Sleeping Beauty-Mediated Gene Therapy for Hemophilia

描述（由申请人提供）：睡美人（SB）是一种转座子系统，已广泛显示其能够介导新基因序列整合到靶细胞和组织的染色体中。在Discovery Genomics，Inc. (DGI)目前，我们正致力于开发用于人类基因治疗的SB转座子系统，其中血友病是我们的主导项目。在最初的II期SBIR研究中，我们证明了SB转座子DNA有效地递送到狗的肝脏。使用单或双球囊导管实现肝脏的全部或部分闭塞，然后快速、高容量逆行输注含DNA的溶液至肝静脉循环中。这些实验的结果（在输注后观察报告基因表达六周）将DGI置于靶向大型动物肝脏的非病毒基因治疗努力的最前沿。基于这些结果和美国食品药品监督管理局最近的反馈，在这项II期竞争性更新申请中，我们提出了进一步的临床前研究，以解决安全性问题，扩大到人类的大小，以及在血友病大型动物模型中的疗效。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，并在猪这一与人类体型相当的大型动物模型中测试这些导管输送DNA的安全性和有效性;（ii）为了延长SB转座子递送至正常狗的肝脏后转基因表达的持续时间，使用DGI独特的犬分泌碱性磷酸酶（cSEAP）报告系统;（iii）将编码犬凝血因子IX（cFIX）的SB转座子递送至cFIX缺陷狗的肝脏，作为SB介导的血友病B基因治疗的大型动物模型，测试cFIX的长期表达和改善的凝血功能。这些研究的结果将为后续向FDA提交使用SB转座子系统治疗血友病B的研究性新药申请以及Discovery Genomics，Inc.的后续增长和商业开发提供必要的临床前数据。 公共卫生相关性：在这项资助申请中，提出了一种新的研究方法，通过使用称为睡美人的整合DNA元件（转座子）的非病毒基因治疗来治疗血友病。该提案中描述的实验将建立在猪中递送睡美人DNA的条件，作为与人类大小相似的动物模型，并在血友病犬中作为治疗人类血友病的模型。

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.