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

(L)我们用携带增强型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地转导人脐血源性CD34+细胞，在MOI5×10(3)Vg/cell时达到最大(99.6±0.1)%。(2)用携带人凝血因子VIII基因的慢病毒载体(SIVhFVIII)转导Cb-CD34+细胞，体外培养24小时后，10(6)个Cb-CD34+细胞产生hFVIII(274.3±20.1 ng)。(3)将转导SIVhFVIII的CB-CD34+细胞(5-10×10(5))移植到NOD/SCID小鼠体内，可成功植入CD34+细胞并产生hFVIII(最小1.2±0.9 ng/m L，最大3.6±0.8 ng/m L)。在小鼠骨髓细胞中也检测到hFVIII基因和抗原的转录本。(4)注射TH…的NOD/SCID小鼠骨髓和脾组织中人造血细胞的谱系标志表达差异无统计学意义更多的SIVhFVIII转导细胞和接受模拟转导细胞的细胞，表明稳定的SIV载体转导人FVIII基因并不改变CB-CD34+细胞在小鼠造血微环境中重新繁殖和扩张的能力。(5)用携带GFP基因的SLVagm载体以剂量依赖的方式转导培养的人脂肪细胞，并在体外用SIVhFVIII转导脂肪细胞高效表达人FVIII(320±39.8 ng/10(6)脂肪细胞/24小时)。(6)在携带LacZ基因的SIV载体体内成功转导脂肪细胞的基础上，将SIVhFVIII基因导入db/db小鼠的脂肪组织。注射后第11天，小鼠血浆中出现短暂的人FVIII抗体(最高浓度为1.8 ng/mL)。第14天，RT-PCR和免疫荧光法也分别在脂肪组织中检测到人FVIII基因和抗原的转录本。提示非致病性SIVhFVIII体外转导的造血干细胞移植不暴露于病毒载体是安全的，可用于血友病A患者的基因治疗，携带人FVIII基因的载体转导脂肪细胞也可用于血友病A的基因治疗。较少

(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