J Gene Med. 2010 Oct;12(10):818-31. doi: 10.1002/jgm.1499.

Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields.

Carnes AE, Luke JM, Vincent JM, Anderson S, Schukar A, Hodgson CP, Williams JA.

Source

Nature Technology Corporation, Lincoln, NE, USA.

Abstract

BACKGROUND:

For safety considerations, regulatory agencies recommend the elimination of antibiotic resistance markers and non-essential sequences from plasmid DNA-based gene medicines. In the present study, we analyzed antibiotic-free (AF) vector design criteria impacting upon bacterial production and mammalian transgene expression.

METHODS:

Both CMV-HTLV-I R RNA Pol II promoter (protein transgene) and murine U6 RNA Pol III promoter (RNA transgene) vector designs were studied. Plasmid production yield was assessed through inducible fed-batch fermentation. RNA Pol II-directed enhanced green fluorescent protein and RNA Pol III-directed RNA expression were quantified by fluorometry and quantitative real-time polymerase chain reaction, respectively, after transfection of human HEK293 cells.

RESULTS:

Sucrose-selectable minimalized protein and therapeutic RNA expression vector designs that combined an RNA-based AF selection with highly productive fermentation manufacturing (>1000 mg/l plasmid DNA) and high-level in vivo expression of encoded products were identified. The AF selectable marker was also successfully applied to convert existing kanamycin-resistant DNA vaccine plasmids gWIZ and pVAX1 into AF vectors, demonstrating a general utility for retrofitting existing vectors. A minimum vector size for high yield plasmid fermentation was identified. A strategy for stable fermentation of plasmid dimers with improved vector potency and fermentation yields up to 1740 mg/l was developed.

CONCLUSIONS:

We report the development of potent high yield AF gene medicine expression vectors for protein or RNA (e.g. short hairpin RNA or microRNA) products. These AF expression vectors were optimized to exceed a newly-identified size threshold for high copy plasmid replication and direct higher transgene expression levels than alternative vectors.

PMID:: 20806425; [PubMed - indexed for MEDLINE]
PMCID:: PMC2959117

Free PMC Article

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Figure 1

Antibiotic-free (AF) selection vectors. (A) RNA-OUT AF selection. Left: In a plasmid-free cell, levansucrase is expressed from a chromosomally-integrated SacB gene, leading to cell death in the presence of sucrose. Right: RNA-OUT (R-OUT) from the plasmid represses translation of the SacB gene, achieving plasmid selection. (B) pINT-λ-PIN-SacB vector. This vector was used to integrate the constitutive promoter (P_C) expressed RNA-IN-SacB selection cassette into the phage λ genomic attachment site of the E. coli TG1 strain. (C) NTC8485 EGFP AF vector, with transient expression enhancers HTLV-I R, VA1 and SV40 enhancer. Optimization of the SV40-CMV boundary (BE 120 bp deletion) resulted in the improved expression NTC8685 vector (Table 1). These vectors incorporated an extended replication origin that included a primosomal assembly site (PAS-BH) and the SV40 enhancer. These sequences (PAS-BH-SV40 backbone) improved manufacturing yields [10]. (D) NTC8485E-U6. (E) NTC8485E-U6 shRNA. shRNA oligonucleotides were annealed and cloned downstream of the mU6 promoter by replacing the HpaI-EcoRI pTac-GFP selection module in NTC8485E-U6. Recombinants were non-fluorescent (parentals were green due to GFP expression) in lacI repressor expressing host strains (e.g. lacI expressing strains DH5α, DH10B, TOP10; lacIq expressing strains XL1Blue, TG1, JM109, SURE, Stbl4) when plated on standard LB media supplemented with IPTG. Recombinants were positively selected in cells lines that do not encode the lacI repressor (e.g. JM108, Stbl2) due to pTac promoter mediated toxic overexpression of parental plasmid-encoded GFP. In these strains essentially 100% of the recovered colonies were recombinant. (F) Annotated map of the kanR NTC7482-41H-HA expression vector.

Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields

J Gene Med. 2010 October;12(10):818-831.

Figure 3

Vectors <3 kb have reduced copy number. The relative fermentation copy number for monomer and dimer plasmid in DH5α (Table 3) versus monomer vector size for: (A) kanR NTC7485 derivatives; and (B) AF NTC8485 derivatives. Monomer kanR or AF plasmids <3 kb had a much lower relative copy number than larger plasmids. The dimer plasmids of the 2.8 and 3.5kb NTC7485 kanR and the 2.1 and 2.7 kb NTC8485 vectors (plotted at the monomer kb size) had up to two fold improved copy number. Since a dimer plasmid also contains 2 fold more DNA, this resulted in dramatically higher plasmid yields than the corresponding monomer plasmids. (C) The inducible fermentation process maintained AF monomer and dimer plasmid stability with all vector sizes and produced high quality supercoiled plasmid. Plasmid DNA was prestained with SYBR Green I (Sigma, St. Louis, MO) and resolved on a TAE agarose gel. Fermentation harvests (see Table 3 for yields) of 2.1 kb NTC8485-U6-shRNA monomer (RF230; lane 1) and 4.2 kb dimer; (RF240; lane 2) and 2.7 kb NTC8485E-U6-shRNA monomer (RF232; lane 3) and 5.4 kb dimer; (RF241; lane 4) are shown. M is marker lanes (1 kb DNA ladder; Invitrogen). (D) Supercoiling linking number analysis of NTC7485-U6-shRNA fermentation samples. Samples were treated with T5 exonuclease to remove nicked plasmid prior to AGE in the presence of chloroquine. Plasmid from 42°C induction (lanes 1 and 4 = 8 h induction; lanes 2, 5 and 7 = 10 h induction) and post-induction 25°C hold stages (lanes 3, 6 and 8) of DH5α fermentations RF226 (lanes 1-3), RF279 (lanes 4-6), and XL1-Blue fermentation RF273 (lanes 7 and 8) are shown. Lanes C1 and C2 are 8 h 42°C induction sample from DH5α fermentation RF226 (without T5 treatment to remove nicked species) with (C2) or without (C1) E. coli DNA gyrase (New England Biolabs, Beverly, MA) treatment [10] to increase negative supercoiling linking number. In this assay negatively supercoiled plasmid topoisomers with higher linking numbers migrated faster. M is marker lanes (1 kb DNA ladder; Invitrogen).

Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields

J Gene Med. 2010 October;12(10):818-831.