Gene Therapy for Blood Protein Deficiencies

血友病B发生在1：30,000男性中，并与终生出血素质有关。虽然静脉注射凝血因子IX可以预防或止血，但这种治疗方法不方便，费用昂贵，而且会传播感染。肝脏基因治疗可以永久纠正血友病的临床表现。逆转录病毒载体(RV)可以导致啮齿动物肝脏凝血因子的长期和治疗水平的表达，目前正用于人类甲型血友病的临床试验。然而，在RV介导的肝脏基因治疗将被常规使用之前，有两个主要问题必须解决：1)寻找获得更高效率的稳定的、没有重大毒性的基因转移的方法；2)寻找在RV介导的肝脏基因治疗的背景下阻断对治疗性基因的免疫反应的方法。这个项目将解决这两个问题。第一个目的是确定将表达犬凝血因子IX(CFIX)基因的RV送入肝脏是否可以减少血友病B犬的出血症状，该血友病B犬是从一个很少产生犬蛋白抗体的群体获得的。这应该使我们能够量化基因表达，而不会出现免疫反应的混乱问题。最初的研究将使用新生狗，因为它们的高基线肝细胞复制水平允许9%的肝细胞转导。随后的研究将使用肝细胞生长因子来诱导年轻成年狗的复制。将对动物进行cFIX水平、抗体产生、出血和其他不良反应的评估。第二个目标将解决轮状病毒介导的肝脏基因治疗的第二个主要问题，即对治疗性基因产品的免疫反应。在这个目标中，我们将尝试通过进行新生基因转移，或者在年轻人进行基因治疗时注射免疫抑制剂，来阻止小鼠对RV转基因从头表达的免疫反应。尽管出于成本考虑，小鼠是最初研究的最佳选择，但在近亲交配的小鼠身上发挥作用的方法有时在远缘繁殖的大型动物中失败。因此，我们将在AIM III中测试任何在小鼠身上起作用的免疫调节方法对正常和血友病B犬的疗效。这个项目的成功可能会带来一种安全、有效和持久的血友病B疗法。

Hemophilia B occurs in 1:30,000 males and is associated with a life-long bleeding diathesis. Although IV injection of Factor IX can prevent or stop bleeding, this treatment is inconvenient, expensive, and can transmit infections. Hepatic gene therapy could permanently correct the clinical manifestations of hemophilia. Retroviral vectors (RV) can result in long-term and therapeutic levels of expression of coagulation factors from the liver in rodents, and are currently being used in a clinical trial for Hemophilia A in humans. However, there are two major problems that must be solved before RV-mediated hepatic gene therapy will be used routinely: 1) identify ways to achieve a higher efficiency of stable gene transfer without major toxicity; and 2) identify methods for blocking an immune response to the therapeutic gene in the context of RV-mediated hepatic gene therapy. This project will address both of these issues. The first aim is to determine if delivery of an RV expressing the canine Factor IX (cFIX) cDNA into the liver can reduce the bleeding manifestations of Hemophilia B dogs obtained from a colony that rarely makes antibodies to the canine protein. This should allow us to quantify gene expression without the confounding issue of an immune response. Initial studies will use neonatal dogs, as their high baseline level of hepatocyte replication allows transduction of 9 percent of liver cells. Subsequent studies will use hepatocyte growth factor to induce replication in young adult dogs. Animals will be evaluated for cFIX levels, development of antibodies, bleeding, and for other adverse effects. The second aim will address the second major problem of RV-mediated hepatic gene therapy, that of immune responses to the therapeutic gene product. In this aim, we will try to block immune responses to the de novo expression of a transgene from an RV in mice by either performing neonatal gene transfer, or by injecting immunoinhibitory agents at the time of gene therapy in young adults. Although mice are optimal for initial studies due to cost considerations, approaches that function in inbred mice sometimes fail in outbred larger animals. We will therefore test any immunomodulatory approaches that function in mice for their efficacy in normal and Hemophilia B dogs in Aim III. Success in this project might lead to a safe, effective, and permanent therapy for Hemophilia B.