Sleeping Beauty-Mediated Gene Therapy for Hemophilia

描述(申请人提供)：睡美人(SB)是一种转座子系统，已被广泛证明能够介导新的基因序列整合到目标细胞和组织的染色体中。在发现基因组公司(DGI)，我们正在努力开发用于人类基因治疗的SB转座子系统，血友病是我们的主导项目。在最初的第二阶段SBIR研究中，我们证明了SB转座子DNA有效地输送到狗的肝脏。用单或双气囊导管完全或部分阻断肝脏，然后快速、大容量地将含有DNA的溶液逆行注入肝静脉循环。这些实验的结果(输液后六周观察到报告基因的表达)使DGI处于以大动物肝脏为靶点的非病毒基因治疗努力的前沿。基于这些结果和最近来自食品和药物管理局的反馈，在这一第二阶段竞争性更新申请中，我们建议进行进一步的临床前研究，以解决安全性、扩大到人类大小以及在大型动物血友病模型中的疗效等问题。具体目标是：(I)开发能够将SB转座子DNA输送到人类肝脏的双气囊导管，并测试这些导管在猪体内作为与人类大小相当的大型动物模型的DNA输送的安全性和有效性；(Ii)利用DGI独特的犬分泌型碱性磷酸酶(CSEAP)报告系统，延长将SB转座子输送到正常狗肝脏后转基因表达的持续时间；(Iii)将编码犬凝血因子IX(CFIX)的SB转座子运送到cFIX缺陷狗的肝脏，测试cFIX的长期表达并改善凝血功能，作为SB介导的血友病B基因治疗的大型动物模型。这些研究的结果将为随后向FDA提交使用SB转座子系统治疗血友病B的研究性新药申请提供必要的临床前数据，并随后发现基因公司的成长和商业发展。 公共卫生相关性：在这项拨款申请中，有人建议开发一种新的方法，通过使用名为睡美人的整合DNA元件(转座子)的非病毒基因疗法来治疗血友病。提案中描述的实验将为在猪身上传递睡美人DNA作为与人类相似的动物模型，在血友病狗身上作为治疗人类血友病的模型创造条件。

DESCRIPTION (provided by applicant): Sleeping Beauty (SB) is a transposon system that has been extensively shown to be capable of mediating integration of new gene sequences into the chromosomes of target cells and tissues. At Discovery Genomics, Inc. (DGI), we are working to develop the SB transposon system for human gene therapy, with hemophilia as our lead project. During initial Phase II SBIR studies, we demonstrated effective delivery of SB transposon DNA to the liver of dogs. Single or double balloon catheters were used to achieve whole or partial occlusion of the liver followed by rapid, high volume retrograde infusion of DNA containing solution into the hepatic venous circulation. Results from these experiments (reporter gene expression was observed for six weeks following infusion) place DGI at the forefront of non-viral gene therapy efforts targeting the liver in large animals. Based on these results and recent feedback from the Food and Drug Administration, in this Phase II competing renewal application we propose further preclinical studies to address issues of safety, scale-up to the size of humans, and efficacy in a large animal model of hemophilia. The Specific Aims are; (i) To develop double balloon catheters capable of delivering SB transposon DNA to the liver in humans and to test the safety and effectiveness of these catheters for DNA delivery in pigs as a large animal model of comparable size to human beings; (ii) To extend the duration of transgene expression after delivery of SB transposons to the liver of normal dogs, using DGI's unique canine secreted alkaline phosphatase (cSEAP) reporter system; (iii) To deliver SB transposons encoding canine clotting factor IX (cFIX) to the liver of cFIX deficient dogs, testing for long-term expression of cFIX and improved clotting function as a large animal model for SB mediated gene therapy of hemophilia B. Results from these studies will provide necessary preclinical data for subsequent submission of an Investigational New Drug application with the FDA for treatment of hemophilia B using the SB transposon system, with subsequent growth and commercial development of Discovery Genomics, Inc. PUBLIC HEALTH RELEVANCE: In this grant application, studies are proposed to develop a new approach for treating hemophilia by non-viral gene therapy using an integrating DNA element (a transposon) called Sleeping Beauty. The experiments described in the proposal will establish conditions for delivery of Sleeping Beauty DNA in pigs as an animal model similar in size to humans, and in hemophilic dogs as a model for treatment of human hemophilia.