Gene Therapy for Blood Protein Deficiencies

血液蛋白缺乏症的基因治疗

• 批准号: 6687743
• 负责人: Katherine P. Ponder
• 金额: $ 26.68万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6687743
• 关键词: Retroviridae; animal genetic material tag; antibody formation; artificial immunosuppression; biotechnology; cell mediated lymphocytolysis test; coagulation factor IX; complementary DNA; cytotoxic T lymphocyte; disease /disorder model; dogs; enzyme linked immunosorbent assay; gene expression; gene therapy; hemophilia B; hemorrhage; human genetic material tag; immunomodulators; laboratory mouse; liver cells; mature animal; newborn animals; nonhuman therapy evaluation; technology /technique development; transfection /expression vector

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.

血友病B发生在1：30,000名男性中，与终身出血素质有关。 尽管IV注射IX可以预防或停止出血，但这种治疗是不方便的，昂贵的，并且可以传播感染。 肝基因疗法可以永久纠正血友病的临床表现。 逆转录病毒载体（RV）可以导致啮齿动物中肝脏凝血因子的长期和治疗水平，目前正在人类的血友病A临床试验中使用。 但是，在常规使用RV介导的肝基因疗法之前，必须解决两个主要问题：1）确定没有重大毒性的稳定基因转移效率更高的方法； 2）确定在RV介导的肝基因治疗的背景下阻止对治疗基因的免疫反应的方法。 该项目将解决这两个问题。第一个目的是确定表达犬IX（CFIX）cDNA的RV是否可以减少从菌落中获得的血友病B狗的出血表现，该菌落很少生产犬蛋白的抗体。 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淆问题。 初步研究将使用新生儿犬，因为它们的高基线肝细胞复制水平允许转导9％的肝细胞。随后的研究将使用肝细胞生长因子在年轻的成年犬中诱导复制。 将评估动物的CFIX水平，抗体的发展，出血和其他不良反应。 第二个目的将解决RV介导的肝基因疗法的第二个主要问题，即对治疗基因产物的免疫反应。 在此目标中，我们将尝试通过进行新生儿基因转移或在年轻人的基因治疗时对小鼠中RV的从头表达进行免疫反应。 尽管由于成本考虑因素，小鼠对于初步研究是最佳的，但在近交小鼠中起作用的方法有时在大型较大的动物中失败。因此，我们将测试任何在小鼠中起作用的免疫调节方法，以在AIM III中在正常和血友病B狗中的功效。 该项目的成功可能会导致对血友病的安全，有效且永久的治疗。