Effect of Methylated Adenine in Plasmid DNA on Transgene Expression in Mice

Ochiai, Hiroshi; Harashima, Hideyoshi; Kamiya, Hiroyuki

doi:10.1248/bpb.28.2019

Cited by 5 publications

(8 citation statements)

References 24 publications

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“…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). This conclusion was supported by extensive QC analysis that demonstrated dcmþ and dcmÀ plasmids were of similar high quality for a variety of quality attributes such as RNA primer removal, % abasic residues, and % supercoiling.…”

Section: Discussionsupporting

confidence: 59%

“…For example, Dam and K12 methylation product, N 6 -methyladenosine, is a wide spread bacterial signature not present in mammalian cells. Oligonucleotides or plasmid DNA containing N 6 -methyladenosine increased cytokine induction (interleukin 12; IL-12) in mice compared to unmethylated control DNA (Tsuchiya et al, 2005), and IL-12 induction was twofold lower with unmethylated plasmid (vs. damþ dcmþ methylated) after IV lipofection delivery to mice (Ochiai et al, 2005). N 6 -methyladenosine responses are cell-type specific, since damÀ dcmÀ unmethylated or damþ dcmþ methylated plasmid was equivalent for macrophage activation (Roberts et al, 2005) or TLR9 independent activation of human neutrophils (Trevani et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

“…By contrast, Allamane et al (2000) reported CMV promoter expression from JM110 produced damÀ dcmÀ plasmid was fourfold reduced in rat and mouse cells compared to JM109 produced damþ dcmþ DNA and twofold lower after in vivo electroporation of mice. However, CMV promoter-driven expression was similar with SCS110 produced damÀ dcmÀ plasmid versus DH5a damþ dcmþ plasmid after IV lipofection delivery to mice despite higher levels of damÀ dcmÀ plasmid nicking and aggregation (Ochiai et al, 2005). This inter-study inconsistency may be due to variable EndA nuclease-mediated plasmid nicking during plasmid purification from the endAþ damÀ dcmÀ JM110 strain.…”

Section: Introductionmentioning

confidence: 94%

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

Carnes

Luke

Vincent

et al. 2010

Biotech & Bioengineering

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Industrial plasmid DNA manufacturing processes are needed to meet the quality, economy, and scale requirements projected for future commercial products. We report development of a modified plasmid fermentation copy number induction profile that increases gene vaccination/therapy vector yields up to 2,600 mg/L. We determined that, in contrast to recombinant protein production, secretion of the metabolic byproduct acetate into the media had only a minor negative effect on plasmid replication. We also investigated the impact of differences in epigenetic dcm methylase-directed cytosine methylation on plasmid production, transgene expression, and immunogenicity. While Escherichia coli plasmid production yield and quality are unaffected, dcm- versions of CMV and CMV-HTLV-I R promoter plasmids had increased transgene expression in human cells. Surprisingly, despite improved expression, dcm- plasmid is less immunogenic. Our results demonstrate that it is critical to lock the plasmid methylation pattern (i.e., production strain) early in product development and that dcm- strains may be superior for gene therapy applications wherein reduced immunogenicity is desirable and for in vitro transient transfection applications such as AAV production where improved expression is beneficial.

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Section: Discussionsupporting

confidence: 59%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 94%

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

Carnes

Luke

Vincent

et al. 2010

Biotech & Bioengineering

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“…In general, transgenes are expressed mainly in the lung by the intravenous administration of lipoplexes, depending on the lipid composition [13][14][15][16]. Alternatively, the spleen is a transgene-expressing organ in the case of Lipofectin [17]. In this study, the hydrodynamics-based administration was also used for plasmid-Lipofectin complexes, since we could deliver the complexes into the mouse liver, the same tissue examined for the naked plasmids.…”

Section: Discussionmentioning

confidence: 99%

The location of the left-handedly curved DNA sequence affects exogenous DNA expression in vivo

Kamiya

Fukunaga

Ohyama

et al. 2007

Archives of Biochemistry and Biophysics

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The intranuclear disposition of a plasmid is extremely important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on plasmid expression were examined in vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were . They also showed that histones bound to the left-handedly curved DNA, and that the TATA box was exposed out of the nucleosome core when the curved DNA was located at appropriate positions [9]. This finding prompted us to examine the effects of this left-handedly curved sequence in vivo. In this study, we investigated the in vivo effects of this sequence on plasmids when delivered in a naked form into mouse liver. We found that the position of the left-handedly curved sequence affected the transgene expression by one order of magnitude, without altering the amount of the exogenous DNAs, suggesting the different accessibility of transcriptional factors to the plasmids in vivo. Similar results were observed when plasmids complexed with cationic lipids were delivered into mouse liver. These results indicate that the left-handedly curved sequence on plasmids also determined 5 transgene expression in vivo, and that controlled interactions between the plasmid and the histones are important for the intranuclear disposition. Materials and Methods MaterialsOligodeoxyribonucleotides were purchased from Sigma Genosys Japan (Ishikari, Japan) in purified forms. The pLHC4/TLN-6, pLHC4/TLN-16, pLHC4/TLN+47, and pST0/TLN-7 plasmids, containing the thymidine kinase (tk) promoter and the luciferase gene (Fig. 1) [9], were amplified in Escherichia coli strain DH5α and purified with a Qiagen (Hilden, Germany) EndoFree Plasmid Mega kit. Hydrodynamics-based injectionPlasmid DNA (20 µg in 2 ml of saline) was injected into the tail vein of male six week-old Balb/c mice within 5 sec [10,11]. The livers were harvested from the injected mice at various time points, and the luciferase activity and the amount of the exogenous DNA were measured, as described below. Luciferase activity 6Livers were minced with scissors and homogenized completely in Lysis Buffer (100 mM Tris-HCl, 2 mM EDTA, 0.1% Triton X-100, pH 7.8). After centrifugation at 13,000 g for 10 min at 4°C, the supernatant was examined for luciferase activity, using a Luciferase Assay Systems kit (Promega, Madison, Wisconsin, USA). Isolation of nuclear DNA and Quantitative PCRLivers were homogenized in phosphate-buffered saline (PBS).After centrifugation at 2,500 g for 5 min at 4°C, the pellet was washed three times with PBS. The pellet was resuspended in DNA Lysis Buffer (100 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl 2 , 0.5% (w/v) IGEPAL-CA630, pH 7.4) [12]. After centrifugation at 1,400 g for 5 min at 4°C, the pellet was washed three times with DNA Lysis Buffer. The intranuclear DNA was extracted with the SepaGene reagent (Sanko Jun-yaku, Tokyo, Japan).Quantitative PCR (Q-PCR) was performed using an ABI 7500 real time PCR system, and SYBR-Green...

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“…This contrasts the results from a separate study wherein CMV promoter expression was 2-fold higher after in vivo electroporation of mice with dam+ dcm+ methylated plasmid compared to dam- dcm- unmethylated plasmid (Allamane et al, 2000). CMV promoter driven expression was also slightly lower with unmethylated plasmid (versus dam+ dcm+ methylated) after IV hydrodynamic lipofection delivery to mice (Ochiai et al, 2005). The CMV promoter contains only 2 dcm and no dam sites; increased expression in mice with methylated DNA may be a secondary effect, perhaps promoter stimulation due to dam methylation-mediated cytokine induction (see Section 3.3.2).…”

Section: Components Affecting Immunogenicitymentioning

confidence: 99%

Plasmid DNA vaccine vector design: Impact on efficacy, safety and upstream production

Williams

Carnes

Hodgson

2009

Biotechnology Advances

155

131

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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 followon 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.

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Effect of Methylated Adenine in Plasmid DNA on Transgene Expression in Mice

Cited by 5 publications

References 24 publications

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

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

The location of the left-handedly curved DNA sequence affects exogenous DNA expression in vivo

Plasmid DNA vaccine vector design: Impact on efficacy, safety and upstream production

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