Affordable Oral Delivery of Human Therapeutic Proteins Bioencapsulated in Plant Cells

项目总结/摘要 目前的蛋白质药物（PD）非常昂贵（前十名PD的价格为1400亿美元，占全球GDP的75%以上）。 全球三分之一日收入低于2美元的人口负担不起任何PD。胰岛素，一种已有50年历史的药物， 即使在发达国家，也负担不起，正如美国最近几例糖尿病患者的死亡所表明的那样。 如此高的成本是由于在昂贵的发酵罐中进行PD生产、纯化、冷处理、冷冻干燥和干燥。 运输/储存、短保质期和无菌递送方法。患者依从性较低， 注射或手术植入的PD。因此，负担得起的易于交付的PD是一个迫切的未满足的需求。 丹尼尔实验室已经解决了这些挑战，开创了一个新的概念，表达PD， 植物叶绿体（每个细胞多达10，000个基因拷贝），导致高水平的PD表达。物在口服 在递送过程中，植物细胞壁通过生物包封保护PD免受胃中的酸/酶的影响。然而，在这方面， 肠道微生物消化植物细胞壁并在肠道中释放PD。当标签与蛋白质药物融合时， 它们有效地穿过肠上皮并被递送到循环系统或免疫系统。PD是 当在环境温度下储存时，在冻干的植物细胞中稳定多年，保持其折叠， 因此，它完全消除了存储/运输所需的冷链。通过之前的R 01 资金（2011-2020年），口服药物递送的挑战性机制问题已经通过评估 在小鼠、大鼠、狗动物疾病模型中口服数千剂PD。 在第二次更新申请中，我们提出优化人血蛋白的表达 (Ace2/Ang 1 -7/IL 10）和食物过敏原（Ara h 1、Ara h 2、Ara h 3、Ara h 6）的关系 密码子优化、抗生素抗性基因的去除、GLP产生、体内药物剂量确定 和增强PD口服递送。在狗和人中，激活肾素-血管紧张素- 醛固酮系统（RAAS）是心力衰竭（HF）的关键神经激素方面和治疗靶点。 口服生物微囊化血管紧张素转换酶-2（ACE 2）和血管紧张素1-7（Ang 1-7）对血管紧张素转化酶-2（ACE 2）和血管紧张素1-7（Ang 1-7）的影响。 将在家犬自发性HF模型中研究植物细胞对RAAS的作用，并与ACE- 血管紧张素转换酶抑制剂（ACEI）或血管紧张素受体阻滞剂（ARB），评估血浆和外泌体RAAS血管紧张素 肽（AP）和超声心动图和临床措施的狗自发性HF。此外，我们将 扩展我们在先前R 01中开发的独特免疫耐受平台，以抑制抗体形成/ 蛋白质替代疗法中的过敏反应，以治疗危及生命的食物过敏。因此，我们将开发 使用与在叶绿体中表达的CTB融合的花生抗原的口服免疫调节疗法 致敏或皮肤致敏小鼠模型。本提案的总体目标是 解决负担得起的PD的迫切未满足的医疗需求，利用前两个R 01的工作来启动 并开发针对心脏病或食物过敏的特定治疗方法。

PROJECT SUMMARY/ABSTRACT Current Protein Drugs (PD) are prohibitively expensive ($140 billion in top ten PDs, >75% of global GDP). One third of global population earning <$2 per day can’t afford any PD. Insulin, a five-decade old drug, is still unaffordable, even in developed countries, as illustrated by several recent deaths of diabetes patients in the US. Such high costs are due to PD production in prohibitively expensive fermenters, purification, cold transportation/storage, short shelf-life and sterile delivery methods. Patient compliance is less for repetitively injected or surgically implanted PDs. Therefore, affordable PDs with ease of delivery is an urgent unmet need. The Daniell laboratory has addressed these challenges by pioneering a novel concept to express PDs in plant chloroplasts (up to 10,000 gene copies per cell), leading to high levels of PD expression. Upon oral delivery, plant cell wall protects PDs from acids/enzymes in the stomach via bio-encapsulation. However, commensal microbes digest plant cell walls and release PDs in the gut. When tags are fused to protein drugs, they efficiently cross the intestinal epithelium and are delivered to the circulatory or immune system. PDs are stable for many years in lyophilized plant cells when stored at ambient temperature, maintaining their folding/ functionality, thereby totally eliminating the cold chain needed for storage/transportation. Through prior R01 funding (2011-2020), challenging mechanistic questions on oral drug delivery have been evaluated through several thousand oral doses of PDs in mice, rat, dog animal disease models. In this second renewal application, we propose to optimize expression of human blood proteins (Ace2/Ang1-7/IL10) and food allergens (Ara h 1, Ara h 2, Ara h 3, Ara h 6) in lettuce chloroplasts through codon optimization, removal of antibiotic resistance genes, GLP production, in planta drug dose determination and enhancement of PD oral delivery. In both dogs and humans, activation of the renin-angiotensin- aldosterone system (RAAS) represents a key neurohormonal aspect of heart failure (HF) and target of therapy. The effect of oral angiotensin converting enzyme-2 (ACE2) and angiotensin 1-7 (Ang 1-7) bioencapsulated in plant cells, on RAAS will be studied in a spontaneous model of HF in domestic dogs, in conjunction with ACE- inhibitor (ACEI) or angiotensin receptor blocker (ARB), evaluation of plasma and exosomal RAAS angiotensin peptides (APs) and echocardiographic and clinical measures of dogs with spontaneous HF. Further, we will extend our unique immune tolerance platform, developed in prior R01s to suppress antibody formation/ anaphylaxis in protein replacement therapy, to treat life-threatening food allergies. Therefore, we will develop oral immune modulatory therapy using peanut antigens fused to CTB expressed in chloroplasts using oral sensitization or skin sensitization mouse models of peanut allergy. The overarching goal of this proposal is to address the urgent unmet medical need of affordable PDs, harnessing work from prior two R01s to launch transformative solutions and develop specific treatments for heart disease or food allergies.