Preclinical development of plant‐based oral immune modulatory therapy for haemophilia B

Abstract

Anti-drug antibody (ADA) formation is a major complication in treatment of the X-linked bleeding disorder haemophilia B (deficiency in coagulation factor IX, FIX). Current clinical immune tolerance protocols are often not effective due to complications such as anaphylactic reactions against FIX. Plant-based oral tolerance induction may address this problem, as illustrated by the recent first regulatory approval of orally delivered plant cells to treat peanut allergy. Our previous studies showed that oral delivery of plant cells expressing FIX fused to the transmucosal carrier CTB (cholera toxin subunit B) in chloroplasts suppressed ADA in animals with haemophilia B. We report here creation of the first lettuce transplastomic lines expressing a coagulation factor, in the absence of antibiotic resistance gene. Stable integration of the CTB-FIX gene and homoplasmy (transformation of~10 000 copies in each cell) were maintained in both T1 and T2 generation marker-free plants. CTB-FIX expression in lyophilized leaves of T1 and T2 marker-free plants was 1.0-1.5 mg/g dry weight, confirming that the marker excision did not affect antigen levels. Oral administration of CTB-FIX to Sprague Dawley rats at 0.25, 1 or 2.5 mg/ kg did not produce overt adverse effects or toxicity. The no-observed-adverse-effect level (NOAEL) is at least 2.5 mg/kg for a single oral administration in rats. Oral administration of CTB-FIX at 0.3 or 1.47 mg/kg either mixed in food or as an oral suspension to Beagle dogs did not produce any observable toxicity. These toxicology studies should facilitate filing of regulatory approval documents and evaluation in haemophilia B patients.

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

“…22 In pulmonary hypertension (PH) disease with disease symptoms similar to those of COVID-19, oral ACE2 attenuates PH with a decrease in right ventricular (RV) hypertrophy, RV systolic pressure, total pulmonary resistance, and pulmonary artery remodeling. 43,57 In contrast to injected truncated (transmembrane deleted) sACE2, 22 full-length oral CTB-ACE2 accumulates in the lungs at 10-fold higher concentrations than in the plasma upon oral delivery of bioencapsulated plant cells, 43,44 offering yet another approach to treat COVID-19 patients.…”

“…Debulking and blocking of viral entry using ACE2 chewing gum CTB has been shown to be a transmucosal carrier and facilitates oral delivery of therapeutic proteins by forming a pentameric structure and binding to gut GM1 epithelial receptors. [43][44][45][46] CTB-ACE2 has the potential to effectively bind to both the GM1 and ACE2 receptor-binding sites located in close proximity on the human cell surface and thereby prevent viral entry into human cells, especially via oral epithelial cells that are enriched with both receptors. 47 In addition, direct binding of ACE2 to the SARS-CoV-2 spike proteins could trap the virus particles and decrease infectivity (Figure 2).…”

“…47 In addition, direct binding of ACE2 to the SARS-CoV-2 spike proteins could trap the virus particles and decrease infectivity (Figure 2). Pentameric insoluble microparticles 43,44 of CTB-ACE2 could facilitate removal of bound viral particles by centrifugation. Therefore, CTB-ACE2 chewing gum is evaluated in this study for its impact on entry and transmission of SARS-CoV-2 (Figure 2).…”

“…In the oral cavity, tongue epithelial cells constitute a large reservoir of ACE2, even more so than buccal and gingival tissues. [43][44][45][46] The ACE2 released upon mastication of the chewing gum serves as a novel approach to diminish virus infection.…”

Contributions of the international plant science community to the fight against infectious diseases in humans—part 2: Affordable drugs in edible plants for endemic and re‐emerging diseases

“…In the case of orally administered products, progress has also been slowed by the challenge of achieving precise antigen doses (Kashima et al, 2016), and the perceived risk that pharmaceutical products could end up in the food chain (Spok et al, 2008). However, the clinical advancement of antigens produced in edible leaves will be encouraged by recent progress in the high-level expression of antigens, the accurate control of antigen doses, the removal of antibiotic resistance genes, the large-scale production of leaves in FDA-approved hydroponic growth facilities, the tightly controlled removal of moisture, batch-to-batch records for regulatory compliance and strict bioburden validation of microbes on the leaf surface accomplished recently (Daniell et al, 2019a(Daniell et al, , 2020(Daniell et al, , 2021Srinivasan et al, 2021;Su et al, 2015;).…”