SOMATIC CELL GENE THERAPY AND NITROGEN FLUX IN UREA CYCLE PATIENTS

先天性肝脏代谢缺陷组仍是一个 治疗不力导致精神发育迟滞的突出原因 战略。基因替代疗法提供了以下理论优势 纠正碱性蛋白质缺乏症。然而，肝细胞的研究进展 定向基因治疗一直受到以下问题的限制 病理生理过程，启动子和载体传递系统的选择， 传递途径，宿主免疫清除，表达持续时间， 小动物和大动物疾病模型的可用性，以及量化 临床疗效测评。这项提案的三个部分试图 使用尿素循环缺陷组模型来解决其中的一些问题 系统。第一个目标是更好地理解病理生理学 尿素循环缺陷患者的相关基因紊乱 和临床严重程度通过体内测量氮通量和 尿失禁，同时也开发了一种未来 动物和人类的活体基因治疗干预。助熔剂 通过尿素循环途径将通过量化 [15/N-酰胺]谷氨酰胺转化为[15/N]尿素。这一流量将是 与遗传状态(纯合、杂合、 半合性)、突变的性质(零突变与亚形突变)以及临床 受影响患者的严重程度(新生儿与后来的表现)， 杂合子家庭成员和正常对照。在第二部分中 研究，第一代(E1a)的相对安全性和有效性 删除)和第二代(删除E1a/E2a和所有编码序列 删除)腺病毒载体将在静脉注射后确定。 在动物体内分娩。此外，允许长期存在的潜在途径 将对转基因表达进行调查。瞬变的功效 用于病毒载体管理的免疫抑制将是 以及Mariner转座子元件在 在宿主哺乳动物基因组中介导转基因整合将是 学习。在第三部分，尿素循环障碍，特别是小鼠 和牛的瓜氨酸血症模型，将被用于 将这些基本发现应用到临床环境中。一种有效的方法 无处不在活性的CAG和肝脏特异的人血清白蛋白的杂交 启动子将在体内进行比较。这些数据将形成临床前 尿素基因治疗I期临床试验设计依据 周期病人。这些结果加在一起也将是更普遍的 适用于肝细胞代谢的其他先天缺陷和 肝细胞产生胞外产物。早期和长期 预计生化校正将大大降低 与这些情况相关的神经系统疾病。

The group of inborn errors of hepatic metabolism continue to be a prominent cause of mental retardation because of ineffective treatment strategies. Gene replacement therapy offers the theoretic advantage of correcting the basic protein deficiency. However, progress in hepatocyte directed gene therapy has been limited by questions involving pathophysiologic processes, choice of promoter and vector delivery system, route of delivery, host immune clearance, duration of expression, availability or small and large animal disease models, and quantitative measures of clinical efficacy. The three parts of this proposal attempt to address some of these issues using the group of urea cycle defects a model system. The first goal is to better understand the pathophysiologic disturbances in patients with urea cycle defects by correlating genotype and clinical severity with in vivo measurement of nitrogen flux and ureagenesis, while also developing a quantitative measure for future in vivo gene therapeutic interventions in both animal and humans. Flux through the urea cycle pathway will be measured by quantifying the conversion of [15/N-amide]glutamine to [15/N] urea. This flux will be correlated with genetic status (homozygosity, heterozygosity, hemizygosity), nature of mutation (null versus hypomorphic), and clinical severity (neonatal versus later presentation) in affected patients, heterozygous family members, and normal controls. In the second part of the study, the relative safety and efficacy of first generation (E1a deleted) and second generation (E1a/E2a deleted and all coding sequence deleted) adenovirus vectors will be determined after intravenous (i.v.) delivery in animals. In addition, potential avenues permitting long term transgene expression will be investigated. The efficacy of transient immunosuppression for the readministration of viral vectors will be evaluated, and the potential use of mariner transposon elements in mediating transgene integration in a host mammalian genome will be studied. In the third part, the urea cycle disorders, specifically murine and bovine models of citrullinemia, will be used a model systems in applying these basic findings to a clinical setting. The efficacy of the hybrid, ubiquitously active, CAG and liver-specific human albumin promoters will be compared in vivo. These data will form the preclinical basis for designing phase I clinical trials involving gene therapy in urea cycle patients. These results together will also be more generally applicable to other inborn errors of hepatocyte metabolism and to the production of extracellular products by hepatocytes. Early and long term biochemical correction would be expected to greatly decrease the great neurologic morbidity associated with these conditions.