Abstract
A large number of studies aimed at the treatment of cancer, autoimmune and metabolic diseases, neurodegenerative disorders, allergic diseases, as well as muscle disorders strengthen the fact that gene therapy could represent an alternative method to treat human diseases where conventional approaches are less effective.
To improve transgene expression from plasmid vectors, DNA nuclear targeting sequences (DTSs) can be introduced in a vector backbone to increase in vivo expression up to 20-fold using electroporation (EP) delivery in muscle tissue. The purpose of this chapter is to represent a step-by-step strategy for the construction of a plasmid vector with enhanced efficiency of nuclear plasmid uptake and the methodic for the in vivo efficiency evaluation of the obtained expression vector.
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References
Iurescia S, Fioretti D, Fazio VM et al (2011) Design and pre-clinical development of epitope-based DNA vaccines against B-cell lymphoma. Curr Gene Ther 11:414–422
Iurescia S, Fioretti D, Pierimarchi P et al (2010) Genetic immunization with CDR3-based fusion vaccine confers protection and long-term tumor-free survival in a mouse model of lymphoma. J Biomed Biotechnol 2010:316069
Rinaldi M, Fioretti D, Iurescia S et al (2008) Anti-tumor immunity induced by CDR3-based DNA vaccination in a murine B-cell lymphoma model. Biochem Biophys Res Commun 370:279–284
Fazio VM, Ria F, Franco E et al (2004) Immune response at birth, long-term immune memory and 2 years follow-up after in-utero anti-HBV DNA immunization. Gene Ther 11:544–551
Rinaldi M, Catapano AL, Parrella P et al (2000) Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by intramuscular injection of plasmid DNA. Gene Ther 7:1795–1801
Rinaldi M, Ria F, Parrella P et al (2001) Antibodies elicited by naked DNA vaccination against the complementary-determining region 3 hypervariable region of immunoglobulin heavy chain idiotypic determinants of B-lymphoproliferative disorders specifically react with patients’ tumor cells. Cancer Res 61:1555–1562
Rinaldi M, Signori E, Rosati P et al (2006) Feasibility of in utero DNA vaccination following naked gene transfer into pig fetal muscle: transgene expression, immunity and safety. Vaccine 24:4586–4591
Fioretti D, Iurescia S, Fazio VM et al (2010) DNA vaccines: developing new strategies against cancer. J Biomed Biotechnol 2010:174378
Fioretti D, Iurescia S, Fazio VM et al (2013) In vivo DNA electrotransfer for immunotherapy of cancer and neurodegenerative diseases. Curr Drug Metab 14:279–290
Luke JM, Vincent JM, Du SX et al (2011) Improved antibiotic-free plasmid vector design by incorporation of transient expression enhancers. Gene Ther 18:334–343
Lam AP, Dean DA (2010) Progress and prospects: nuclear import of nonviral vectors. Gene Ther 17:439–447
Wagstaff KM, Jans DA (2007) Nucleo-cytoplasmic transport of DNA: enhancing non-viral gene transfer. Biochem J 406:185–202
Miller AM, Munkonge FM, Alton EW et al (2009) Identification of protein cofactors necessary for sequence-specific plasmid DNA nuclear import. Mol Ther 17:1897–1903
Li S, MacLaughlin FC, Fewell JG et al (2001) Muscle-specific enhancement of gene expression by incorporation of SV40 enhancer in the expression plasmid. Gene Ther 8:494–497
Dean DA, Dean BS, Muller S et al (1999) Sequence requirements for plasmid nuclear import. Exp Cell Res 253:713–722
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Fioretti, D., Iurescia, S., Rinaldi, M. (2014). Enhancement of Plasmid-Mediated Transgene Expression. In: Rinaldi, M., Fioretti, D., Iurescia, S. (eds) DNA Vaccines. Methods in Molecular Biology, vol 1143. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0410-5_2
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DOI: https://doi.org/10.1007/978-1-4939-0410-5_2
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Publisher Name: Humana Press, New York, NY
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