PHARMACOLOGICAL & GENE THERAPY OF ARPKD--FROM CELL TO ANIMAL

描述：(直接取自申请)这项研究的目标是开发和测试常染色体隐性遗传性多囊肾病(ARPKD)的治疗方法，这些治疗方法专门针对疾病发病机制中的关键过程而设计。有待检验的总体假设是，阻断或靶向ARPKD中表皮生长因子(EGF)-转化生长因子-α(TGF-α)表皮生长因子受体(EGFR)轴的异常将导致囊性肾脏疾病的结构和功能的改善。多个实验室公布的数据表明，囊性小管上皮细胞中EGFR表达的改变在人类和小鼠ARPKD以及常染色体显性遗传性多囊肾病(ADPKD)中是常见的。虽然建议的具体项目将集中在ARPKD和人类ARPKD细胞的动物模型上，但在本研究过程中开发的治疗方法可能也适用于ADPKD。研究将在原代和永生化细胞系、器官培养和完整动物中进行。将利用药理学、遗传学和细胞生物学技术来实现以下特定目的：1.开发旨在阻断ARPKD中EGF-TGFα-EGFR轴活性的治疗策略。需要检验的具体假设是：a.)抑制EGFR活性可持续改善ARPKD患者的肾功能，且无毒性；抑制生物活性配体(EGF/TGFpha)的产生和/或加工对ARPKD：和c.具有治疗效果。)联合抑制EGFR活性和EGF/TGFα的加工/产生比单独使用任何一种治疗方法更有效，毒性更小。建议研究中使用的方法包括EGFR酪氨酸激酶抑制剂、改变配体产生的药物和反义寡核苷酸的体外/体内试验。2.开发ARPKD基因治疗的新策略。需要检验的具体假设是：a.)(B)疾病发展后的基因替换可以改变ARPKD的疾病进展；通过利用EGF/TGFpha/EGFR轴的元件来增加基因传递，可以提高基因治疗的有效性和特异性；针对疾病发展后的关键致病过程的基因治疗可以改变ARPKD的疾病进展。这些研究的方法包括脂质体介导的基因转移，腺相关病毒介导的基因转移，以及利用EGFR配体或抗体通过受体介导的内吞作用传递基因。这些研究将提供新的科学见解和信息，可能有助于设计人类ARPKD和ADPKD的特定治疗方法。

Description: (Taken directly from the application) The objective of this research is to develop and test therapies for autosomal recessive polycystic kidney disease (ARPKD), which are specifically designed to target key processes in disease pathogenesis. The overall hypothesis to be tested is that interruption or therapeutic targeting of abnormalities in the epidermal growth factor (EGF)-transforming growth factor alpha (TGF-alpha) epidermal growth factor receptor (EGFR) axis in ARPKD will result in structural and functional improvement in cystic kidney disease. Published data from a number of laboratories demonstrate that altered EGFR expression in cystic tubular epithelial cells is common to human and murine ARPKD as well as autosomal dominant polycystic kidney disease (ADPKD). Although the specific projects proposed will focus on animal models of ARPKD and human ARPKD cells, therapies developed during the course of this study may be generalizable to ADPKD as well. Studies will be conducted in primary and immortalized cell lines, organ culture and intact animals. Pharmacologic, genetic and cell biology techniques will be employed to accomplish the following Specific Aims: 1. Develop therapeutic strategies aimed at interrupting EGF-TGFalpha-EGFR axis activity in ARPKD. Specific hypotheses to be tested are: a.) Inhibition of EGFR activity results in sustained improvement of renal function without toxicity in ARPKD; b.) Inhibition of the production and/or processing of biologically active ligands (EGF/TGFalpha ) has therapeutic effectiveness in ARPKD: and c.) Combined inhibition of EGFR activity and processing/production of EGF/TGFalpha is more effective and less toxic than either therapy given alone. Methods to be utilized in the proposed studies include in vitro/vivo trials of EGFR tyrosine kinase inhibitors, agents which alter ligand production, and antisense oligonucleotides. 2. Develop new strategies for gene therapy in ARPKD. Specific hypotheses to be tested are: a.) Gene replacement following disease development can alter disease progression in ARPKD; b.) Effectiveness and specificity of gene therapy can be improved by utilizing elements of the EGF/TGFalpha /EGFR axis to augment gene delivery; and c.) Genetic therapy directed at targeting key pathogenic processes following disease development can alter disease progression in ARPKD. Methods to be utilized in the proposed studies include liposome mediated gene transfer, adeno-associated virus-mediated gene transfer, and the use of EGFR-ligand or antibody to deliver genes by receptor mediated endocytosis. These studies will provide new scientific insights and information which may be useful in the design of specific treatments for human ARPKD and ADPKD.