Sleeping Beauty Gene Therapy from Liver to BOECs

描述（由申请人提供）：使用病毒载体的基因疗法在治疗各种类型的血友病方面取得了有限的成功。宿主反应和载体系统的独特特性使得难以实现持久的长期转基因表达。通过替代疗法治疗血友病的另一个并发症是产生凝血因子的抑制性抗体。事实上，这促使使用rFVIIa通过血小板表面的组织因子（TF）非依赖性FX激活来诱导止血。本研究项目的主要目的是评估非病毒性睡美人转座子（SB-Tn）载体在因子IX缺乏症小鼠模型中的基因治疗。这一目的检验了我们的假设，即FIX、rFVIIa和可溶性TF（sTF）转基因的单独或联合表达将改善疾病表型。第一个具体目标旨在优化细胞培养中的（i）凝血因子IX、工程化rFVIIa和sTF的最大转座和持续基因表达的Tn设计;以及（ii）通过几种不同的配体部分通过脱唾液酸糖蛋白受体向肝细胞的非病毒递送系统。第二个具体目标是评估最佳Tn载体/递送系统促进Tn插入和体内持续转基因表达的能力。将通过相关代谢参数监测和定量FIX、rFVIIa和/或sTF表达的体内治疗作用。将针对转座效率和安全性优化给药方案、递送媒介物和施用途径。第三个具体目标是评估血液生长内皮细胞（BOEC）作为通过SB转座的转基因表达载体的潜在用途。将在BOEC中单独和成对组合检查表达FIX、TF和/或rFVIIa的不同SB-Tn的转座和表达特征。将离体工程化的BOEC输注回血友病B小鼠，并分析其长期表达和对FIX缺陷动物出血素质的影响。本研究提案的主要目标是：（i）优化Tn设计和非病毒递送系统，通过SB介导的基因转移提供治疗水平的FIX、rFVIIa和/或sTF;（ii）确定纠正凝血障碍动物模型的最佳体内条件。|血友病B;和（iii）表征BOEC作为持续转基因表达的自体载体的潜力。

DESCRIPTION (provided by applicant): Gene therapy using viral vectors has had limited success in the treatment of the various types of hemophilia. Both the host response and the unique characteristics of the vector systems have made it difficult to achieve persistent long-term transgene expression. An additional complication in the treatment of hemophilia by replacement therapy is the development of inhibitory antibodies to the clotting factor. In fact, this has prompted the use of rFVIIa in an effort to induce hemostasis via tissue factor (TF) independent FX activation at the platelet surface. The main objective of this research project is to evaluate the nonviral Sleeping Beauty transposon (SB-Tn) vector for gene therapy in a mouse model of factor IX deficiency. This objective tests our hypothesis that separate or combined expression of FIX, rFVIIa and soluble TF (sTF) transgenes will improve the disease phenotype. The first specific aim is designed to optimize in cell culture (i) the Tn design for maximal transposition and persistent gene expression of the coagulation factors IX, an engineered rFVIIa and sTF; and (ii) the nonviral delivery systems to hepatocytes via the asialoglycoprotein receptor by several different ligand moieties. The second specific aim evaluates the capacity of the best Tn vectors/delivery systems to promote Tn insertion and persistent transgene expression in vivo. The therapeutic effects in vivo for expression of FIX, rFVIIa, and/or sTF will be monitored and quantitated by the relevant metabolic parameters. The dosing regimen, the delivery vehicle and route of administration will be optimized for efficiency of transposition and safety. The third specific aim is designed to evaluate the potential use of blood outgrowth endothelial cells (BOECs) as a vehicle of transgene expression via SB transposition. The transposition and expression characteristics of the different SB-Tns expressing FIX, TF and/or rFVIIa will be examined in BOECs both individually and in pair wise combinations. The ex vivo engineered BOECs will be infused back into hemophilia B mice, and analyzed for long-term expression and effect on the bleeding diathesis of the FIX deficient animals. The major goals of this research proposal are to (i) optimize Tn designs and nonviral delivery systems that provide therapeutic levels of FIX, rFVIIa and/or sTF with SB-mediated gene transfer; (ii) identify the best in vivo conditions for correction of the clotting disorder in animal models of | hemophilia B; and (iii) characterize the BOECs potential as an autologous vehicle for persistent transgene expression.