Live microbial therapeutics for enzyme replacement therapy against homocystinuria

用于治疗同型半胱氨酸尿症的酶替代疗法的活微生物疗法

• 批准号: 10384712
• 负责人: Joseph Moeller Schinaman
• 金额: $ 30.69万
• 依托单位: PETRI BIO LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10384712
• 关键词: Accounting; Adherence; Amino Acids; Betaine; Catalysis; Characteristics; Clinical; Cloning; Connective Tissue; Cystathionine; Cystathionine beta-Synthase; Cysteine; Data; Dependence; Detection; Development; Diet; Dietary Supplementation; Dietary intake; Disease; Disease model; Doctor of Philosophy; Eating; Ensure; Enzymes; Escherichia coli; Exhibits; Future; Genes; Genetic Diseases; Goals; Homocysteine; Homocystinuria; Human; Human Microbiome; Impairment; In Vitro; Incidence; Lens dislocation; Lyase; Measures; Metabolic; Metabolic Diseases; Methionine; Methods; Modeling; Mutate; Mutation; Outcome; Phase; Plasma; Preclinical Drug Development; Production; Property; Pyridoxal Phosphate; Reaction; Recombinant Proteins; Reporting; Risk; Safety; Serine; Stroke; Surveys; Symptoms; Testing; Therapeutic; Tissues; Toxic effect; Transgenes; Transgenic Organisms; Vascular Diseases; Vitamin B6; Western Blotting; bacterial vector; base; biomaterial compatibility; diagnostic assay; dietary restriction; efficacy testing; enzyme replacement therapy; enzyme therapy; expression vector; eye center; gastrointestinal; gut microbiome; human subject; in silico; in vitro Assay; in vitro testing; in vivo; lead candidate; lens; microbial; mouse model; novel strategies; programs; screening; stroke risk; success; therapeutic enzyme; therapeutic protein; thrombotic; thrombotic complications; treatment strategy; tumorigenesis; uptake; vector

Project Summary The goal of this proposal is to continue the development of a synthetic live bacterial therapeutic for homocystinuria, an inborn metabolic disorder leading to accumulation of the amino acid methionine, and results in dramatically increased risk of stroke and other thrombotic conditions. Petri Bio’s approach will be capable of breaking down methionine in the gut to reduce or eliminate dependence on a methionine restricted diet and result in decreased plasma and tissue homocysteine resulting in superior clinical outcomes. The condition is estimated to occur at an incidence of 1 in 250,000, however some reports indicate a potential incidence of 1 in 65,000 when accounting for the current imprecise diagnostic assays and often subtle symptoms which may evade clinical detection until they become severe or obvious, such as lens detachment from the center of the eye. Cystathionine beta-synthase (CBS), the gene mutated in classical homocystinuria, is localized at a key regulatory branch point in the eukaryotic methionine cycle. CBS catalyzes a pyridoxal 5′-phosphate dependent beta-replacement reaction condensing serine and homocysteine (Hcy) into cystathionine that is subsequently converted to cysteine in a reaction catalyzed by cystathionine-γ-lyase (CGL, EC 4.4.1.1). Inactivation of CBS by mutation results in classical homocystinuria (HCU) which in human subjects, is characterized by a range of connective tissue disturbances including marfanoid habitus and lens dislocation, intellectual impairment, and a dramatically increased incidence of vascular disorders particularly thromboembolic complications such as stroke. Treatment strategies for pyridoxine non-responsive HCU typically attempt to lower plasma and tissue levels of Hcy by a combination of restricting dietary intake of the Hcy precursor methionine and dietary supplementation with trimethylglycine, more commonly referred to as betaine. Petri Bio, Inc. has developed a novel strategy for enzyme therapy, employing prokaryotic strains compatible with the human gut microbiome to serve as expression vectors for therapeutic protein administration. After in silico screening of bacterially-derived methionases for a number of desirable characteristics of therapeutic enzymes, ten have been cloned, expressed, and shown to reduce methionine concentrations in vitro. During this Phase I program, we will extend these studies by screening hundreds of bacterially-derived methionases in silico and subsequently cloning, expressing, and testing in vitro methionine catalysis capabilities. In vitro tests will be undertaken to measure methionase activity of these bacterial strains. After optimization of strains with high methionase activity, we will evaluate their ability to reduce methionine concentrations in vivo, as well as ameliorate the effects of methionine accumulation in a murine model of HCU. Future studies will optimize the bacterial methionase transgenes to ensure maximum activity and biocompatibility as well as select a lead candidate bacterial strain for preclinical drug development.

项目摘要 这项提案的目标是继续开发一种合成的活细菌治疗剂， 高胱氨酸尿症，一种导致氨基酸甲硫氨酸积累的先天性代谢紊乱， 中风和其他血栓性疾病的风险显著增加。Petri Bio的方法将能够 分解肠道中的甲硫氨酸以减少或消除对甲硫氨酸限制饮食的依赖， 导致血浆和组织同型半胱氨酸降低，从而导致上级临床结果。病情 估计发生率为1/250，000，但一些报告显示潜在发生率为1/250，000。 65，000当考虑到目前不精确的诊断测定和通常可能 避免临床检测，直到它们变得严重或明显，例如透镜从中心脱离。 眼睛胱硫醚β-合酶（CBS），在经典同型胱氨酸尿症的基因突变，是定位在一个关键的 真核生物蛋氨酸循环中的调节分支点。CBS催化吡哆醛5′-磷酸依赖性 β-置换反应将丝氨酸和同型半胱氨酸（Hcy）缩合为胱硫醚，随后 在由胱硫醚-γ-裂解酶（CGL，EC 4.4.1.1）催化的反应中转化为半胱氨酸。通过以下方法灭活CBS 突变导致经典的同型胱氨酸尿症（HCU），其在人类受试者中的特征在于一系列的 结缔组织障碍，包括马凡样体质和透镜脱位、智力障碍和 血管疾病的发病率显著增加，特别是血栓栓塞并发症，如中风。 吡哆醇无反应HCU的治疗策略通常试图降低吡哆醇的血浆和组织水平。 通过限制Hcy前体蛋氨酸的饮食摄入和饮食补充相结合来治疗Hcy 与三甲基甘氨酸，更通常地称为甜菜碱。Petri Bio，Inc.开发了一种新的策略， 酶疗法，采用与人类肠道微生物组相容的原核菌株， 用于治疗性蛋白质施用的表达载体。在计算机筛选细菌衍生的 甲硫氨酸酶具有许多治疗酶的理想特性，已经克隆了10种甲硫氨酸酶，表达， 并显示在体外降低甲硫氨酸浓度。在第一阶段计划中，我们将 通过计算机筛选数百种细菌衍生的甲硫氨酸酶，随后克隆，表达， 并测试体外甲硫氨酸催化能力。将进行体外试验以测量甲硫氨酸酶 这些菌株的活性。在优化具有高甲硫氨酸酶活性的菌株之后，我们将评估其 降低体内蛋氨酸浓度的能力，以及改善蛋氨酸积累的影响 在HCU小鼠模型中。未来的研究将优化细菌甲硫氨酸酶转基因，以确保最大的 活性和生物相容性以及选择用于临床前药物开发的主要候选菌株。