Affordable oral proinsulin bioencapsulated in plant cells regulates blood sugar levels similar to natural insulin.

Diabetes Mellitus is a silent epidemic affecting >500 million, which claimed 6.7 million lives in 2021, a projected increase of >670% in <20 years old in the next two decades but insulin is unaffordable for the large majority of the globe. Therefore, we engineered proinsulin in plant cells to facilitate oral delivery. Stability of the proinsulin gene and expression in subsequent generations, after removal of the antibiotic-resistance gene, was confirmed using PCR, Southern and western blots. Proinsulin expression was high (up to 12 mg/g DW or 47.5% of total leaf protein), stable up to one year after storage of freeze-dried plant cells at ambient temperature and met FDA regulatory requirements of uniformity, moisture content and bioburden. GM1 receptor binding, required for uptake via gut epithelial cells was confirmed by pentameric assembly of CTB-Proinsulin. IP insulin injections (without C peptide) in STZ mice rapidly decreased blood glucose level leading to transient hypoglycemia, followed by hepatic glucose compensation. On the other hand, other than the 15-min lag period of oral proinsulin (transit time required to reach the gut), the kinetics of blood sugar regulation of oral CTB-Proinsulin in STZ mice was very similar to naturally secreted insulin in healthy mice (both contain C-peptide), without rapid decrease or hypoglycemia. Elimination of expensive fermentation, purification and cold storage/transportation should reduce cost and increase other health benefits of plant fibers. The recent approval of plant cell delivery of therapeutic proteins by FDA and approval of CTB-ACE2 for phase I/II human clinical studies augur well for advancing oral proinsulin to the clinic.

Debulking different Corona (SARS-CoV-2 delta, omicron, OC43) and Influenza (H1N1, H3N2) virus strains by plant viral trap proteins in chewing gums to decrease infection and transmission.

Because oral transmission of SARS-CoV-2 is 3-5 orders of magnitude higher than nasal transmission, we investigated debulking of oral viruses using viral trap proteins (CTB-ACE2, FRIL) expressed in plant cells, delivered through the chewing gum. In omicron nasopharyngeal (NP) samples, the microbubble count (based on N-antigen) was significantly reduced by 20 µg of FRIL (p < 0.0001) and 0.925 µg of CTB-ACE2 (p = 0.0001). Among 20 delta or omicron NP samples, 17 had virus load reduced below the detection level of spike protein in the RAPID assay, after incubation with the CTB-ACE2 gum powder. A dose-dependent 50% plaque reduction with 50-100 ng FRIL or 600-800 µg FRIL gum against Influenza strains H1N1, H3N2, and Coronavirus HCoV-OC43 was observed with both purified FRIL, lablab bean powder or gum. In electron micrographs, large/densely packed clumps of overlapping influenza particles and FRIL protein were observed. Chewing simulator studies revealed that CTB-ACE2 release was time/dose-dependent and release was linear up to 20 min chewing. Phase I/II placebo-controlled, double-blinded clinical trial (IND 154897) is in progress to evaluate viral load in saliva before or after chewing CTB-ACE2/placebo gum. Collectively, this study advances the concept of chewing gum to deliver proteins to debulk oral viruses and decrease infection/transmission.

Decrease in Angiotensin-Converting Enzyme activity but not concentration in plasma/lungs in COVID-19 patients offers clues for diagnosis/treatment.

Although several therapeutics are used to treat coronavirus disease 2019 (COVID-19) patients, there is still no definitive metabolic marker to evaluate disease severity and recovery or a quantitative test to end quarantine. Because severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects human cells via the angiotensin-converting-enzyme 2 (ACE2) receptor and COVID-19 is associated with renin-angiotensin system dysregulation, we evaluated soluble ACE2 (sACE2) activity in the plasma/saliva of 80 hospitalized COVID-19 patients and 27 non-COVID-19 volunteers, and levels of ACE2/Ang (1-7) in plasma or membrane (mACE2) in lung autopsy samples. sACE2 activity was markedly reduced (p < 0.0001) in COVID-19 plasma (n = 59) compared with controls (n = 27). Nadir sACE2 activity in early hospitalization was restored during disease recovery, irrespective of patient age, demographic variations, or comorbidity; in convalescent plasma-administered patients (n = 45), restoration was statistically higher than matched controls (n = 22, p = 0.0021). ACE2 activity was also substantially reduced in the saliva of COVID-19 patients compared with controls (p = 0.0065). There is a strong inverse correlation between sACE2 concentration and sACE2 activity and Ang (1-7) levels in participant plasmas. However, there were no difference in membrane ACE2 levels in lungs of autopsy tissues of COVID-19 (n = 800) versus other conditions (n = 300). These clinical observations suggest sACE2 activity as a potential biomarker and therapeutic target for COVID-19.

Debulking SARS-CoV-2 in saliva using angiotensin converting enzyme 2 in chewing gum to decrease oral virus transmission and infection.

To advance a novel concept of debulking virus in the oral cavity, the primary site of viral replication, virus-trapping proteins CTB-ACE2 were expressed in chloroplasts and clinical-grade plant material was developed to meet FDA requirements. Chewing gum (2 g) containing plant cells expressed CTB-ACE2 up to 17.2 mg ACE2/g dry weight (11.7% leaf protein), have physical characteristics and taste/flavor like conventional gums, and no protein was lost during gum compression. CTB-ACE2 gum efficiently (>95%) inhibited entry of lentivirus spike or VSV-spike pseudovirus into Vero/CHO cells when quantified by luciferase or red fluorescence. Incubation of CTB-ACE2 microparticles reduced SARS-CoV-2 virus count in COVID-19 swab/saliva samples by >95% when evaluated by microbubbles (femtomolar concentration) or qPCR, demonstrating both virus trapping and blocking of cellular entry. COVID-19 saliva samples showed low or undetectable ACE2 activity when compared with healthy individuals (2,582 versus 50,126 ΔRFU; 27 versus 225 enzyme units), confirming greater susceptibility of infected patients for viral entry. CTB-ACE2 activity was completely inhibited by pre-incubation with SARS-CoV-2 receptor-binding domain, offering an explanation for reduced saliva ACE2 activity among COVID-19 patients. Chewing gum with virus-trapping proteins offers a general affordable strategy to protect patients from most oral virus re-infections through debulking or minimizing transmission to others.

Colloidal Gels with Tunable Mechanomorphology Regulate Endothelial Morphogenesis.

Endothelial morphogenesis into capillary networks is dependent on the matrix morphology and mechanical properties. In current 3D gels, these two matrix features are interdependent and their distinct roles in endothelial organization are not known. Thus, it is important to decouple these parameters in the matrix design. Colloidal gels can be engineered to regulate the microstructural morphology and mechanics in an independent manner because colloidal gels are formed by the aggregation of particles into a self-similar 3D network. In this work, gelatin based colloidal gels with distinct mechanomorphology were developed by engineering the electrostatic interaction mediated aggregation of particles. By altering the mode of aggregation, colloidal gels showed either compact dense microstructure or tenuous strand-like networks, and the matrix stiffness was controlled independently by varying the particle fraction. Endothelial Cell (EC) networks were favored in tenuous strand-like microstructure through increased cell-matrix and cell-cell interactions, while compact dense microstructure inhibited the networks. For a given microstructure, as the gel stiffness was increased, the extent of EC network was reduced. This result demonstrates that 3D matrix morphology and mechanics provide distinct signals in a bidirectional manner during EC network formation. Colloidal gels can be used to interrogate the angiogenic responses of ECs and can be developed as a biomaterial for vascularization.