鼠造血微环境中重新聚集和扩增的能力。(5)用携带GFP基因的SlVagm载体以剂量依赖性方式转导培养的人脂肪细胞，用SIVhFVIII转导脂肪细胞可在体外高效表达人FVIII（320±39.8 ng/10(6)个脂肪细胞/24小时）。(6)以携带lacZ基因的SIV载体在小鼠体内成功转导脂肪细胞为基础，用SIVhFVIII转导db/db小鼠的脂肪组织。注射后第11天，小鼠血浆中短暂出现人FVIII（最高1.8 ng/mL）。第14天，分别用RT-PCR和免疫荧光法在脂肪组织中检测人FVIII转基因转录本和抗原转录本。这些数据表明，在不暴露于病毒载体的情况下，通过非致病性SIVhFVIII移植体外转导的造血干细胞是安全的，并且可能适用于血友病A患者的基因治疗，并且携带人类FVIII基因的载体转导脂肪细胞也可能适用于血友病A基因治疗。少