Gene Therapy for Blood Protein Deficiencies

B型血友病男性发病率为1:30 000，与终生出血素质有关。虽然静脉注射因子IX可以预防或止血，但这种治疗不方便，费用昂贵，并可能传播感染。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中凝血因子的长期和治疗水平的表达，目前正在用于人类血友病a的临床试验。然而，在常规使用rv介导的肝脏基因治疗之前，必须解决两个主要问题：1)确定实现稳定基因转移效率更高且无重大毒性的方法；2)在rv介导的肝脏基因治疗中，确定阻断对治疗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因子IX (cFIX) cDNA的RV传递到肝脏是否可以减少血友病B犬的出血表现，这些血友病B犬是从很少产生犬蛋白抗体的菌落中获得的。这将使我们能够量化基因表达，而不会出现免疫反应的混淆问题。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在年轻成年犬中诱导复制。将评估动物的cFIX水平、抗体的发展、出血和其他不良反应。第二个目标将解决rv介导的肝脏基因治疗的第二个主要问题，即对治疗性基因产物的免疫反应。在这个目标中，我们将尝试通过进行新生儿基因转移或在年轻成人基因治疗时注射免疫抑制剂来阻断小鼠对RV转基因从头表达的免疫反应。尽管由于成本考虑，小鼠是初始研究的最佳选择，但在近亲繁殖的小鼠中起作用的方法有时在近亲繁殖的大型动物中失败。因此，我们将在Aim III中测试任何在小鼠中起作用的免疫调节方法对正常和B型血友病犬的功效。这个项目的成功可能会为血友病B带来安全、有效和永久的治疗方法。

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.