Oral Delivery of Bioencapsulated Proteins Across Blood–Brain and Blood–Retinal Barriers

Abstract

Delivering neurotherapeutics to target brain-associated diseases is a major challenge. Therefore, we investigated oral delivery of green fluorescence protein (GFP) or myelin basic protein (MBP) fused with the transmucosal carrier cholera toxin B subunit (CTB), expressed in chloroplasts (bioencapsulated within plant cells) to the brain and retinae of triple transgenic Alzheimer's disease (3×TgAD) mice, across the blood-brain barriers (BBB) and blood-retinal barriers (BRB). Human neuroblastoma cells internalized GFP when incubated with CTB-GFP but not with GFP alone. Oral delivery of CTB-MBP in healthy and 3×TgAD mice shows increased MBP levels in different regions of the brain, crossing intact BBB. Thioflavin S-stained amyloid plaque intensity was reduced up to 60% by CTB-MBP incubation with human AD and 3×TgAD mice brain sections ex vivo. Amyloid loads were reduced in vivo by 70% in hippocampus and cortex brain regions of 3×TgAD mice fed with bioencapsulated CTB-MBP, along with reduction in the ratio of insoluble amyloid β 42 (Aβ42) to soluble fractions. CTB-MBP oral delivery reduced Aβ42 accumulation in retinae and prevented loss of retinal ganglion cells in 3×TgAD mice. Lyophilization of leaves increased CTB-MBP concentration by 17-fold and stabilized it during long-term storage in capsules, facilitating low-cost oral delivery of therapeutic proteins across the BBB and BRB.

Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells

“…15,45,46 In addition, elimination of highly expensive purification, cold storage, transportation, and sterile injections significantly reduces their costs. Although we have not optimized the codons for FVIII expression in the current investigation, it has been reported that protein expression can reach up to 70% of the total leaf protein under optimal conditions.…”

Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells

“…8,45 Antigen-CTB-GM1 complexes then traffic retrograde through the Trans-Golgi network into the lumen of the endoplasmic reticulum. 35,[46][47][48] Because of the furin cleavage site, proteolytic cleavage occurs in the Trans-Golgi network, so that FIX antigen can be exocytosed and released into extracellular fluid, whereas CTB is retained intracellularly. 8,35,46 The orally delivered FIX is identical in amino acid sequence to the mature recombinant FIX used for systemic delivery, but lacks glycosylation and g-carboxylation (and therefore does not have coagulation activity and cannot restore hemostasis).…”

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…CNTB fusion proteins expressed in chloroplasts form pentameric structures that are highly resistant to detergents, and this hampers solubilization due to tight interactions between CNTB monomers, mediated by 30 hydrogen bonds, seven salt bridges, and hydrophobic interactions (Miyata et al, 2012). In our previous studies (Boyhan and Daniell, 2011;Kwon et al, 2013;Kohli et al, 2014;Shil et al, 2014), multimeric forms exist even after treatment with DTT, detergents (SDS), and boiling. Also, acid (pH 2) could not completely dissociate CNTB pentamers due to the reformation of multimeric structures.…”

“…A wide range of proteins, from very small antimicrobial peptides (Lee et al, 2011) or hormones (Boyhan and Daniell, 2011;Kwon et al, 2013) to very large proteins encoded by bacterial, viral, fungal, animal, and human genes, have been expressed successfully in plant chloroplasts (DeGray et al, 2001;Daniell et al, 2009;Verma et al, 2010;Shenoy et al, 2014;Sherman et al, 2014;Shil et al, 2014). Most importantly, expressed proteins are highly stable when lyophilized plant cells are stored at ambient temperature (Kwon et al, 2013;Lakshmi et al, 2013;Kohli et al, 2014;Jin and Daniell, 2015). Therefore, oral delivery of proinsulin or exendin-4 reduced blood sugar levels similar to injected proteins (Boyhan and Daniell, 2011;Kwon et al, 2013).…”

“…Furthermore, ocular inflammation caused by decreased activity of the protective axis of the renin-angiotensin system was improved significantly . Likewise, oral delivery of myelin basic protein reduced Ab plaques in advanced mouse and human Alzheimer's brains (Kohli et al, 2014). Delivery of coagulation factors to hemophilic mice induced oral tolerance and suppressed inhibitor formation and anaphylaxis Sherman et al, 2014;Wang et al, 2015a).…”