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.

高氨血症是一个临床问题，对中枢神经系统造成严重后果。它通常是由肝病，遗传的代谢疾病和各种毒素引起的。氨产生的主要来源是产生的肠子，然后扩散到门户血液中。如果由于肝病或遗传性酶促缺陷，肝脏无法将模棱两可的氨转化为尿素，则氨将进入大脑上毒性的全身循环。该项目的目的是开发一种针对旨在在目标环境中起作用的高症治疗的新型形式。这种方法将输送到肠道的流体细菌基因过度表达精氨酸的生物合成酶，以便可以合成大量精氨酸。我们的假设是，驻留在富含氨的环境中的表达酶会捕获氨并将其遮盖以使其无毒。将大量氮掺入精氨酸中的大肠杆菌菌株已经成功地设计了。 这些细菌将被输送到高氨学动物模型SPF-ASH小鼠的肠道上。我们将研究肠道中工程细菌的生存力，定殖和解剖分布。这种治疗方法对宿主对氨代谢的代谢作用将得到研究。由于厌氧菌细菌细菌脆弱的片段比大肠杆菌比大肠杆菌比大肠杆菌中的大小数量更大，因此将测试各种受调节的启动子在肠道环境中的精氨酸生物合成基因的最佳表达，这是我们的最终型细菌治疗。我们目前正在完成精氨酸生物合成基因及其在该生物体中的调节。 B. fragilis将被设计为过量生产精氨酸，然后将其用于将氨气捕获在SPF-ASH小鼠的肠中。将研究精氨酸基因的精氨酸在转化后的脆弱芽孢杆菌中的表达，其高症小鼠与工程大肠杆菌一样。该项目的长期目标是在实验动物中证明了该疗法的疗效和安全性后，在其高氨血症的人类中测试这种疗法。还有许多其他遗传和获得的疾病可能适合细菌疗法。该项目将更好地理解这种治疗方法的希望和挑战。