Preclinical Study for Hemophilia Gene and Cell Therapy in Model Mice and in Non-human Primates

血友病是一种X连锁遗传性出血性疾病，由凝血因子VIII(FVIII)基因或凝血因子IX(FIX)基因突变引起，进而导致FVIII或FIX缺陷。血友病的基因治疗在老鼠和猕猴身上进行了研究。在血友病A小鼠体内，携带犬FVIII基因的AAV8载体在肝特异性Hcr/α1-抗胰蛋白酶增强子/启动子复合体的调控下，可在不受免疫抑制的情况下实现犬FVIII的高效、长期表达，提示FVIII在肝脏中的特异性表达可实现对FVIII的免疫耐受。由于人FVIII在小鼠循环中的半衰期较短，并形成了抗人FVIII的中和抗体，因此在血友病A小鼠体内很难在治疗水平上表达人FVIII。我们可以在血友病A小鼠体内用携带人FVIII基因的AAV8载体在肝特异性Hcr/α1-…的控制下在治疗水平上表达人FVIII更多的抗胰蛋白酶增强子/启动子复合体。在GPIBα启动子的控制下，将携带FVIII基因的SIV载体转导的造血干细胞移植到血友病A小鼠体内，导致FVIII的血小板特异性表达，使血友病A小鼠在剪尾时免于出血。类似的以活化形式表达FVII(FVIIa)的方法也可以使血友病A小鼠即使在FVIII中和抗体存在的情况下也能在夹尾时避免出血，这表明凝血因子的血小板特异性表达可能是一种治疗出血性疾病的基因疗法。AAV载体血清型1、8和9型已被证明在治疗水平上表达FIX，甚至在小鼠中表达超正常水平。用猕猴进行了血友病B基因治疗的临床前研究。将携带突变FIX基因的AAV载体1、8和9型分别注入猕猴体内，研究突变FIX T262a的表达情况。所有注射AAV1载体的猕猴、1只AAV8血清型猕猴和1只AAV9载体注射猕猴的FIX治疗水平均持续1年，未见不良反应。而注射AAV8或AAV9载体的猕猴FIX T262a基因在其他猕猴体内的表达均未达到治疗水平。先前存在的针对野生型AAV的中和抗体被认为对AAV载体介导的基因转移具有抑制作用。这些数据表明，AAV载体介导的肌肉和肝脏基因转移是治疗血友病的一种安全和有前途的基因治疗方法。较少

Hemophilia is an X-linked inherited bleeding disorder caused by mutations in the factor VIII (FVIII) gene or the factor IX (FIX) gene which in turn create deficiency of FVIII or FIX. Gene therapy for hemophilia is studied in mice and in macaques. High and long term expression of canine FVIII without immune suppression was achieved with the AAV8 vector carrying the canine FVIII gene under the control of the liver specific HCR/α1-antitrypsin enhancer/promoter complex in hemophilia A mice, suggesting that immune tolerance to FVIII can be achieved with specific expression of FVIII in the liver. Because of the short half-life of human FVIII in the mouse circulation and neutralizing antibody formation to human FVIII, it had been difficult to express human FVIII at the therapeutic level in hemophilia A mice. We could express human FVIII at the therapeutic level in hemophilia A mice with administration of the AAV8 vector carrying the human FVIII gene under control of the liver specific HCR/α1- … More antitrypsin enhancer/promoter complex. Transplantation of hematpoietic stem cells transduced with the SIV vector carrying the FVIII gene under control of the GPIbα promoter in hemophilia A mice resulted in platelet-specific expression of FVIII that rescued hemophilia A mice from bleeding upon tail clipping. The similar approach to express FVII in the activated form (FVIIa) could also rescue hemophilia A mice from bleeding upon tail clipping even in the presence of neutralizing antibody to FVIII, suggesting that the platelet-specific expression of coagulation factors can be a genetic therapy for bleeding disorders. AAV vectors serotypes 1, 8, and 9 had been shown to express FIX at the therapeutic levels and even at the super normal levels in mice. A preclinical study of gene therapy for hemophilia B was conducted using macaques. The AAV vector serotypes 1, 8, and 9 carrying the macaque FIX gene that could express mutant macaque FIX T262A, which could be quantified by the human FIX specific monoclonal antibody, were administered into macaques and expression of mutant FIX T262A was studied. The therapeutic levels of FIX persisted in all AAV1 vector-injected macaques, one macaque of serotype AAV8 vector-injected group, and one macaque of AAV9 vector-injected group for one year without adverse effect. However, expression of macaque FIX T262A in other macaques with AAV8 or AAV9 vector injection did not reach the therapeutic levels. Presence of pre-existing neutralizing antibody against wild-type AAV was thought to function inhibitory to AAV vector-mediated gene transfer. These data suggested that AAV vector-mediated gene transfer to the muscle and to the liver is a safe and promising genetic therapy for hemophilia. Less