Expression of cholera toxin B?￠????proinsulin fusion protein in lettuce and tobacco chloroplasts ?￠???? oral administration protects against development of insulitis in non?￠????obese diabetic mice

摘要

Lettuce and tobacco chloroplast transgenic lines expressing the cholera toxin B subunit?￠????human proinsulin (CTB?￠????Pins) fusion protein were generated. CTB?￠????Pins accumulated up to ~16% of total soluble protein (TSP) in tobacco and up to ~2.5% of TSP in lettuce. Eight milligrams of powdered tobacco leaf material expressing CTB?￠????Pins or, as negative controls, CTB?￠????green fluorescent protein (CTB?￠????GFP) or interferon?￠????GFP (IFN?￠????GFP), or untransformed leaf, were administered orally, each week for 7????weeks, to 5?￠????week?￠????old female non?￠????obese diabetic (NOD) mice. The pancreas of CTB?￠????Pins?￠????treated mice showed decreased infiltration of cells characteristic of lymphocytes (insulitis); insulin?￠????producing ???2?￠????cells in the pancreatic islets of CTB?￠????Pins?￠????treated mice were significantly preserved, with lower blood or urine glucose levels, by contrast with the few ???2?￠????cells remaining in the pancreatic islets of the negative controls. Increased expression of immunosuppressive cytokines, such as interleukin?￠????4 and interleukin?￠????10 (IL?￠????4 and IL?￠????10), was observed in the pancreas of CTB?￠????Pins?￠????treated NOD mice. Serum levels of immunoglobulin G1 (IgG1), but not IgG2a, were elevated in CTB?￠????Pins?￠????treated mice. Taken together, T?￠????helper 2 (Th2) lymphocyte?￠????mediated oral tolerance is a likely mechanism for the prevention of pancreatic insulitis and the preservation of insulin?￠????producing ???2?￠????cells. This is the first report of expression of a therapeutic protein in transgenic chloroplasts of an edible crop. Transplastomic lettuce plants expressing CTB?￠????Pins grew normally and transgenes were maternally inherited in T 1 progeny. This opens up the possibility for the low?￠????cost production and delivery of human therapeutic proteins, and a strategy for the treatment of various other autoimmune diseases.