Alan Escher

The goal of immunoregulatory DNA vaccination is the antigen- and tissue-specific suppression of pathological inflammation that underlies immune-mediated inflammatory disorders like autoimmune diseases and allograft rejection. Recent patents and patent applications have applied immunoregulatory DNA vaccines in rodent model systems and human clinical trials using plasmid DNA coding for autoantigens such as insulin and glutamic acid decarboxylase for type 1 diabetes, myelin-associated proteins for multiple sclerosis, and heat-sock protein 60 for rheumatoid arthritis. In these cases, the objective is to induce a homeostatic-like regulatory immune response to suppress pathological inflammation. In addition, patent applications have disclosed the use of DNA vaccines encoding the pro-inflammatory MIF cytokine and the CD25 IL-2 receptor subunit to interfere with the inflammatory process. Approaches have also been taken to improve DNA vaccination efficacy, including covalent modification of plasmid DNA, engineering secretion of vaccine-encoded antigen, and co-delivery of DNA coding for anti-inflammatory cytokines, a mutant co-stimulatory molecule, a growth factor, or a pro-apoptotic protein. Furthermore, a patent application has disclosed the use of a DNA vaccine previously shown to treat successfully an autoimmune disease to prolong allograft survival. Taken together, these patents and patent applications indicate a promising bench-to-bedside potential for immunoregulatory DNA vaccination applied to autoimmune diseases and allograft rejection.

We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild‐type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light‐emission properties similar to that of unfused Renilla luciferase and GFP. In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells. Simian COS‐7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase–GFP fusion protein. Renilla luciferase activity was measured from COS‐7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy. Data indicated that the SRUC–GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity. This fusion protein could be a useful marker for the study of protein secretion in mammalian cells. Copyright © 2000 John Wiley & Sons, Ltd.

Type 1 diabetes results from the destruction of insulin producing beta cells by the immune system. Several immunization methods based on autoantigenic proteins or peptides have been used to prevent autoimmune diabetes in the non-obese diabetic (NOD) mouse. In the work presented here, a gene-based approach was taken for a similar purpose. Intramuscular injection ofnaked plasmid DNA was used to determine the efficacy of genes encoding different versions of a beta cell associated autoantigen for prevention of cyclophosphamide accelerated diabetes in female NOD mice. Four-week old animals received injections of DNA encoding either intracellular or secreted human glutamic acid decarboxylase (GAD), an enzyme found in beta cells and whose recognition by the immune system is thought to be necessary for disease onset.