NTC8684 plasmid无抗生素筛选哺乳动物DNA疫苗蛋白酶体靶向表达载体基因治疗质粒 BioVector NTCC质粒载体菌种细胞基因保藏中心

NTC8684 plasmid无抗生素筛选哺乳动物DNA疫苗蛋白酶体靶向表达载体基因治疗质粒

NTC8681,NTC8682,NTC8684and NTC8685 plasmids are antibiotic-free vectors optimized to combine maximal eukaryotic gene expression with superior bacterial manufacturing yields. These plasmids were specifically designed as safe minimalized antibiotic-free selection vectors for theexpression of recombinant proteins in mammalian cells. This may be for protein production, gene therapy or induction of neutralizing immune responses by genetic immunization. The vectors combine minimal prokaryotic sequences including an antibiotic-free sucroseselectable marker. The vectors also contain a novel chimeric promoter that directs superior mammalian cell expression(Luke et al.2009, 2011). The vectors are available in four versions. NTC8681 targets encoded protein to the endosomeusing an optimized human lysosomal-associated membrane protein 1 (Lamp1) targeting tag. NTC8682 targets encoded protein into the secretory pathwayusing an optimized tissue plasminogen activator (TPA) signal peptide. NTC8684 targets proteins to the proteosome by fusion C-terminal to a destabilizing UbiquitinA76tag. NTC8685 expresses encoded protein ‘native’ without targeting sequences.The vectors were designed to be responsive to Food and Drug Administration (FDA) regulatory guidance’s regarding DNA Vaccine vector composition (FDA 1996, FDA 2007; reviewed in Williams et al. 2009a). All sequences that were not essential for Escherichia coliplasmid replication or mammalian cell expression of the target gene were eliminated. Synthetic eukaryotic mRNA leader and terminators were utilized in the vector design to limit DNA sequence homology with the human genome to reduce the possibility of chromosomal integration. The vectors encode a consensus Kozak translation initiation sequence and ATG start codon. Target gene expression is driven from an optimized chimeric promoter-intron (SV40-CMV-HTLV-1 R synthetic intron).The boundary between the CMV promoter and the SV40 enhancerhas been optimized resulting in dramatically improved expression in mammalian cells (Luke et al. 2011). Thischimeric CMV promoter achieves significantly higher expression levels than traditional human cytomegalovirus (CMV) promoter based vectors (Luke et al.2009, 2011).NTC8681, NTC8682, NTC8684 and NTC8685 vectors also incorporate the adenoviral serotype 5 VA RNAI (VA1) transient expression enhancer. VA1 further improves eukaryotic expression without affecting Escherichia coliproduction yields.

NTC8681, NTC8682, NTC8684, NTC8685 Targeting Expression Vectors4Antibiotic-free SelectionAntibiotic-resistance markers, typically kanamycin resistance (KanR), allow selective retention of plasmid DNA during bacterial fermentation and are the most commonly utilized selectable markers.To ensure safety, however, regulatory agencies recommend elimination of antibiotic-resistance markers from therapeutic and vaccine plasmid DNA vectors. The presence of an antibiotic resistance gene in the plasmid backbone is considered undesirable by regulatory agencies, due to the potential transfer of antibiotic resistance to endogenous microbial flora and the potential activation and transcription of the genes from mammalian promoters after cellular incorporation into the genome(Reviewed in Williams et al.2009a).NTC has designedan antibiotic-free selection system

Vectors with this selection system incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a counter-selectable marker (SacB) from the host chromosome (selection host DH5α attλ::P5/6 6/6-RNA-IN-SacB, catR). SacBencodes a levansucrase, which is toxic in the presence of sucrose. Plasmid selection is achieved in the presence of sucrose.NTC8685 vector production yields > 1 g/L were verified in Escherichia colifermentation culture (Carnes et al.2010)

In summary, the NTC8681, NTC8682, NTC8684 and NTC8685 vectors offer the following advantages• Highestlevel expression in a wide range of mammalian cells using: 1) an optimized chimeric CMV-HTLV-I promoter; 2) Adenoviral VA RNAI transient expression enhancer; and 3) SV40 enhancer (Fig. 1)• Antibiotic-free selection in Escherichia colihost• Choice of Escherichia coliproduction strain for higher immunogenicity (dcm+NTC4862) orhigher expression (dcm-NTC48165;Fig. 1) (Carnes et al. 2011)• Superior Escherichia coliplasmid production yields using optimized vector backbone• Optional intracellular antigen targetingN-terminal and C terminal LAMP1 tag forendosomaltargeting (NTC8681) N-terminal TPA signal peptide tag for secretion targeting(NTC8682)N-terminal destabilizing Ubiquitin tagfor proteosome targeting (NTC8684) • Simultaneous cloninginto NTC8682, NTC8684 and NTC8685 vectorsthrough use of compatibleprecision cloning cassettes• Small vectors for more efficient transfection • Compliance with regulatory guidance (i.e. Reduced size, elimination of homology to human genomic DNA, elimination of antibiotic resistance marker)



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