Gene therapy for hemophilia and its preclinical study in cynomolgus macaques

在血友病A小鼠中使用重组腺相关病毒（AAV）载体研究血友病A的基因治疗。当将携带犬因子VIII（FVIII）基因的AAV 1载体注射至血友病A小鼠时，FVIII水平增加至治疗水平。通过静脉注射携带FVIII基因的AAV 8载体，血友病A小鼠中的FVIII水平增加至正常和超常水平。未观察到明显的副作用，如肝功能障碍。这些数据表明，使用携带FVIII基因的AAV载体可以实现安全有效的基因治疗。携带FVIII基因的SIV载体用于扩增造血干细胞以进行血小板特异性基因表达。通过移植携带位于GPIb启动子下游的FVIII基因的SIV载体转导的血友病A小鼠造血干细胞，在受体血友病A小鼠中检测到FVIII ...更多信息 血小板中的干细胞，并从血小板中释放，导致剪尾后有效止血，而移植模拟转染干细胞的对照血友病A小鼠由于出血而死亡。在小鼠中使用AAV载体研究血友病B的基因治疗。通过在小鼠体内注射携带位于派-1启动子下游的人FIX基因的AAV载体来实现因子IX（FIX）的组织特异性表达。在注射部位肌纤维周围的内皮细胞中观察到人FIX表达。注射在派-1启动子下游携带人FIX基因的AAV载体的小鼠中的人FIX表达高于注射在CMV启动子下游携带人FIX基因的AAV载体（对照载体）的小鼠中的人FIX表达，并且持续超过1年。在用TGFβ1刺激后，在注射在派-1启动子下游携带人FIX基因的AAV载体的小鼠中，人FIX表达增加约1.5倍，而在注射对照AAV载体的小鼠中未观察到人FIX表达增加。在LPS施用后，在注射在派-1启动子下游携带人FIX基因的AAV载体的小鼠中人FIX表达增加，而在注射对照载体的小鼠中人FIX降低。这些数据表明基于派-1启动子的载体对于内皮细胞特异性表达的有利性质。在食蟹猴中研究了FIX基因的肌肉定向转移用于血友病B基因治疗。人FIX的氨基酸序列与猕猴FIX的氨基酸序列高度同源，然而，当用携带人FIX基因的AAV载体在猕猴中表达人FIX时，人FIX对猕猴具有免疫原性，导致人FIX的中和抗体形成。为了研究长期FIX转基因表达，将携带修饰的猕猴FIX基因的AAV载体注射至猕猴，所述修饰的猕猴FIX基因表达可由人FIX特异性单克隆抗体检测的突变体FIX。用该载体实现了长期治疗性FIX表达，并且没有观察到明显的不良反应，表明用AAV载体可以实现血友病B的有效基因治疗的可能性。少

Gene therapy for hemophilia A was studied using recombinant adeno-associated virus (AAV) vectors in hemophilia A mice. When the AAV1 vectors carrying the canine factor VIII (FVIII) gene were im-injected to hemophilia A mice, FVIII levels were increased to the therapeutic levels. With iv-injection of the AAV8 vectors carrying the FVIII gene, the FVIII levels in hemophilia A mice were increased to the normal and the supernormal levels. No apparent side effects such as liver dysfunction were observed. These data suggested the possibility that the safe and efficient gene therapy can be achieved using the AAV vectors carrying the FVIII gene. The SIV vectors carrying the FVIII gene were used to transduce the hematopoietic stem cells for platelet-specific gene expression. By transplantation of the hemophilia A mouse hematopoietic stem cells transduced with the SIV vectors carrying the FVIII gene located in the downstream of the GPIb promoter, FVIII was detected in recipient hemophilia A mouse … More platelets and was released from platelets, resulting in efficient hemostasis upon tail clipping whereas the control hemophilia A mice with transplantation of mock transfected stem cells died because of bleeding. Gene therapy for hemophilia B was studied using AAV vectors in mice. The tissue specific expression of factor IX (FIX) was achieved with im-injection of AAV vectors carrying the human FIX gene located in the downstream of the PAI-1 promoter in mice. Human FIX expression was observed in the endothelial cells surrounding muscle fibers at the injected sites. Human FIX expression in mice with injection of AAV vectors carrying the human FIX gene in the downstream of the PAI-1 promoter was higher than that in mice with injection of AAV vectors carrying the human FIX gene in the downstream of the CMV promoter (control vector) and continued more than 1 year. Upon stimulation with TGFβ1, human FIX expression increased approximately 1.5-fold in the mice with injection of AAV vectors carrying the human FIX gene in the downstream of the PAI-1 promoter while no increase of human FIX expression was observed in the mice with injection of the control AAV vectors. Human FIX expression increased in the mice with injection of AAV vectors carrying the human FIX gene in the downstream of the PAI-1 promoter upon LPS administration, while human FIX in the mice with the control vector injection decreased. These data suggested the advantageous nature of the PAI-1 promoter-based vectors for endothelial cell specific expression. The muscle-directed transfer of FIX gene was studied in cynomolgus macaques for hemophilia B gene therapy. The amino acid sequence of human FIX is highly homologous to that of macaque FIX, however, human FIX is immunogenic to macaques causing the neutralizing antibody formation to human FIX when human FIX is expressed in macaques with the AAV vectors carrying the human FIX gene. To study the long term FIX transgene expression, the AAV vectors carrying the modified macaque FIX gene, that expressed mutant FIX detectable by the human FIX specific monoclonal antibody, was injected to macaques. The long-term therapeutic FIX expression was achieved with this vector and no apparent adverse effects were observed, suggesting the possibility that the effective gene therapy for hemophilia B can be achieved with the AAV vectors. Less