Sleeping Beauty Gene Therapy from Liver to BOECs

从肝脏到 BOEC 的睡美人基因治疗

• 批准号: 7252464
• 负责人: CLIFFORD John STEER
• 金额: $ 35.44万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7252464
• 关键词: Animal Model; Animals; Antibodies; Asialoglycoprotein Receptor; Autologous; Back; Blood; Blood Coagulation Disorders; Blood Coagulation Factor; Blood Platelets; Cell Line; Cells; Characteristics; Coagulation Process; Complication; Condition; Cultured Cells; Data; Defect; Development; Disease susceptibility; Dose; Endothelial Cells; Engineering; Factor IX; Factor X; Gene Delivery; Gene Expression; Gene Silencing; Gene Transduction Agent; Gene Transfer; Genomics; Goals; Hemophilia A; Hemophilia B; Hemorrhage; Hemostatic function; Hepatocyte; Human; Immune response; In Vitro; Infusion procedures; Injection of therapeutic agent; Kidney; Ligands; Liver; Long-Term Effects; Mediating; Metabolic; Metabolic Diseases; Methods; Methylation; Monitor; Mus; Patients; Play; Primary carcinoma of the liver cells; Replacement Therapy; Research Personnel; Research Project Grants; Research Proposals; Role; Route; Safety; Site; Sleeping Beauty; Standards of Weights and Measures; Surface; System; Technology; Testing; Therapeutic; Therapeutic Effect; Thromboplastin; Transgenes; Transgenic Mice; Transgenic Organisms; Treatment Protocols; Viral Vector; design; disease phenotype; gene replacement; gene therapy; improved; in vivo; insight; mouse model; non-viral gene therapy; novel; programs; recombinant FVIIa; success; tool; transgene expression; vector

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.

描述（由申请人提供）：使用病毒载体的基因治疗在治疗各种类型的血友病方面取得的成功有限。宿主反应和载体系统的独特特征都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凝血因子抑制性抗体的产生。事实上，这促使人们使用 rFVIIa 来通过血小板表面组织因子 (TF) 独立的 FX 激活来诱导止血。该研究项目的主要目的是评估非病毒睡美人转座子 (SB-Tn) 载体在因子 IX 缺乏小鼠模型中的基因治疗效果。该目的检验了我们的假设，即 FIX、rFVIIa 和可溶性 TF (sTF) 转基因的单独或组合表达将改善疾病表型。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、工程化 rFVIIa 和 sTF 的最大转座和持久基因表达； (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。第二个具体目标评估最佳 Tn 载体/递送系统促进 Tn 插入和体内持久转基因表达的能力。将通过相关代谢参数监测和定量FIX、rFVIIa和/或sTF表达的体内治疗效果。给药方案、递送载体和给药途径将针对转位效率和安全性进行优化。第三个具体目标是评估血液生长内皮细胞 (BOEC) 作为通过 SB 转座进行转基因表达的载体的潜在用途。将在BOEC中单独地和成对组合地检查表达FIX、TF和/或rFVIIa的不同SB-Tns的转座和表达特征。离体工程化的 BOEC 将被输回血友病 B 小鼠中，并分析其长期表达及其对 FIX 缺陷动物出血素质的影响。该研究计划的主要目标是 (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) 确定纠正动物模型中凝血障碍的最佳体内条件 |血友病B； (iii) 描述 BOEC 作为持续转基因表达的自体载体的潜力。