Pathobiology of Cystic Fibrosis-Related Diabetes in a Ferret Model

雪貂模型中囊性纤维化相关糖尿病的病理学

基本信息

批准号：
8468701
负责人：
Zoe Stewart
金额：
$ 14.75万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-08 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8468701
关键词：
Abdomen Address Adenoviruses Adult Age Alkaline Phosphatase Animal Model Animals Antibiotics Attenuated Award Basic Science Bicarbonates Birth Caucasians Caucasoid Race Cell Culture Techniques Cell Lineage Cell physiology Cells Characteristics Chloride Channels Clinical Commit Complementary DNA Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Defect Development Diabetes Mellitus Disease Disease Progression Doctor of Philosophy Ductal Duodenum Effectiveness Endocrine Epithelial Cells Epithelium Excision Exhibits Exposure to Fellowship Ferrets Fibrocystic Disease of Pancreas Foundations Functional disorder Funding Gallbladder Gene Delivery Gene Expression Gene Transduction Agent Genetic Glucagon Glucose Goals Growth Health Histopathology Human Hyperglycemia IL8 gene Infection Inflammation Mediators Insulin Interleukin-1 Intestines Islets of Langerhans K-Series Research Career Programs Knock-out Laboratories Laboratory Research Lead Leadership Learning Life Life Expectancy Liver Luciferases Lung Lung diseases Measures Mediating Medical Mentors Mentorship Methods Modeling Molecular Molecular Genetics Mus Neonatal Nutritional Onset of illness Operative Surgical Procedures Oral Organ Outcome Study Pancreas Pancreatic Diseases Pancreatic duct Pancreatic enzyme Pathway interactions Patients Perinatal mortality demographics Phenotype Physicians Physiology Predisposition Process Proton Pump Inhibitors Quality of life Recombinants Records Regulator Genes Reporter Genes Research Research Personnel Residencies Satellite Viruses Scientist Secure Serotyping Serum Severities Symptoms Systems Development Techniques Testing Therapeutic Time Training Transplantation Tropism Variant Viral Vector Weight Gain Work Writing adeno-associated viral vector apical membrane authority base blood glucose regulation career cell type clinical care cohort cystic fibrosis patients effective therapy gastrointestinal gastrointestinal function gene replacement gene replacement therapy gene therapy glucose metabolism improved in vivo inhibitor/antagonist intestinal epithelium lipid metabolism nutrition programs response skills success therapy development tool undergraduate student vector

项目摘要

DESCRIPTION (provided by applicant): I have been committed to a career in basic science since my first exposure to research as an undergraduate student. After completing an MD/PhD program, I went on to pursue surgical residency and then a fellowship in abdominal transplant surgery. To achieve my goal of becoming a successful independent researcher, I recently entered the laboratory of Dr. John Engelhardt, a national authority on the molecular pathophysiology of cystic fibrosis (CF) and strategies to utilize recombinant adenovirus-associated viruses (rAAV) as gene therapy vectors. Dr. Engelhardt has proven track records in both mentoring physician- scientists and securing research funding, and his guidance will be a critical determinant of my success in developing my independent research laboratory. Under Dr. Engelhardt's mentorship, I am focused on combining my basic science training with my medical background to tackle an important clinical question that directly impacts my patients, namely studying the pathophysiology of cystic fibrosis-related diabetes (CFRD). This is a clinically important question, as CF is the most common lethal autosomal recessive disease in Caucasians, resulting from defects in the cystic fibrosis transmembrane conductance regulator (CFTR) channel. This K-award will allow critical protected time for me to fully develop my independent research career. During the period of this award, I intend to: 1) expand my skills in molecular genetics, 2) master the techniques relevant to CF research and learn to generate pathophysiologically important hypotheses about CFRD, 3) develop skill in the implementation of rAAV gene therapies, 4) strengthen my writing and leadership/mentoring skills, and 5) solidify preliminary data for my first R01 application. In CF patients, pancreatic disease leads to diabetes mellitus in up to 50% of adult patients, resulting in reduced life expectancy. Progress in elucidating the pathophysiology of CFRD has been hindered by the lack of an animal model. Dr. Engelhardt's laboratory has recently developed a CF-knockout ferret - the first animal model that develops CFRD. However, CF ferrets have high perinatal mortality from abnormal intestinal and pancreatic function, altered glucose and lipid metabolism, and lung infections. Elevating GI pH appears to attenuate nutritional abnormalities, as the use of proton-pump inhibitors (PPIs) normalizes weight gain during the neonatal period. The CF ferret model appears to be a robust system for the development of CF pancreatic therapies and is the only animal model available for the study of CFRD. We hypothesize that effective treatment of CFRD will lead to reduced nutritional complications in CF ferrets, increased long-term survival, and reduced severity of lung disease. We will test this hypothesis by carrying out the following specific aims: 1) Identify the defects underlying the pancreatic and intestinal pathophysiologies associated with CFRD in the ferret CF model and 2) Establish a CFTR gene replacement therapy that will improve pancreatic function in CF ferrets. To address the goals of Aim 1, we will use standard pancreatic endocrine functional tests to confirm that a subset of CF ferrets develop CFRD and characterize the disease pathophysiology. We will determine if CFTR-mediated bicarbonate (HCO3-) secretion by the pancreatic duct and/or duodenal epithelial cells is reduced, and if so, how this impacts CFRD phenotypic severity. We will also evaluate the activity of compensatory, non-CFTR-based HCO3- secretory pathways using CFTR inhibitors to determine if these channels might influence the severity and/or disease onset of CFRD. To address the goals of Aim 2, we will adapt methods developed for the mouse (application of rAAV vectors encoding the WT ferret CFTR cDNA) for in vivo gene delivery to the pancreas in CFTR-/- ferrets. We will assess the effectiveness of pancreatic CFTR gene replacement in the CFTR-/- ferrets by evaluating serum glucose, insulin, glucagon, endocrine pancreatic function, and pancreatic duct cell physiology. We will also assess the impact of pancreatic CFTR gene therapy by evaluating ferret growth and nutritional parameters in the presence and absence of PPIs and pancreatic enzymes. Finally, we will evaluate the impact of pancreatic CFTR gene therapy on the progression of early pulmonary disease progression in CF ferrets following removal of antibiotics. These studies will assess lung inflammatory mediators (IL-1, IL-8, and TNFa), lung bacterial colonization, and lung histopathology. The major expected outcomes of this study are: 1) a more comprehensive characterization of endocrine pancreas abnormalities in the new CF ferret model, 2) an understanding of the basis of phenotypic variation in CFTR-associated pancreatic disease observed in both the CF ferrets and humans, and 3) the development of a pancreatic gene replacement approach that will aid in dissecting pathophysiology and treatment of CFRD. These results are expected to have a significant positive impact on the field of CF research.

描述（由申请人提供）：自从我首次作为一名本科生的研究接触以来，我一直致力于基础科学的职业。完成MD/博士学位计划后，我继续进行外科手术居留权，然后在腹部移植手术中获得研究金。为了实现成为一名成功独立研究人员的目标，我最近进入了约翰·恩格哈特（John Engelhardt）博士的实验室，约翰·恩格哈特（John Engelhardt）博士是囊性纤维化分子病理生理学（CF）（CF）的国家权威，并利用重组腺病毒相关病毒（RAAV）作为基因治疗量。恩格哈特（Engelhardt）博士在指导医师科学家和确保研究资金方面已经证明了往绩记录，他的指导将是我成功发展独立研究实验室的重要决定因素。在恩格哈特（Engelhardt）博士的指导下，我专注于将基础科学培训与我的医学背景相结合，以解决一个重要的临床问题，该问题直接影响了我的患者，即研究与囊性纤维化相关糖尿病（CFRD）的病理生理学。这是一个临床上重要的问题，因为CF是高加索人中最常见的致死性常染色体隐性疾病，这是由于囊性纤维化跨膜电导调节剂（CFTR）通道的缺陷所致。这个K-award将为我提供关键的保护时间，以充分发展我的独立研究生涯。在该奖项期间，我打算：1）扩展我在分子遗传学方面的技能，2）掌握与CF研究相关的技术，并学会产生有关CFRD的病理生理学上重要的假设，3）在实施Raav Gene Therapies的实施方面，4）4）增强我的写作和领导能力的技能，以及5）固定的Prelimialify Prelimiary Datial Insimalion My My My My r0 r0 r0 r0 r0 r0。在CF患者中，胰腺疾病会导致多达50％的成年患者导致糖尿病，导致预期寿命降低。缺乏动物模型，阻碍了阐明CFRD病理生理学的进展。恩格哈特（Engelhardt）博士的实验室最近开发了一种CF -KNOCKOUT雪貂，这是开发CFRD的第一个动物模型。然而，CF雪貂的肠道和胰腺功能异常，葡萄糖和脂质代谢改变以及肺部感染具有较高的围产期死亡率。随着质子 - 泵抑制剂（PPI）的使用使新生儿期间的体重增加正常，升高的GI pH似乎会减弱营养异常。 CF雪貂模型似乎是用于开发CF胰腺疗法的强大系统，也是唯一可用于研究CFRD的动物模型。我们假设CFRD的有效治疗将导致CF雪貂的营养并发症减少，长期存活率增加以及肺部疾病严重程度降低。我们将通过执行以下特定目的来检验这一假设：1）确定与雪貂CF模型中与CFRD相关的胰腺和肠道病理生理的缺陷和2）建立CFTR基因替代疗法，该疗法将改善CF Ferrets中的胰腺功能。为了满足AIM 1的目标，我们将使用标准的胰腺内分泌功能测试来确认CF雪貂的一部分会发展为CFRD并表征疾病的病理生理学。我们将确定胰管和/或十二指肠上皮细胞的CFTR介导的碳酸氢盐（HCO3-）分泌是否减少，如果是这样，这如何影响CFRD表型严重程度。我们还将使用CFTR抑制剂评估基于代偿性的，非CFTR的HCO3分泌途径的活性，以确定这些通道是否可能影响CFRD的严重性和/或疾病发作。为了满足AIM 2的目标，我们将适应为小鼠开发的方法（使用编码WT雪貂CFTR cDNA的RAAV载体的应用），用于体内基因递送到CFTR - / - 雪貂的胰腺。我们将通过评估血清葡萄糖，胰岛素，胰高血糖素，内分泌胰腺功能和胰腺管细胞生理学来评估CFTR - / - 雪貂中胰腺CFTR基因替代的有效性。我们还将通过在PPIS和胰腺酶的存在下评估雪貂生长和营养参数来评估胰腺CFTR基因治疗的影响。最后，我们将评估胰腺CFTR基因治疗对去除抗生素后CF雪貂早期肺部疾病进展的进展的影响。这些研究将评估肺部炎症介质（IL-1，IL-8和TNFA），肺细菌定殖和肺组织病理学。这项研究的主要预期结果是：1）在新的CF雪貂模型中，内分泌胰腺异常的更全面表征，2）对CFTR相关胰腺疾病的基础的理解，在CF雪貂和人类中都观察到了pather path的pather path path path path path path ot path path path ot path path path ot path path path the pancrestion的发展。 CFRD。预计这些结果将对CF研究领域产生重大积极影响。