Gene Therapy for Blood Protein Deficiencies

血友病B发生在1：30，000的男性中，并与终身出血素质相关。 虽然IV注射因子IX可以预防或止血，但这种治疗不方便，昂贵，并且可能传播感染。 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）可导致啮齿动物肝脏中凝血因子的长期和治疗水平表达，目前正用于人类血友病A的临床试验。 然而，在RV介导的肝基因治疗将被常规使用之前，有两个主要问题必须解决：1）确定实现稳定基因转移的更高效率而没有主要毒性的方法;和2）确定在RV介导的肝基因治疗的背景下阻断对治疗性基因的免疫应答的方法。 本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递送到肝脏中是否可以减少从很少产生犬蛋白抗体的菌落获得的血友病B犬的出血表现。 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转导9%的肝细胞。后续研究将使用肝细胞生长因子在年轻成年犬中诱导复制。 将评价动物的cFIX水平、抗体形成、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应答。 在这一目标中，我们将尝试通过进行新生儿基因转移或在年轻成人中进行基因治疗时注射免疫抑制剂来阻断小鼠中RV转基因从头表达的免疫反应。 尽管出于成本考虑，小鼠是初始研究的最佳选择，但在近交系小鼠中起作用的方法有时会在远交系较大动物中失败。因此，我们将在Aim III中检测在小鼠中起作用的任何免疫调节方法在正常和血友病B犬中的有效性。 该项目的成功可能为血友病B提供安全、有效和永久的治疗。

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.