Gene therapy for hemophilia and its preclinical study in cynomolgus macaques

用重组腺相关病毒(AAV)载体对血友病A小鼠进行基因治疗。将携带犬凝血因子VIII(FVIII)基因的AAV1载体注射到血友病A小鼠体内后，FVIII水平上升到治疗水平。静脉注射携带FVIII基因的AAV8载体后，血友病A小鼠的FVIII水平升高到正常和超常水平。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。用携带FVIII基因的SIV载体转导造血干细胞表达血小板特异性基因。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更多的血小板和从血小板释放，导致有效止血的尾巴，而对照血友病A小鼠移植模拟转基因干细胞死于出血。利用AAV载体对血友病B进行了基因治疗研究。将携带人FIX基因的AAV载体注射到PAI-1启动子下游的小鼠体内，实现FIX的组织特异性表达。在注射部位的肌纤维周围的内皮细胞中观察到人FIX的表达。在PAI-1启动子下游注射携带人FIX基因的AAV载体的小鼠的人FIX表达高于在CMV启动子下游注射人FIX基因的小鼠(对照载体)，并持续1年以上。在转化生长因子β-1刺激下，在PAI-1启动子下游注射携带人FIX基因的AAV载体的小鼠，人FIX的表达增加约1.5倍，而注射对照AAV载体的小鼠，人FIX的表达没有增加。在PAI-1启动子下游注射携带人FIX基因的AAV载体的小鼠在注射脂多糖后，人FIX的表达增加，而注射对照载体的小鼠的人FIX表达减少。这些数据提示了基于PAI-1启动子的载体对内皮细胞特异性表达的有利性质。本文研究了FIX基因在食蟹猴体内的肌肉定向转移，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，但人FIX对猕猴具有免疫原性，当人FIX在携带人FIX基因的AAV载体中表达时，会产生对人FIX的中和抗体。为了研究FIX转基因的长期表达，将携带修饰的FIX基因的AAV载体注射到猕猴体内，获得了人FIX特异性单抗可检测到的突变型FIX。该载体实现了血友病B的长期治疗性FIX表达，未观察到明显的不良反应，提示AAV载体有可能实现血友病B的有效基因治疗。较少

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