Improved antibiotic-free plasmid vector design by incorporation of transient expression enhancers

Abstract

Methods to improve plasmid-mediated transgene expression are needed for gene medicine and gene vaccination applications. To maintain a low risk of insertional mutagenesis-mediated gene activation, expression-augmenting sequences would ideally function to improve transgene expression from transiently transfected intact plasmid, but not from spurious genomically integrated vectors. We report herein the development of potent minimal, antibiotic-free, high-manufacturing-yield mammalian expression vectors incorporating rationally designed additive combinations of expression enhancers. The SV40 72 bp enhancer incorporated upstream of the cytomegalovirus (CMV) enhancer selectively improved extrachromosomal transgene expression. The human T-lymphotropic virus type I (HTLV-I) R region, incorporated downstream of the CMV promoter, dramatically increased mRNA translation efficiency, but not overall mRNA levels, after transient transfection. A similar mRNA translation efficiency increase was observed with plasmid vectors incorporating and expressing the protein kinase R-inhibiting adenoviral viral associated (VA)1 RNA. Strikingly, HTLV-I R and VA1 did not increase transgene expression or mRNA translation efficiency from plasmid DNA after genomic integration. The vector platform, when combined with electroporation delivery, further increased transgene expression and improved HIV-1 gp120 DNA vaccine-induced neutralizing antibody titers in rabbits. These antibioticfree vectors incorporating transient expression enhancers are safer, more potent alternatives to improve transgene expression for DNA therapy or vaccination.

Plasmid DNA fermentation strain and process‐specific effects on vector yield, quality, and transgene expression

“…Transfected dcmþ and dcmÀ plasmid DNA have similar expression levels in primary human dendritic cells (Luke et al, 2010) and a dcmÀ KGF plasmid expression vector directs potent production of biologically active KGF after in vivo delivery to the porcine dermis (Aaron Tabor, personal communication). The isogenic dcmþ and dcmÀ strains utilized herein also eliminated possible strain to strain variation in plasmid quality attributes such as % supercoiling or RNA primer removal, as was the case with previous studies of damÀ dcmÀ DNA (Allamane et al, 2000;Lai et al, 2008;Ochiai et al, 2005).…”

Antibiotic-free production of a herpes simplex virus 2 DNA vaccine in a high yield cGMP process

“…3 This vector is specifically designed as a safe, minimalized, and antibiotic-free selection vector, which offers superior expression of recombinant proteins in mammalian cells, and is designed to comply with US Food and Drug Administration (FDA) and European Medicines Agency (EMA) regulatory guidance. The Two DNa vaccine plasmids encoding herpes simplex virus type 2 (hsV-2) glycoprotein D, NTc8485-O2-gD2 and NTc8485-O2-UgD2tr, were produced at large scale under current good manufacturing practice (cGMP) for use in a Phase I human clinical trial.…”

Molecular-Genetic Imaging of Cancer

“…Some enhancers have been found to be located at over 100,000 base pairs distant from the gene that they enhance or within the intron (Pennacchio et al, 2013). Strong enhancers have been successfully added to expression vectors, including the human cytomegalovirus (CMV) immediate-early enhancer (Boshart et al, 1985; Gruh et al, 2008) and simian virus 40 (SV40) enhancer (Haas, Ramanujam, Chandrasekharappa, & Subramanian, 1991; Luke et al, 2011). Enhancers from certain gene promoters have also been used to increase promoter-dependent, tissue-specific expression of a reporter.…”