RECEPTOR MEDIATED GENE THERAPY OF HEMOPHILIAS

血友病的临床治疗因病毒感染而变得复杂 混合血浆的污染和抗体的产生。 血友病A和B都是性连锁隐性疾病，所以基因 增强应该纠正这些缺陷。为了成功地进行基因治疗 对于血友病来说，使用无创、有效的方法是至关重要的 把正常的基因拷贝送到肝脏。这也是可取的 为了避免使用病毒序列，因为反复给药 候选基因可能是必需的。功能基因已经被引入 靶向去唾液酸糖蛋白在体外和体内进入肝细胞 肝脏的受体。我们建议用两种经典疗法来治疗狗狗 血友病(凝血因子VIII缺乏，血友病A)和圣诞病 (凝血因子IX缺乏，血友病B)使用基因转移的方法 我们实验室开发的靶向去唾液酸糖蛋白受体的药物。 这项技术的主要创新涉及DNA的缩合 使用共价连接的多L赖氨酸转变为单分子形式 与配体结合，即半乳糖。将DNA复合体注射到系统中 成年动物的静脉将有效地靶向肝脏，而不需要 用于侵入性手术，如肝部分切除术。使用这个 技术，我们已经传递了一个包含来自 PEPCK基因(-460至+73)，与 人凝血因子IX结构基因在成年大鼠肝脏中的表达 并发现转基因的表达时间长达140天( 实验持续时间)。转基因仍留在肝脏中 主教派。此外，转基因的转录可以通过以下方式诱导 给老鼠喂一种不含碳水化合物的饮食。我们建议延长 这些研究通过将犬因子IX(CFIX)基因复合体引入到 血友病B犬肝脏半乳糖化L-赖氨酸 将DNA复合体注射到头静脉。中的cFIX水平 血液将通过使用ELISA法和WESTERN法的连续测量进行监测 分析；功能活动将通过测量 全血凝血时间的相对变化及改良激活 部分凝血活酶时间。只有在我们展示了成功之后 用cFIX治疗患有血友病B的狗的实验，我们会吗？ 将人因子基因导入慢性阻塞性肺疾病犬的尝试 血友病A。最后，我们将研究在 通过测试替代配体将DNA靶向到猪的肝脏的技术 老鼠。将进行测试的其他潜在改进包括使用 各种替代推动者，试图增加 转基因在肝脏中的表达及包装系统的改变 用来浓缩DNA。本提案中描述的程序包括 治疗血友病的承诺，值得进一步 考虑作为这些疾病的基因治疗的替代方法 疾病。

The clinical management of hemophilia has become complicated by viral contamination of pooled plasma and the development of antibodies. Hemophilia A and B are both sex-linked recessive disorders, so that gene augmentation should correct the defects. For successful gene therapy of the hemophilias, it is critical to use non-invasive, efficient methods that deliver normal copies of the gene to the liver. It is also desirable to avoid the use of viral sequences since repeated administration of the candidate gene may be necessary. Functional genes have been introduced into hepatocytes in vitro and in vivo by targeting the asialoglycoprotein receptor of the liver. We propose to treat dogs with either classic hemophilia (Factor VIII deficiency, Hemophilia A) and Christmas disease (Factor IX deficiency, Hemophilia B) using an approach to gene transfer developed in our laboratory that targets the asialoglycoprotein receptor. The major innovation in this technique involves the condensation of DNA into a unimolecular form using poly-L-lysine, which is covalently attached to a ligand, i.e. galactose. The DNA complex injected into a systemic vein of an adult animal will efficiently target the liver without the need for invasive procedures, such as partial hepatectomy. Using this technique, we have delivered a chimeric gene containing the promoter from the P-enolpyruvate carboxykinase (PEPCK) gene (-460 to +73), linked to the structural gene for human Factor IX (hFIX) into the livers of adult rats and found prolonged expression of the transgene for up to 140 days (the duration of the experiment). The transgene remained in the liver as an episome. In addition, transcription of the transgene can be induced by feeding the rats a diet devoid of carbohydrate. We propose to extend these studies by introducing the canine Factor IX (cFIX) gene complexes to galactosylated poly-L-lysine into the livers of dogs with Hemophilia B by injecting the DNA complex into the cephalic vein. The level of cFIX in the blood will be monitored by serial measurements using ELISA and Western analysis; the functional activity will be determined by measuring the relative change in the whole blood clotting time and modified activated partial thromboplastin time. Only after we have demonstrated the success of experiments with cFIX treatment of dogs with Hemophilia B, will we attempt to introduce the human Factor VIII (hFVIII) gene into dogs with Hemophilia A. Finally, we will examine potential improvements in the technique by testing alternative ligands to target the DNA to the liver of rats. Other potential improvements that will be tested include the use of a variety of alternative promoters in an attempt to increase the expression of the transgene in the liver and to alter the packaging system used to condense the DNA. The procedure described in this proposal has promise for the treatment of hemophilias and deserves further consideration as an alternative approach to gene therapy of these diseases.