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in 1990 , the direct gene transfer of plasmid dna into mouse muscle in vivo without the need for a special delivery system was demonstrated . furthermore , intramuscular inoculation with plasmid dna encoding reporter genes induced protein expression within the muscle cells .
this study provided evidence for the idea that naked dna could be delivered in vivo to direct protein expression .
subsequently , a further study reported the gene expression a year or more after intramuscular injection of plasmid dna . since these initial studies
, many more experiments have been carried out to evaluate different factors that determine the efficiency of gene transfer and immunogenicity of plasmid dna .
furthermore , plasmid dna has been used to immunise against a variety of diseases ( known as dna vaccination ) .
alternatively , plasmid dna has been used to treat genetic diseases and similar factors may affect the efficacy of this gene therapy .
dna vaccines usually consist of plasmid vectors ( derived from bacteria ) that contain heterologous genes ( transgenes ) inserted under the control of a eukaryotic promoter , allowing protein expression in mammalian cells .
an important consideration when optimising the efficacy of dna vaccines is the appropriate choice of plasmid vector .
the basic requirements for the backbone of a plasmid dna vector are a eukaryotic promoter , a cloning site , a polyadenylation sequence , a selectable marker and a bacterial origin of replication .
a strong promoter may be required for optimal expression in mammalian cells . for this , some promoters derived from viruses such as cytomegalovirus ( cmv ) or simian virus 40 ( sv40 ) have been used .
a cloning site downstream of the promoter should be provided for insertion of heterologous genes , and inclusion of a polyadenylation ( polya ) sequence such as the bovine growth hormone ( bgh ) or sv40 polyadenylation sequence provides stabilisation of mrna transcripts .
the most commonly used selectable markers are bacterial antibiotic resistance genes , such as the ampicillin resistance gene . however , since the ampicillin resistance gene is precluded for use in humans , a kanamycin resistance gene is often used .
finally , the escherichia coli cole1 origin of replication , which is found in plasmids such as those in the puc series , is most often used in dna vaccines because it provides high plasmid copy numbers in bacteria enabling high yields of plasmid dna on purification .
various reports have described the strength of promoter / enhancers or other transcriptional elements in dna vaccines ( see table 1 ) [ 5 - 20 ] . in general ,
virally - derived promoters have provided greater gene expression in vivo than other eukaryotic promoters . in particular , the cmv immediate early enhancer - promoter ( known as the cmv promoter ) has often been shown to direct the highest level of transgene expression in eukaryotic tissues when compared with other promoters .
for example , in one study a plasmid expressing human immunodeficiency virus type 1 ( hiv-1 ) gag / env under the regulation of the cmv promoter / enhancer was compared to a comparable plasmid utilising the endogenous akv murine leukemia long terminal repeat .
analysis of the immune responses in macaques injected with the plasmids showed that the cmv - containing plasmid elicited higher gag- and env - specific humoral and t - cell proliferative responses , reflecting the greater transcriptional activity of the cmv promoter .
furthermore , it has been demonstrated that inclusion of the cmv intron a improved the level of expression of transgenes expressed by the cmv promoter or other promoter / enhancers .
it is thought that the beneficial effect of introns on expression is primarily due to an enhanced rate of polyadenylation and/or nuclear transport associated with rna splicing .
however , some widely used virally - derived promoters , such as the cmv promoter , may not be suitable for some gene therapy applications since treatment with interferon- or tumour necrosis factor- may inhibit transgene expression from dna vaccines containing these promoters .
for example , the desmin promoter / enhancer , which controls expression of the muscle - specific cytoskeletal protein desmin , was used effectively to drive expression of the hepatitis b surface antigen priming both humoral and cellular immunity against the antigen .
these responses were shown to be of a comparable magnitude to those in mice immunised with comparable dna vaccines containing the cmv promoter .
other tissue - specific promoters that have been studied include the creatine kinase promoter , also specific to muscle cells , and the metallothionein and 1,24-vitamind(3)(oh)(2 ) dehydroxylase promoters , both of which are specific to keratinocytes .
comparison of promoters used in dna expression studies in vitro and in vivo since the rate of transcriptional initiation is generally increased by the use of strong promoter / enhancers , the rate of transcriptional termination may become rate - limiting .
in addition , the efficiency of primary rna transcript processing and polyadenylation is known to vary between the polyadenylation sequences of different genes .
thus , the polyadenylation sequence used within a dna vaccine may also have significant effects on transgene expression .
for example , it was demonstrated that the commonly used sv40 polyadenylation sequence was less efficient than the minimal rabbit -globin and bovine growth hormone polyadenylation sequences in mouse liver , although addition of a second sv40 enhancer downstream of the sv40 polyadenylation signal did increase expression to a level comparable to the other signals .
therefore , it is possible that the strategy of inserting a second sv40 enhancer downstream of a sv40 polyadenylation sequence may be utilised in the construction of more efficient vectors .
sequences flanking the aug initiator codon within mrna influence its recognition by eukaryotic ribosomes . as a result of studying the conditions required for optimal translational efficiency of expressed mammalian genes ,
it has been proposed that this defined translational inititiating sequence ( gcca / gccaugg ) should be included in vertebrate mrnas located around the initiator codon .
it has also been suggested that efficient translation is obtained when the -3 position contains a purine base or , in the absence of a purine base , a guanine is positioned at + 4 .
therefore , the expression level of these genes might be increased by the insertion of a kozak sequence .
codon bias is observed in all species , and the use of selective codons in genes often correlates with gene expression efficiency . in general , taxonomically - close organisms , such as e. coli and salmonella enterica serovar typhimurium , for example , use similar codons for their protein synthesis whereas taxonomically - distant organisms , such as e. coli and saccharomyces cerevisiae , utilise very different codons .
nagata et al .   studied the effect of codon optimisation for mammalian cells of cytotoxic t - lymphocyte ( ctl ) epitopes derived from the intracellular bacterium , listeria monocytogenes , and the parasite plasmodium yoelii , and reported that the codon optimisation level of the genes correlated well with translational efficiency in mammalian cells .
the greatest deviation from random codon usage in an organism occurs in the most highly expressed genes as a result of selection for codons that maximise translational efficiency .
thus , differences between codon usage in a heterologous gene and the host organism may affect expression . to improve expression of human immunodeficiency virus type 1 gp120 from a dna vaccine vector , andr et al . generated a synthetic gp120 sequence in which most of the wild - type codons were replaced with codons from
the resulting construct showed increased in vitro expression of gp120 compared to the wild - type sequence .
in addition , significantly increased antibody titres and ctl reactivity were observed following administration of the vector containing the synthetic sequence .
similarly , a dna vaccine vector encoding a synthetic epitope of listeriolysin o with mammalian codon usage showed higher translation efficiency than a vector containing the wild - type sequence in murine cells .
furthermore , the first dna vaccine was capable of inducing specific cd8 t cells able to confer partial protection against challenge with l. monocytogenes where the second dna vaccine could not .
a number of other studies have reported that increased immune responses may be obtained by dna vaccination with a transgene sequence with optimised codon usage .
in this review the methodologies by which antigen expression has been optimised to date , i.e. optimisation of vector and transgene sequences , have been discussed .
it is clear that transgene expression may be increased through the use of optimised promoters and polya sequences .
however , in some circumstances it may be necessary to optimise dna vaccines to produce reduced transgene expression .
for example , the weaker sv40 promoter has been used rather than the cmv promoter to drive expression of antigens that induce cell death upon overexpression .
such tissue - specific expression systems may be able to produce stable expression by reducing the probability of inducing an immune response to the transgene
. it may be possible to design vectors for gene therapeutic purposes that avoid inducing unwanted immune responses against the encoded antigen by using tissue - specific promoters .
restricting the site of expression of genes should minimise the risks related to aberrant expression of a gene product .
furthermore , it should be possible to develop expression systems where gene products are only expressed in the critical cell types for dna vaccination or gene therapy , for example , dendritic cells ( dcs ) . as a better understanding of the proteins
whose expression is limited to dcs is obtained , novel expression systems will be generated . finally , through increased knowledge of the regulation of expression of antigens , it is now possible to produce multivalent systems whereby multiple antigens may be expressed from a single dna vaccine vector .
it is clear that the optimisation of antigen expression is an important consideration in dna vaccine vector design .
however , it is important to recognise that other aspects of vector design may influence the efficacy of the vaccine / gene therapy .
a rational approach to improve the efficacy of dna vaccination or gene therapy would optimise the : ( i ) vector backbone dna sequence ; ( ii ) transgene sequence ; ( iii ) co - expression of stimulatory sequences ; ( iv ) delivery system used for the vector ; ( v ) targeting of the vector for appropriate immune stimulation .
the backbone of a dna vaccine vector could be further modified to enhance immunogenicity via the manipulation of the dna to include certain sequences , so that the dna itself will have an adjuvantising effect .
dna vaccine vectors contain many cpg motifs ( consisting of unmethylated cpg dinucleotides flanked by two 5 ' purines and two 3 ' pyrimidines ) that , overall , induce a th1-like pattern of cytokine production , and are thought to account for strong ctl responses frequently seen following dna vaccination .
it is possible to augment responses to dna vaccine vectors by incorporating cpg motifs into the dna backbone of the plasmid . alternatively ,
immune responses may be modulated or enhanced by the co - expression of stimulatory molecules or cytokines   or through the use of localisation or secretory signals [ 47 - 49 ] , or ligand fusions [ 50 - 54 ] to direct antigens to sites appropriate for immune modulation .
finally , a variety of routes of administration of dna vaccines have been studied , including intramuscular , intradermal , subcutaneous , intravenous , intraperitoneal , oral , vaginal , intranasal and , more recently , non - invasive delivery to the skin ( reviewed by gurunathan et al . ) .
the approaches outlined above will together allow for the rational and optimised design for dna vaccines and gene therapy vectors .
the ability to improve antigen expression through the use of optimisation of regulatory elements , kozak sequences and codon usage is highlighted in this review , as part of this rational approach .
dna vaccination is a relatively recent development in vaccine methodology . 
 it is now possible to undertake a rational step - by - step approach to dna vaccine design . 
 strategies may include the incorporation of immunostimulatory sequences in the backbone of the plasmid , co - expression of stimulatory molecules , utilisation of localisation / secretory signals , and utilisation of the appropriate delivery system , for example . 
 however , another important consideration is the utilisation of methods designed to optimise transgene expression . in this review 
 we discuss the importance of regulatory elements , kozak sequences and codon optimisation in transgene expression .