Chloroplast transformation for bioencapsulation and oral delivery using the immunoglobulin G fragment crystallizable (Fc) domain

Proinsulin Like Growth Factor (prolGF1) and myostatin (Mstn) regulate muscle regeneration when intravenously delivered. We set out to test if chloroplast bioencapsulated forms of these proteins may serve as a non-invasive means of drug delivery through the digestive system. We created tobacco (Nicotiana tabacum) plants carrying GFP-Fc1, proIGF-I-Fc1, and Mstn-Fc1 fusion genes, in which fusion with the immunoglobulin G Fc domain improved both protein stability and absorption in the small intestine. No transplastomic plants were obtained with the Mstn-Fc1 gene, suggesting that the protein is toxic to plant cells. proIGF-I-Fc1 protein levels were too law to enable in vivo testing. However, GFP-Fc1 accumulated at a high level, enabling evaluation of chloroplast-made Fc fusion proteins for oral delivery. Tobacco leaves were lyophilized for testing in a mouse system. We report that the orally administered GFP-Fc fusion protein (5.45 μg/g GFP-Fc) has been taken up by the intestinal epithelium cells, evidenced by confocal microscopy. GFP-Fc subsequently entered the circulation where it was detected by ELISA. Data reported here confirm that chloroplast expression and oral administration of lyophilized leaves is a potential delivery system of therapeutic proteins fused with Fc, with the advantage that the proteins may be stored at room temperature.