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的成功有限。宿主的反应和载体系统的独特特性都使得转基因的持续、长期表达变得困难。替代疗法治疗血友病的另一个并发症是凝血因子的抑制性抗体的产生。事实上，这促使了rFVIIa的使用，试图通过组织因子(TF)非依赖于血小板表面的FX激活来诱导止血。本研究的主要目的是评价非病毒睡美人转座子(SB-TN)载体在凝血因子IX缺乏的小鼠模型中的基因治疗作用。这一目标验证了我们的假设，即单独或联合表达FIX、rFVIIa和可溶性转铁蛋白(STF)转基因将改善疾病的表型。第一个具体目标是在细胞培养中优化(I)TN设计，以实现凝血因子IX、工程rFVIIa和STF的最大转座和持久基因表达；以及(Ii)通过几种不同的配体部分通过去唾液酸糖蛋白受体向肝细胞的非病毒递送系统。第二个具体目的是评估最好的TN载体/递送系统在体内促进TN插入和持续转基因表达的能力。体内对FIX、rFVIIa和/或STF表达的治疗效果将通过相关代谢参数进行监测和量化。将优化给药方案、给药工具和给药路线，以提高换位效率和安全性。第三个特定目的是评估血液生长内皮细胞(BOECs)通过SB转座作为转基因表达载体的潜在用途。表达FIX、Tf和/或rFVIIa的不同SB-TNS的转座和表达特征将在BOEC中单独和成对组合进行检测。将体外培养的BOECs回输到血友病B小鼠体内，分析其长期表达情况及对FIX缺陷动物出血素质的影响。这项研究建议的主要目标是：(I)优化TN设计和非病毒输送系统，通过SB介导的基因转移提供治疗性水平的FIX、rFVIIa和/或STF；(Ii)确定纠正血友病B动物模型中凝血障碍的最佳体内条件；以及(Iii)表征BOECs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.