THERAPY FOR HYPERAMMONEMIA WITH GENE ENGINEERED BACTERIA

用基因工程细菌治疗高氨血症

• 批准号: 6442587
• 负责人: Mendel Tuchman
• 金额: $ 10.62万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6442587
• 关键词: Bacteroides; Escherichia coli; aminoacid biosynthesis; ammonia; arginine; bacterial genetics; biotechnology; biotherapeutic agent; blood chemistry; colon; detoxification; enteric bacteria; gene expression; gene therapy; laboratory mouse; microorganism metabolism; microorganism population study; nitrogen metabolism; transfection /expression vector

Hyperammonemia is a clinical problem with severe consequences to the central nervous system. It is usually caused by liver disease, inherited metabolic disorders and various toxins. The main source of ammonia production is the bowel where it is generated and then diffuses into the portal blood. If the liver is unable to convert ambiguous ammonia to urea due to liver disease or an inherited enzymatic defect, the ammonia enters the systemic circulation exerting toxicity on the brain. The goal of this project is to develop a novel form off therapy for hyperammonemia aimed to act within aimed the intestinal environment. This approach will deliver into the bowel's lumen bacterial genes over-expressing arginine biosynthesis enzymes so that large amounts of arginine can be synthesized. Our hypothesis is that the expressed enzymes, residing within an ammonia rich environment, will trap ammonia and covert it to arginine rendering it non toxic. Escherichia coli strains incorporating large amounts of nitrogen into arginine have already been successfully engineered. These bacteria will be delivered to the bowel of a hyper- ammonemic animal model, the spf-ASH mouse. We will study the viability, colonization and anatomical distribution of the engineered bacteria in the bowel. The metabolic effects of this treatment of this treatment on the host with respect to ammonia metabolism will be investigated. Various regulated promoters will be tested for optimal expression of arginine biosynthesis genes in the bowel's environment As the anaerobic bacteria Bacteroides fragilis is orders of magnitudes more numerous within the colonic bacterial flora than E. coli, it is our ultimate vehicle for bacterial therapy. We are currently completing the identification of arginine biosynthesis genes and their regulation in this organism. B. fragilis will be engineered to over-produce arginine and will then be used to trap ammonia in the intestine of the spf-ASH mouse. The expression of arginine of arginine genes in the transformed B. fragilis will be investigated and their hyperammonemic mice as with engineered E. coli. The long term goal of this project is to test this therapy in humans with hyperammonemia after its efficacy and safety have been demonstrated in laboratory animals. There are many other inherited and acquired conditions which may be amenable to bacterial therapy. This project will provide better understanding of the promise and challenges of such therapeutic approach.

高氨血症是一种对中枢神经系统造成严重后果的临床问题。它通常是由肝脏疾病、遗传性代谢紊乱和各种毒素引起的。氨产生的主要来源是肠道，氨在肠道中产生，然后扩散到门静脉血液中。如果由于肝脏疾病或遗传性酶缺陷，肝脏无法将模糊的氨转化为尿素，氨就会进入体循环，对大脑产生毒性。该项目的目标是开发一种针对高氨血症的新型疗法，旨在针对肠道环境发挥作用。这种方法将过度表达精氨酸生物合成酶的细菌基因输送到肠腔中，从而可以合成大量精氨酸。我们的假设是，表达的酶存在于富含氨的环境中，会捕获氨并将其转化为精氨酸，使其无毒。将大量氮掺入精氨酸的大肠杆菌菌株已被成功改造。 这些细菌将被输送到高氨血症动物模型 spf-ASH 小鼠的肠道。我们将研究工程细菌在肠道中的活力、定植和解剖分布。将研究这种处理对宿主在氨代谢方面的代谢影响。将测试各种受调节的启动子，以确保精氨酸生物合成基因在肠道环境中的最佳表达。由于厌氧细菌脆弱拟杆菌在结肠细菌菌群中的数量比大肠杆菌多几个数量级，因此它是我们细菌治疗的最终载体。我们目前正在完成精氨酸生物合成基因的鉴定及其在该生物体中的调控。脆弱拟杆菌将被改造为过量产生精氨酸，然后用于捕获 spf-ASH 小鼠肠道中的氨。将研究转化的脆弱拟杆菌及其高氨血症小鼠中精氨酸或精氨酸基因的表达，就像工程化的大肠杆菌一样。该项目的长期目标是在实验动物中证明该疗法的有效性和安全性后，在患有高氨血症的人类中测试该疗法。还有许多其他遗传性和后天性疾病可能适合细菌治疗。该项目将让人们更好地了解这种治疗方法的前景和挑战。