Plasmid DNA vaccine vector design: impact on efficacy, safety and upstream production - PubMed (original) (raw)
Review
Plasmid DNA vaccine vector design: impact on efficacy, safety and upstream production
James A Williams et al. Biotechnol Adv. 2009 Jul-Aug.
Abstract
Critical molecular and cellular biological factors impacting design of licensable DNA vaccine vectors that combine high yield and integrity during bacterial production with increased expression in mammalian cells are reviewed. Food and Drug Administration (FDA), World Health Organization (WHO) and European Medical Agencies (EMEA) regulatory guidance's are discussed, as they relate to vector design and plasmid fermentation. While all new vectors will require extensive preclinical testing to validate safety and performance prior to clinical use, regulatory testing burden for follow-on products can be reduced by combining carefully designed synthetic genes with existing validated vector backbones. A flowchart for creation of new synthetic genes, combining rationale design with bioinformatics, is presented. The biology of plasmid replication is reviewed, and process engineering strategies that reduce metabolic burden discussed. Utilizing recently developed low metabolic burden seed stock and fermentation strategies, optimized vectors can now be manufactured in high yields exceeding 2 g/L, with specific plasmid yields of 5% total dry cell weight.
Figures
Figure 1
DNA Vaccine Vector production and use flowchart.
Figure 2
A. Annotated pDNAVACCUltra-1 (pNTCUltra-1) restriction map. Critical features such as the mRNA leader (UTR), chimeric SV40-CMV promoter, kanR gene, and functional domains in the origin (PAS-BL, RNAII promoter, RNase H cleavage site) are indicated. This vector targets a fusion antigen (antigens 1 and 2) for proteosomal degradation by fusion to the C-terminus of Ubiquitin A76. B. Plasmid DNA replication. For the leading strand, a RNA polymerase transcript (RNAII) either forms a replication driving R-loop (RNA:DNA hybrid) with the origin (left) or a nonproductive interaction with the antisense RNAI copy number regulator (right). The R-loop is cleaved by RNase H, the resulting 3′ OH serving as a primer for DNA pol I DNA synthesis. Replication of the leading strand exposes a phiX174 type primosome assembly site on the lagging strand (PAS-BL), which binds primosomal proteins (see Section 4.1). Lagging strand replication initiated at PAS-BL terminates at a termination site (ter) within the origin. Leading strand replication proceeds around the plasmid, also stopping at the ter site. DNA Pol I and DNA ligase are required after Pol III mediated leading and lagging strand replication to remove primers and seal nicks, respectively. DNA gyrase then negatively supercoils the resultant covalently closed circular (CCC) plasmid.
Figure 3
Antibiotic Free Bird Flu DNA vaccine vector. Vector specific features such as the CMV-HTLV-1 R-U5 derived mRNA leader (UTR) chimeric SV40-CMV promoter, and RNA selectable marker are indicated.
Figure 4
Insert design flowchart. See Section 12 for a detailed discussion.
Figure 5
A. Inducible fed-batch plasmid production fermentation process. B. E. coli strain DH5α + pNTCUltra1 plasmid inducible fed-batch fermentation growth and plasmid yield profiles. Fermentation was shifted from 30 to 42°C at 29hrs to induce plasmid production, plasmid yield reached 2130 mg/L.
References
- Allamane S, Jourdes P, Ratel D, Vicat JM, Dupre I, Laine M, Berger F, Benabid AL, Wion D. Bacterial DNA methylation and gene transfer efficiency. Biochem Biophys Res Commun. 2000;276:1261–4. -PubMed
- August JT, Pardoll DM, Guarnieri FG. Lysosomal targeting of immunogens. 5633234. 1997.
- Azzoni AR, Ribeiro SC, Monteiro GA, Prazeres DMF. The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression. J Gene Med. 2007;9:392–402. -PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical