Recent advances in chromatographic purification of plasmid DNA for gene therapy and DNA vaccines: A review

Abdulrahman, Ahmed; Ghanem, Ashraf

doi:10.1016/j.aca.2018.04.001

Cited by 45 publications

(21 citation statements)

References 82 publications

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“…Other methods should also be used to provide more accurate results. Real-time quantitative PCR targeting the pTRKH3 plasmid could be used to assess plasmid yield (Duarte et al 2019), while uorescence (Levy et al 2000) or chromatography (Abdulrahman et al 2018) based methods could allow for more accurate estimation of the relative abundance of the produced plasmids isoforms.…”

Section: Discussionmentioning

confidence: 99%

One-plasmid based CRISPR-Cas9 editing for gene deletion in Lactococcus lactis

Santos¹,

Monteiro²,

Prazeres³

et al. 2021

Preprint

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Lactococcus lactis strains are promising cell factories and delivery vehicles of plasmid DNA and recombinant protein for therapeutic applications. However, the limited yields of recombinant molecules obtained with these bacteria limits their wide applicability. Genome engineering of this host may solve the problem. However, the current genome editing toolbox available for L. lactis is either too laborious or incapable of large edits, limiting the scope of strain editing experiments. In this work, the basis for a one-plasmid CRISPR-Cas9 based genome editing plasmid was developed and tested. The new plasmid (pTCas9dO) adapted from the pKCcas9dO plasmid was used to delete 657 bp of the lactococcal nuclease nth of L. lactis subsp. lactis LMG19460, with the aim of improving yield and quality of plasmid DNA replicated in this strain. Although deletion mutants were successfully generated, plasmid curing was unsuccessful. Thus, further modifications are required before the plasmid is truly applicable for genome editing experiments. Unexpectedly, the generated deletion mutants generated a roughly 40% decrease in plasmid yield alongside with a decrease in the quality of produced pDNA.

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Section: Discussionmentioning

confidence: 99%

One-plasmid based CRISPR-Cas9 editing for gene deletion in Lactococcus lactis

Santos¹,

Monteiro²,

Prazeres³

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…While this resin does work, the quality and yield of the end product are frequently low. Separately, size-exclusion, hydrophobic interaction, and reversed-phase chromatography have also been used to purify DNA, but they are not optimal for size fractionation [32][33][34][35]. Instead, they appear to be better suited to the purification of plasmid DNA.…”

Section: Pol Scientificmentioning

confidence: 99%

High-yield purification of exceptional-quality, single-molecule DNA substrates

Bianco

2021

J Biol Methods

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Single-molecule studies involving DNA or RNA, require homogeneous preparations of nucleic acid substrates of exceptional quality. Over the past several years, a variety of methods have been published describing different purification methods but these are frustratingly inconsistent with variable yields even in the hands of experienced bench scientists. To address these issues, we present an optimized and straightforward, column-based approach that is reproducible and produces high yields of substrates or substrate components of exceptional quality. Central to the success of the method presented is the use of a non-porous anion exchange resin. In addition to the use of this resin, we encourage the optimization of each step in the construction of substrates. The fully optimized method produces high yields of a hairpin DNA substrate of exceptional quality. While this substrate is suitable for single-molecule, magnetic tweezer experiments, the described method is readily adaptable to the production of DNA substrates for the majority of single-molecule studies involving nucleic acids ranging in size from 70–15000 bp.

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“…), and several studies have shown that DNA vaccines don’t effectively deliver antigen to antigen-presenting cells (APCs) after i.m. Therefore, this leads to a strong immune response that can’t be induced [ 7 , 8 ]. Additionally, DNA vaccine has also been limited in clinical applications due to i.m., high dose, low bioavailability, and immunogenicity [ 9 ].…”

Section: Introductionmentioning

confidence: 99%

Intranasal immunization with O-2′-Hydroxypropyl trimethyl ammonium chloride chitosan nanoparticles loaded with Newcastle disease virus DNA vaccine enhances mucosal immune response in chickens

et al. 2021

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Background There has been a great interest in developing strategies for enhancing antigen delivery to the mucosal immune system as well as identifying mucosal active immunostimulating agents. To elevate the potential of O-2ʹ-Hydroxypropyl trimethyl ammonium chloride chitosan (O-2ʹ-HACC) as an adjuvant and mucosal immune delivery carrier for DNA vaccine, we prepared the O-2ʹ-HACC loaded with Newcastle disease virus (NDV) F gene plasmid DNA and C3d6 molecular adjuvant (O-2ʹ-HACC/pFDNA microparticles). Results The O-2ʹ-HACC/pFDNA exhibited a regular spherical morphology with a particle size of 202.3 ± 0.52 nm, a zeta potential of 50.8 ± 8.21 mV, encapsulation efficiency of 90.74 ± 1.10%, and a loading capacity of 49.84 ± 1.20%. The plasmid DNA could be sustainably released from the O-2ʹ-HACC/pFDNA after an initial burst release. Intranasal vaccination of chickens immunized with O-2ʹ-HACC/pFDNA not only induced higher anti-NDV IgG and sIgA antibody titers but also significantly promoted lymphocyte proliferation and produced higher levels of IL-2, IL-4, IFN-γ, CD4+, and CD8 + T lymphocytes compared with the NDV commercial live attenuated vaccine. Intranasal delivery of the O-2ʹ-HACC/pFDNA enhanced humoral, cellular, and mucosal immune responses and protected chickens from the infection of highly virulent NDV compared with the intramuscular delivery. Conclusions Collectively, our findings indicated that the O-2ʹ-HACC could be used as a vaccine adjuvant and delivery system for mucosal immunity and have an immense application promise. Graphic Abstract

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Recent advances in chromatographic purification of plasmid DNA for gene therapy and DNA vaccines: A review

Cited by 45 publications

References 82 publications

One-plasmid based CRISPR-Cas9 editing for gene deletion in Lactococcus lactis

One-plasmid based CRISPR-Cas9 editing for gene deletion in Lactococcus lactis

High-yield purification of exceptional-quality, single-molecule DNA substrates

Intranasal immunization with O-2′-Hydroxypropyl trimethyl ammonium chloride chitosan nanoparticles loaded with Newcastle disease virus DNA vaccine enhances mucosal immune response in chickens

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