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.
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 beta-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 beta-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 beta-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.
Ruhlman
Tracey
T
University of Central Florida, Department of Molecular Biology and Microbiology, Biomolecular Science, Orlando, FL 32816-2364, USA.
Ahangari
Raheleh
R
Devine
Andrew
A
Samsam
Mohtahsem
M
Daniell
Henry
H
D000284:Administration, Oral; D000818:Animals; D002736:Chloroplasts / Q000737:chemistry*; D002772:Cholera Toxin / Q000008:administration & dosage*; D003921:Diabetes Mellitus, Experimental / Q000150:complications*; D007249:Inflammation / Q000150:complications / Q000517:prevention & control*; D007328:Insulin / Q000008:administration & dosage*; D018545:Lactuca / Q000737:chemistry*; D051379:Mice; D010936:Plant Extracts / Q000008:administration & dosage*; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage*; D014026:Nicotiana / Q000737:chemistry*
D007328:Insulin; D010936:Plant Extracts; D011993:Recombinant Fusion Proteins; C400103:cholera toxin B-insulin conjugate; D002772:Cholera Toxin