Live microbial therapeutics for enzyme replacement therapy against homocystinuria

项目摘要 这项提议的目标是继续开发一种合成的活细菌治疗药物 同型半胱氨酸尿症，一种导致氨基酸蛋氨酸积累的先天代谢紊乱，以及结果 显著增加中风和其他血栓形成的风险。PETERN BIO的方法将能够 分解肠道中的蛋氨酸以减少或消除对蛋氨酸限制饮食的依赖 结果降低血浆和组织同型半胱氨酸，从而获得更好的临床结果。条件是 估计发病率为1/250,000，然而，一些报告表明，潜在的发病率为1/250,000 65,000，原因是目前的诊断分析不准确，而且往往是微妙的症状，可能 逃避临床检测，直到它们变得严重或明显，如晶状体脱离中心 眼睛。同型半胱氨酸尿症中突变的基因--胱硫醚-β-合成酶(CBS)定位于一个关键点 真核生物蛋氨酸循环中的调控分支点。CBS催化5‘-磷酸吡哆醛依赖 β-取代反应将丝氨酸和同型半胱氨酸(Hcy)缩合成胱硫氨酸，随后 在胱硫醚-γ-裂解酶(cgl，EC 4.4.1.1)催化的反应中转化为半胱氨酸。CBS的失活方式 突变导致典型的同型半胱氨酸尿症(HCU)，在人类受试者中，其特征是 结缔组织紊乱，包括马凡氏样习性和晶状体脱位，智能障碍和 血管疾病的发生率急剧增加，特别是中风等血栓栓塞性并发症。 吡哆醇无反应HCU的治疗策略通常试图降低血浆和组织中 通过限制同型半胱氨酸前体蛋氨酸的饮食摄入量和饮食补充剂的组合来实现 与三甲基甘氨酸，更通常被称为甜菜碱。PETRI Bio，Inc.开发了一种新的战略，用于 酶疗法，使用与人类肠道微生物组相容的原核菌株作为 用于治疗性蛋白给药的表达载体。细菌衍生菌的电子筛选后 对于甲硫酶的一些理想特性的治疗酶，已克隆，表达， 并在体外显示能降低蛋氨酸浓度。在此第一阶段计划中，我们将延长这些 通过在硅胶中筛选数百种细菌来源的蛋氨酸酶并随后克隆、表达、 并在体外测试蛋氨酸的催化能力。将进行体外试验以测定蛋氨酸酶 这些细菌菌株的活性。在对甲硫氨酸酶活性较高的菌株进行优化后，我们将对它们的 降低体内蛋氨酸浓度的能力，以及改善蛋氨酸积聚的效果 在HCU的小鼠模型中。未来的研究将优化细菌蛋氨酸酶转基因，以确保最大限度地 活性和生物兼容性，以及为临床前药物开发选择领先的候选细菌菌株。

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.