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 这项开拓者奖的申请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盐与宿主细胞内的氧化还原水平结合起来。线粒体 功能障碍与许多疾病有关，包括但不限于衰老、癌症、神经变性和 糖尿病线粒体电子传递链（ETC）功能障碍是线粒体损伤的标志之一 疾病和新兴的研究表明，由ETC功能障碍引起的升高的NADH/NAD+比率可以 导致还原性压力。其他人最近的工作已经证明，融合蛋白的系统递送 包括细菌乳酸氧化酶（LOX）和过氧化氢酶（CAT），可以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，从而减轻细胞内的还原应激。 线粒体然而，全身递送细菌酶以修复线粒体功能障碍可能面临 几个挑战：（1）LOX和CAT酶是免疫原性的，（2）酶对蛋白酶敏感 （3）LOX和CAT酶具有短的血清半衰期，因此需要重复使用。 注射以维持治疗效果。因为乳酸和丙酮酸可以交换 在肠腔、循环和外周组织之间，我们建议工程化益生菌菌株，E.杆菌 Nissle（EcN），以在胃肠道中表达融合酶LOXCAT，从而将乳酸转化为丙酮酸 口服后。值得注意的是，EcN在人类中有着长期的安全记录，并且是一个受欢迎的开始。 工程治疗微生物的努力。在E. 我们将在EcN中开发一个合成的负反馈环，具有大的动态范围来感知和响应 导致血液中乳酸盐含量升高我们假设这种方法不仅可以解决上述问题- 上述问题与细菌酶在血液中的全身性递送有关，但也将使 一个新的系统，配备了传感器，遗传电路，和输出基因所必需的管理， 以时间和剂量控制的方式来控制LOXCAT融合酶。为了降低拟议工作的风险，我们 已经验证了LOX和CAT酶在EcN中的表达，在细菌中设计了荧光素酶报告基因， 允许无创体内追踪，并进行理论计算来预测可行性。建筑 根据我们的初步数据，我们将首先优化天然乳酸盐响应电路，以感知生理反应。 在一个实施方案中，将确定乳酸盐的浓度范围，并且将鉴定驱动LOXCAT表达的先导回路（目标1）。接下来， 我们将在野生型小鼠中检测工程化EcN的药代动力学、生物分布和安全性。最后 将在线粒体功能障碍的小鼠模型中通过复合物的损失来评价治疗功效 I亚基Ndufs 4（Aim 2）。这项提案的成功完成不仅会设计出一部小说 线粒体功能障碍的平台，但我们也将开发出一种创新的方法来调节 作为一种手段，询问代谢物和疾病之间的因果关系。