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 ...