Sleeping Beauty-Mediated Gene Therapy for Hemophilia

描述（由申请人提供）：A型血友病是由凝血因子VIII （FVIII）缺乏引起的出血性疾病。这种疾病以x连锁隐性方式遗传。在美国有15000名受影响的个体。血友病A的治疗是基于频繁的重组FVIII（重组）。然而，这种治疗的高昂费用（严重病例每年高达10万美元）和血液循环中FVIII水平的变化损害了这种治疗的有效性。FVIII基因疗法是一种更有效和更具成本效益的疾病治疗方法。我们建议在肝脏非病毒FVIII基因转移和表达的II期研究中继续开发睡美人（SB）转座子系统，作为a型血友病的治疗方法。该方法基于我们对FVIII缺陷小鼠肝脏中SB转座子介导的FVIII基因表达的长期I期研究结果。我们假设FVIII基因同样可以在大型动物（最终是人类）的肝脏中传递和表达，从而提供FVIII的长期和治疗性表达。这些目标分为四个具体目标。在Aim 1中，我们将测试转座子和转座酶成分通过导管后直接递送到肝循环后，sb介导的报告基因和FVIII基因在小鼠肝脏中的长期表达。在目标2中，我们将确定使用DNA， DNA-多阳离子复合物或其他DNA复合物将转座子递送到肝脏的功效。然后，这些条件将用于Aim 3，以测试sb介导的报告基因在狗肝脏中的长期表达，作为体内基因转移的大型动物模型。在Aim 4中，正常狗的实验结果将用于评估SB系统在血友病a狗模型中的转位和犬FVIII基因的长期表达，测试是否改善凝血和纠正出血性疾病。在每个阶段，我们将检查动物组织的不良事件的组织病理学指征。这些临床前研究的结果将使DGI向FDA提交新药研究申请（IND），随后启动一项临床试验，测试SB系统FVIII基因治疗在人类受试者中的有效性。技术创新：睡美人是一种新的基因转移系统，在基因治疗中具有潜在的应用前景。这项技术所应用的遗传病细分市场的市场规模约为58亿美元。

DESCRIPTION (provided by applicant): Hemophilia A is a bleeding disorder caused by the absence of clotting factor VIII (FVIII). This disease is inherited in an X-linked recessive manner. There are 15,000 affected individuals in the U.S. Treatment of Hemophilia A is based on frequent delivery of recombinant FVIII (recombinate). However, the high expense of this treatment (as much as $100,000 per year in severe cases) and variable levels of FVIII maintained in the circulation compromise the effectiveness of this therapy. FVIII gene therapy represents a more efficacious and cost-effective treatment of the disease. We propose continued development of the Sleeping Beauty (SB) transposon system in a Phase II study of non-viral FVIII-gene transfer and expression in the liver as a therapeutic approach for hemophilia A. This approach is based on results from our Phase I studies of long-term, SB transposon-mediated FVIII gene expression in livers of FVIII-deficient mice. We hypothesize that the FVIII gene can similarly be delivered and expressed in the livers of larger animals (ultimately, humans), providing long-term and curative expression of FVIII. These goals are addressed in four Specific Aims. In Aim 1 we will test for SB-mediated long-term expression of reporter genes and the FVIII gene in the livers of mice after delivery of transposon and transposase components directly to the hepatic circulation via retroductal delivery. In Aim 2 we will determine the efficacy of using DNA, DNA-polycation complexes or other DNA complexes for delivery of transposons to liver. These conditions will then be used in Aim 3 to test for SB-mediated long-term expression of reporter genes in the livers of dogs as a large animal model for in vivo gene transfer. In Aim 4, results from the experiments in normal dogs will be used to evaluate the SB system for transposition and long-term expression of the canine FVIII gene in a dog model of hemophilia A, testing for improved clotting and correction of the bleeding disorder. At every stage we will examine animal tissues for histopathological indications of adverse events. Results from these preclinical studies will position DGI for submission of an Investigational New Drug application (IND) to the FDA, with subsequent initiation of a clinical trial testing the effectiveness of the SB system FVIII gene therapy in human subjects. Technological Innovation: Sleeping Beauty is a novel gene transfer system with potential application in gene therapy. The market size for the inherited-diseases market niche, to which this technology applies is about $5.8 billion.