Title

Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells

Authors

Authors

K. C. Kwon; R. Nityanandam; J. S. New;H. Daniell

Comments

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Abbreviated Journal Title

Plant Biotechnol. J.

Keywords

type 2 diabetes; bioencapsulation; lyophilization; therapeutic peptides; molecular farming; plant-made biopharmaceuticals; TOXIN B-SUBUNIT; CHOLERA-TOXIN; TOBACCO CHLOROPLASTS; TRANSGENIC; CHLOROPLASTS; DIABETES-MELLITUS; SMALL-INTESTINE; GLP-1 RECEPTOR; PROTEIN; PEPTIDE; PROINSULIN; Biotechnology & Applied Microbiology; Plant Sciences

Abstract

Glucagon-like peptide (GLP-1) increases insulin secretion but is rapidly degraded (half-life: 2min in circulation). GLP-1 analogue, exenatide (Byetta) has a longer half-life (3.34h) with potent insulinotropic effects but requires cold storage, daily abdominal injections with short shelf life. Because patients with diabetes take >60000 injections in their life time, alternative delivery methods are highly desired. Exenatide is ideal for oral delivery because insulinotropism is glucose dependent, with reduced risk of hypoglycaemia even at higher doses. Therefore, exendin-4 (EX4) was expressed as a cholera toxin B subunit (CTB)fusion protein in tobacco chloroplasts to facilitate bioencapsulation within plant cells and transmucosal delivery in the gut via GM1 receptors present in the intestinal epithelium. The transgene integration was confirmed by PCR and Southern blot analysis. Expression level of CTB-EX4 reached up to 14.3% of total leaf protein (TLP). Lyophilization of leaf material increased therapeutic protein concentration by 12- to 24-fold, extended their shelf life up to 15months when stored at room temperature and eliminated microbes present in fresh leaves. The pentameric structure, disulphide bonds and functionality of CTB-EX4 were well preserved in lyophilized materials. Chloroplast-derived CTB-EX4 showed increased insulin secretion similar to the commercial EX4 in beta-TC6, a mouse pancreatic cell line. Even when 5000-fold excess dose of CTB-EX4 was orally delivered, it stimulated insulin secretion similar to the intraperitoneal injection of commercial EX4 but did not cause hypoglycaemia in mice. Oral delivery of the bioencapsulated EX4 should eliminate injections, increase patient compliance/convenience and significantly lower their cost.

Journal Title

Plant Biotechnology Journal

Volume

11

Issue/Number

1

Publication Date

1-1-2013

Document Type

Article

Language

English

First Page

77

Last Page

86

WOS Identifier

WOS:000312887700008

ISSN

1467-7644

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