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，已被广泛证明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)，我们正致力于开发用于人类基因治疗的SB转座子系统，血友病是我们的主要项目。在最初的II期SBIR研究中，我们证明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，大容量逆行注入含有DNA的溶液到肝静脉循环中。这些实验的结果（在输注后观察报告基因表达6周）将DGI置于针对大型动物肝脏的非病毒基因治疗的前沿。基于这些结果和最近来自美国食品和药物管理局的反馈，在这一II期竞争性续期申请中，我们建议进一步进行临床前研究，以解决安全性、扩大到人类规模以及在大型血友病动物模型中的有效性问题。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球囊导管，并作为与人类大小相当的大型动物模型，在猪身上测试这些导管输送DNA的安全性和有效性；（ii）利用DGI独有的犬分泌碱性磷酸酶（cSEAP）报告系统，延长SB转座子递送至正常犬肝脏后的转基因表达时间；（iii）将编码犬凝血因子IX （cFIX）的SB转座子传递到cFIX缺陷犬的肝脏，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随后向FDA提交使用SB转座子系统治疗血友病B的新药研究申请提供必要的临床前数据。随着Discovery Genomics， Inc.的后续增长和商业发展。

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.