Li et al. showed that activation of serum activation element (SRE activation binding site) at the CMV/SkA promoter region using SRF co-expression technique not only enhance the transgene expression, but also maintained the expression up to 21 days [58]. Using DNA shuffling technique, Wright et al. have created chimeric promoter originated from two human and two nonhuman primate strains of CMV [49]. Screening assays indicated 2-fold increased reporter gene expression

compared to wild-type promoters. Although an initial screen for activity can be done in vitro, in vivo attempt would be challenging. Only with appropriate screen in place, novel AT13387 mouse artificial promoter that outperforms existing endogenous sequence, in terms of both safety levels and duration of expression can be identified. Transgene expression is generally higher if introns are included in the vector backbone downstream of the promoter. Intron, as part of an mRNA leader augments promoter effect for expression of therapeutic gene in vivo [59] and [60]. Usually, plasmid expression for mammalian cells uses intron A from human CMV [61]. Here too, synthetic intron can be designated with the aid of bioinformatics to avoid existing sequences in CMV-infected person. Synthetic intron can enhance mRNA production. Short synthetic intron with efficient spliceable-site can expedite mature mRNA production and transportation from nucleus to the cytoplasm [62]. Therefore, vectors

harboring it stand a better chance to overcome mRNA accumulation barrier, in Cyclopamine datasheet comparison to vectors with endogenous introns. For example, synthetic intron, Ivs8 has been proven safe without causing any mutagenesis to the host [63] and [64]. A synthetic intron consisting a polynucleotide fragment splice site of a sarcoplasmic/endoplasmic reticulum calcium ATPase gene and a fragment contains at least a portion of a 5′UTR of a casein gene, can increase RNA transport and stability [65]. Signal sequence facilitates extra-cellular secretion of the vaccine peptide. This 15–30 amino acids encoded signal placed upstream of the therapeutic

gene often derived from human α-1-antichymotrypsin precursor (ACT) and tissue plasminogen activator (TPA) [66] and [67]. However, immunological cross-reaction can happen when signal peptides no (SP) fuse to immunogen, especially when those peptides are administered alone as a gene vaccine which in turn activates protective immunity against microbial pathogen [68]. Prior screening using statistical methods like the Hidden Markov Model should be considered to avoid undesired immune responses from signal peptide. This modelling is used as prediction methods to generate artificial SP sequences by creating a multiple alignment of a comprehensive set of known human secretory signal peptides [69]. This termination signal is positioned downstream of the therapeutic gene and often derived from bovine growth hormone, SV40 or β-globin genes.