Engineering Probiotics to Sense and Respond to the Intracellular Redox Imbalance towards Mitochondrial Dysfunction

工程益生菌可感知和响应细胞内氧化还原失衡导致线粒体功能障碍

• 批准号: 10303309
• 负责人: Jiahe Li
• 金额: $ 2.97万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303309
• 关键词: Address; Adenosine Triphosphate; Aging; Award; Bacteria; Benchmarking; Biodistribution; Blood; Blood Circulation; Bolus Infusion; Carrier Proteins; Cells; Chimeric Proteins; Chromosomes; Complex; Coupled; Coupling; DNA; Data; Diabetes Mellitus; Disease; Disease Marker; Dose; Drug Kinetics; Electron Transport; Elements; Engineered Probiotics; Engineering; Enzymes; Escherichia coli; Face; Feedback; Frequencies; Functional disorder; Gastrointestinal tract structure; Gene Mutation; Genes; Genetic; Genetic Diseases; Half-Life; Health; Human; Hydrogen Peroxide; Hypoxia; Inflammatory Bowel Diseases; Inherited; Injections; Intervention; Intestines; Kidney Failure; Knockout Mice; Lactic Acidosis; Lead; Liver diseases; Luciferases; Malignant Neoplasms; Metabolic Diseases; Metabolism; Microbe; Mitochondria; Mitochondrial Diseases; Modeling; Mus; NADH; Nature; Nerve Degeneration; Nuclear; Oral; Oral Administration; Output; Oxidases; Oxidation-Reduction; Oxygen; Patients; Peptide Hydrolases; Peripheral; Physiological; Play; Probiotics; Pyruvate; Reporter; Rewards; Risk Factors; Robot; Role; Safety; Serum; Survival Rate; Symptoms; System; Technology; Therapeutic; Therapeutic Effect; Tissues; Translations; Treatment Efficacy; Water; Wild Type Mouse; Work; base; catalase; dosage; gene therapy; high reward; high risk; immunogenic; in vivo; innovation; mitochondrial dysfunction; mouse model; novel; novel strategies; oxidation; pre-clinical; promoter; public health relevance; repaired; safety engineering; sensor; stress reduction; success; therapeutic enzyme

Abstract This Trailblazer Award application will enable a smart bio-robot to ameliorate mitochondrial dysfunctions by coupling a common mitochondrial disease marker, lactate, to the redox levels inside host cells. Mitochondrial dysfunction is associated with many diseases including, but not limited to, aging, cancer, neurodegeneration and diabetes. The dysfunction of mitochondrial electron transport chain (ETC) is one of the hallmarks of mitochondrial diseases and emerging studies show that the elevated NADH/NAD+ ratio resulting from ETC dysfunction can lead to reductive stress. Recent work by others have demonstrated that systemic delivery of a fusion protein comprising bacterial lactate oxidase (LOX) and catalase (CAT), can convert lactate to pyruvate in the blood, which is coupled to lower the intracellular ratio of NADH/NAD+, and thereby mitigating reductive stress in mitochondria. However, systemic delivery of bacterial enzymes to repair mitochondrial dysfunction can face several challenges: (1) LOX and CAT enzymes are immunogenic, (2) enzymes are susceptible to protease degradation in the blood and (3) LOX and CAT enzymes have short serum half-life, therefore requiring repeated injections to sustain the therapeutic effects. Motivated by the fact that lactate and pyruvate can exchange between the gut lumen, circulation and peripheral tissues, we propose to engineer the probiotic strain, E. coli Nissle (EcN), to express the fusion enzyme LOXCAT in the gastrointestinal tract to convert lactate to pyruvate following oral administration. Notably, EcN has a long track record of safety in humans, and is a popular starting point for engineered therapeutic microbe efforts. Building on naturally derived lactate-responsive elements in E. coli, we will develop a synthetic negative feedback loop in EcN with a large dynamic range to sense and respond to elevated levels of lactate in the blood. We hypothesize that this approach will not only address the above- mentioned problems associated with systemic delivery of bacterial enzymes in the blood, but will also enable a new system that is armed with the sensors, genetic circuits, and output genes necessary for administration of the LOXCAT fusion enzyme in a temporally and dosage-controlled manner. To derisk the proposed work, we have validated the expression of LOX and CAT enzymes in EcN, engineered a luciferase reporter in bacteria to allow for noninvasive in vivo tracking, and performed theoretical calculations to predict the feasibility. Building on our preliminary data, we will first optimize the natural lactate-responsive circuit to sense a physiological concentration range of lactate, and the lead circuit will be identified to drive LOXCAT expression (Aim 1). Next, we will examine pharmacokinetics, biodistribution and safety of engineered EcN in wild-type mice. Finally, the therapeutic efficacy will be evaluated in a mouse model of mitochondrial dysfunction via the loss of the complex I subunit Ndufs4 (Aim 2). The successful completion of this proposal will not only have engineered a novel platform for mitochondria dysfunction, but we will have also developed an innovative approach to modulate metabolites in the circulation as a means to interrogate causal relationships between metabolites and diseases.

摘要 这项开拓者奖的应用程序将使智能生物机器人能够通过以下方式改善线粒体功能障碍 将常见的线粒体疾病标志物乳酸与宿主细胞内的氧化还原水平相结合。线粒体 功能障碍与许多疾病有关，包括但不限于衰老、癌症、神经退行性变和 糖尿病。线粒体电子传递链(ETC)功能障碍是线粒体功能障碍的标志之一 疾病和新出现的研究表明，由ETC功能障碍引起的NADH/NAD+比值升高可以 导致减少压力。其他人最近的研究表明，融合蛋白的系统性递送 由细菌乳酸氧化酶(LOX)和过氧化氢酶(CAT)组成，可将血液中的乳酸转化为丙酮酸， 其偶联以降低细胞内NADH/NAD+的比率，从而减轻大鼠体内的还原应激 线粒体。然而，全身输送细菌酶来修复线粒体功能障碍可能会面临 几个挑战：(1)LOX和CAT酶具有免疫原性，(2)酶对蛋白酶敏感 血液中的降解和(3)LOX和CAT酶的血清半衰期很短，因此需要重复 以维持治疗效果的注射。其动机是乳酸和丙酮酸可以交换 在肠腔、循环和周围组织之间，我们建议改造益生菌菌株E.Coli. Nissle(ECN)，在胃肠道表达融合酶LOXCAT，将乳酸转化为丙酮酸 口服给药后。值得注意的是，ECN在人体安全方面有很长的记录，是一个受欢迎的开始 为工程治疗微生物努力的重点。建立在E。 Coli，我们将在ECN中开发一个具有大动态范围的合成负反馈环路来感知和响应 导致血液中乳酸水平升高。我们假设，这种方法不仅将解决上述问题- 提到了与血液中细菌酶的系统传递有关的问题，但也将使 一种新的系统，配备了给药所需的传感器、基因电路和输出基因 LOXCAT融合酶以时间和剂量控制的方式。为了降低拟议工作的风险，我们 验证了LOX和CAT酶在ECN中的表达，在细菌中设计了一个荧光素酶报告程序来 考虑到无创的体内跟踪，并进行了理论计算，以预测其可行性。建房 根据我们的初步数据，我们将首先优化自然乳酸反应回路，以感知生理上的 乳酸的浓度范围，将确定引导回路来驱动LOXCAT的表达(目标1)。下一首, 我们将研究基因工程ECN在野生型小鼠体内的药代动力学、生物分布和安全性。最后， 治疗效果将在线粒体功能障碍的小鼠模型中通过丢失复合体进行评估 I亚基Ndufs4(目标2)。这一提议的成功完成不仅将策划一部小说 线粒体功能障碍的平台，但我们也将开发一种创新的方法来调节 循环中的代谢物，作为研究代谢物和疾病之间因果关系的一种手段。