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

(l)We用携带增强型绿色荧光蛋白基因的猴免疫缺陷病毒agmTYO 1（SIVagm）载体以剂量依赖的方式有效地转导人脐血（CB）-CD 34+细胞，在MOI 5 × 10（3）vg/细胞时达到最大值（99.6±0.1%）。(2)用携带人凝血因子VIII基因的基于SIVagm的慢病毒载体（SIVhFVIII）转导CB-CD 34+细胞后，在体外孵育24小时期间，从10（6）个CB-CD 34+细胞产生hFVIII（274.3±20.1 ng）。(3)将SIVhFVIII-转导的CB-CD 34+细胞（5-10 X 10（5））移植到NOD/SCID小鼠中导致CD 34+细胞的成功植入和hFVIII的产生（最小1.2±0.9 ng/mL，最大3.6±0.8 ng/mL）体内至少60天。在小鼠骨髓细胞中也检测到hFVIII基因和抗原的转录。(4)在NOD/SCID小鼠中，骨髓和脾脏中人类造血细胞的谱系标志物表达没有显著差异。 ...更多信息 e SIVhFVIII转导细胞和接受模拟转导细胞的那些，表明用SIV载体稳定的人FVIII基因转导不会改变CB-CD 34+细胞在小鼠造血微环境中重新增殖和扩增的能力。(5)以剂量依赖性方式用携带GFP基因的SlVagm载体转导培养的人脂肪细胞，并且用SIVhFVIII转导脂肪细胞导致体外人FVIII的有效表达（320± 39.8ng/10（6）脂肪细胞/24小时）。(6)基于携带lacZ基因的SIV载体在小鼠体内成功转导脂肪细胞，用SIVhFVIII转导db/db小鼠的脂肪组织。在注射后第11天，小鼠血浆中短暂出现人FVIII（最大值为1.8 ng/mL）。在第14天，通过RT-PCR和免疫荧光分别在脂肪组织中检测到人FVIII转基因和抗原的转录物。这些数据表明，在不使受试者暴露于病毒载体的情况下，通过非致病性SIVhFVIII离体转导的造血干细胞的移植是安全的，并且可能适用于血友病A患者的基因治疗，并且用携带人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

(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