Gene therapy for hemophilia and its preclinical study in cynomolgus macaques

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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