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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的该开拓者奖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记物乳酸与宿主细胞内的氧化还原水平耦合。线粒体功能障碍与许多疾病相关，包括但不限于衰老、癌症、神经退行性疾病和糖尿病。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。疾病和新兴研究表明，ETC 功能障碍导致 NADH/NAD+ 比率升高可以导致减少压力。其他人最近的工作表明，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，它可以降低细胞内 NADH/NAD+ 的比例，从而减轻细胞内的还原应激线粒体。然而，全身递送细菌酶来修复线粒体功能障碍可能面临几个挑战：(1) LOX 和 CAT 酶具有免疫原性，(2) 酶对蛋白酶敏感 (3) LOX 和 CAT 酶的血清半衰期短，因此需要重复注射以维持治疗效果。受到乳酸和丙酮酸可以交换这一事实的启发在肠腔、循环和外周组织之间，我们建议改造益生菌菌株，大肠杆菌 Nissle (EcN)，在胃肠道中表达融合酶 LOXCAT，将乳酸转化为丙酮酸口服给药后。值得注意的是，EcN 在人类安全方面有着悠久的记录，并且是一个受欢迎的起点工程治疗微生物努力的重点。以大肠杆菌中天然衍生的乳酸反应元素为基础。大肠杆菌，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可以解决上述问题提到了与血液中细菌酶的全身输送相关的问题，但也将使新系统配备了管理所需的传感器、遗传电路和输出基因 LOXCAT融合酶以时间和剂量控制的方式。为了消除拟议工作的风险，我们验证了 EcN 中 LOX 和 CAT 酶的表达，在细菌中设计了荧光素酶报告基因允许无创体内跟踪，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响应电路以感知生理乳酸浓度范围，并且将确定驱动 LOXCAT 表达的先导电路（目标 1）。下一个，我们将检查工程化 EcN 在野生型小鼠中的药代动力学、生物分布和安全性。最后，将通过复合物的丢失在线粒体功能障碍的小鼠模型中评估治疗效果 I 亚基 Ndufs4（目标 2）。该提案的成功完成不仅将设计出一个新颖的线粒体功能障碍的平台，但我们还将开发一种创新方法来调节循环中的代谢物作为询问代谢物与疾病之间因果关系的手段。