Affordable Oral Delivery of Human Therapeutic Proteins Bioencapsulated in Plant Cells

项目摘要/摘要 目前的蛋白质药物(PD)贵得令人望而却步(排名前十的蛋白质药物价值1400亿美元，占全球GDP的75%)。 全球三分之一的人口每天收入2美元，负担不起任何PD。胰岛素，一种有50年历史的药物，仍然 负担不起，即使在发达国家也是如此，美国最近几例糖尿病患者死亡就证明了这一点。 如此高的成本是由于在令人望而却步的昂贵的发酵罐中生产钯、净化、冷藏 运输/储存、保质期短和无菌运送方式。患者的依从性因重复而减少 注射或手术植入的PDS。因此，易于交付、负担得起的PDS是一个迫切的未得到满足的需求。 Daniell实验室开创了一种表达PD的新概念，从而解决了这些挑战 植物叶绿体(每个细胞高达10,000个基因拷贝)，导致PD的高水平表达。在口头上 通过生物包裹，植物细胞壁保护PD免受胃中的酸/酶的伤害。然而， 共生微生物消化植物细胞壁，并在肠道中释放PDs。当标签与蛋白质药物融合时， 它们有效地穿过肠道上皮，并被输送到循环系统或免疫系统。PDS是 在常温下储存的冻干植物细胞可稳定多年，保持其折叠/ 功能，从而完全消除了储存/运输所需的冷链。至之前的R01 资助(2011-2020年)，具有挑战性的口服给药机制问题通过以下方式进行了评估 在小鼠、大鼠、狗的动物疾病模型中口服几千剂量的PDS。 在第二次更新应用中，我们建议优化人类血液蛋白的表达 (ACE2/Ang1-7/IL10)和食物过敏原(Ara h1，Ara h 2，Ara h 3，Ara h 6)通过 密码子优化、抗生素耐药基因去除、GLP生产、植物用药剂量确定 增强PD口服给药效果。在狗和人身上，肾素-血管紧张素-1的激活- 醛固酮系统(RAAS)是心力衰竭(HF)的一个重要的神经激素方面，也是治疗的靶点。 口服血管紧张素转换酶-2(ACE2)和血管紧张素-1-7(Ang-1-7)对血管紧张素转换酶-2和血管紧张素-7的影响 RAAS上的植物细胞将在家犬自发性心力衰竭模型中进行研究，并与ACE- 血管紧张素受体拮抗剂(ARB)或血管紧张素转换酶抑制剂(ACEI)对血浆和外体RAAS血管紧张素的评价 自发性心衰犬的多肽、超声心动图和临床测量。此外，我们还将 扩展我们在之前的R01中开发的独特的免疫耐受平台，以抑制抗体形成/ 蛋白质替代疗法中的过敏反应，用于治疗危及生命的食物过敏。因此，我们将发展 花生抗原与叶绿体表达的CTB融合的口服免疫调节治疗 花生过敏小鼠模型的致敏或皮肤致敏。这项提议的首要目标是 解决负担得起的PDS的紧急未得到满足的医疗需求，利用之前两款R01推出的工作 变革性的解决方案，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.