The immunogenic properties of plasmid DNA and recombinant adenovirus (Ad) encoding the carcinoembryonic antigen (CEA) were examined in mice by measuring both the amplitude and type of immune response, and the immunogenicity of codon usage optimized cDNA encoding CEA (CEAopt) was assessed both in C57Bl/6 and CEA transgenic mice. Vectors were injected into quadriceps muscle either alone or in combination, and plasmid DNA was electroporated to enhance gene expression efficiency and immunogenicity. Injection of plasmid pVIJ/CEA followed by Ad-CEA boost elicited the highest amplitude of both CD4 1 and CD8 1 T-cell response to the target antigen, measured by both IFNc-ELIspot assay and intracellular staining. Vectors carrying cDNA of CEAopt expressed a greater amount of the CEA protein than their wild-type counterparts, and this enhanced expression was associated with greater immunogenicity. Both CD4 1 and CD8 1 T-cell epitopes were mapped in the C-terminal portion of the protein. In CEA transgenic mice, only immunization based on repeated injections of pVIJ/ CEAopt followed by Ad-CEAopt was able to elicit a CEA-specific CD8 1 T-cell response, whereas the wild-type vectors did not break tolerance to this target antigen. MC38-CEA tumor cells injected s.c. in CEA transgenic mice vaccinated with CEAopt vectors exhibited delayed growth kinetics. These studies demonstrate that this type of genetic vaccine is highly immunogenic and can break tolerance to CEA tumor antigen in CEA transgenic mice. ' 2005 Wiley-Liss, Inc.Key words: CEA; DNA electroporation; adenovirus Despite improvements in prevention, early detection and treatment, the possibility of curing many cancer patients remains elusive. Thus, cancer continues to be a largely unmet medical need for which more efficient therapeutic strategies must be developed. 1 Particular attention has been given to active specific immunotherapy of cancer, whereby patients are immunized against antigens presented by tumor cells. In fact, experimental and clinical evidence have demonstrated the critical role played by the cellular and humoral responses in controlling tumor growth and metastasis. 2 Many of these therapies are specifically targeted to tumorassociated antigens among which carcinoembryonic antigen (CEA) is a frequent example due to its ectopic and deregulated expression in a large percentage of adenocarcinomas. 3 Human CEA is the prototypic member of the human CEA family, a group of highly glycosylated homotypic/heterotypic cell surface intracellular adhesion molecules, and part of the immunoglobulin gene superfamily. CEA is widely used as human tumor marker, is expressed mostly in the gastrointestinal tract and is overexpressed in many human cancers, including epithelial tumors originating from the gastrointestinal tract, lung, thyroid, breast, prostate, cervix and ovaries. 4 CEA has been the focus of extensive preclinical and clinical investigation aimed at developing a CEA-specific vaccine with a therapeutic impact on tumor progression. 5 In this context, genetic vacc...

Electrogene transfer (EGT) of plasmid DNA into skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. We report here that preinjecting hyaluronidase (HYAse) significantly increases the gene transfer efficiency of muscle EGT. Three constructs encoding mouse erythropoietin (pCMV/mEPO), secreted alkaline phosphatase (pCMV/SeAP), and luciferase (pGGluc) were electroinjected intramuscularly in BALB/c mice and rabbits with and without HYAse pretreatment. Preinjection 1 or 4 hr before EGT increased EPO gene expression by about 5-fold in mice and maintained higher gene expression than plasmid EGT alone. A similar increment in gene expression was observed on pretreatment with HYAse and electroinjection of pCMV/mEPO into rabbit tibialis muscle. The increment of gene expression in rabbits reached 17-fold on injection of plasmid pCMV/SeAP and 24-fold with plasmid pGGluc. Injection of a plasmid encoding beta-galactosidase (pCMV/beta gal/NLS) and subsequent staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside indicated that HYAse increased the tissue area involved in gene expression. No irreversible tissue damage was observed on histological analysis of treated muscles. HYAse is used in a variety of clinical applications, and thus the combination of HYAse pretreatment and muscle EGT may constitute an efficient gene transfer method to achieve therapeutic levels of gene expression.

We have investigated the efficacy of a gene transfer strategy based on plasmid DNA electroinjection for the correction of anemia associated with renal failure. An expression plasmid encoding the rat erythropoietin (EPO) cDNA under the control of the CMV promoter as constructed and utilized for this work. Electroinjection of pCMV/rEPO in different rat muscles yielded sustained and long-term EPO production and secretion. The muscle-produced EPO corrected the anemia in five of six nephrectomized rats, used as a model of renal failure. The efficiency of muscle transduction was comparable in rats and mice injected with equivalent amounts of DNA per kilogram of body weight. These results demonstrate that gene electrotransfer can be applied to produce therapeutically significant levels of erythropoietin in chronic renal failure.

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