Minicircle‐DNA production by site specific recombination and protein–DNA interaction chromatography

Mayrhofer, Peter; Blaesen, Markus; Schleef, Martin; Jechlinger, Wolfgang

doi:10.1002/jgm.1243

Cited by 91 publications

(74 citation statements)

References 59 publications

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“…It should be noted that given their clinical utility, a novel method involving affinity-based chromatography has been developed for obtaining highly purified mC DNA for gene therapy and vaccination, enabling safe production at an industrial scale [33,62,63]. Together, these factors highlight the critical advantages offered by mC DNA vector technology in conjunction with magnetofection, for clinical translational applications involving genetically engineered neural transplant cells (summarized in Fig.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy

Fernandes

Chari

2016

Journal of Controlled Release

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Please cite this article as: Alinda R. Fernandes, Divya M. Chari, Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy, Journal of Controlled Release (2016Release ( ), doi: 10.1016Release ( /j.jconrel.2016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

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Section: Accepted M Manuscriptmentioning

confidence: 99%

Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy

Fernandes

Chari

2016

Journal of Controlled Release

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“…Furthermore, the low amount of pDNA detected 3 months after injection at the muscle site could be the result of inefficient transfection, or transfected myofiber elimination after an immune cell infiltration in response to CpG sequences found in prokaryotic sequences (that is, the plasmid backbone used here). [40][41][42] The long-term maintenance of rAAV sequences in WBCs provides an easily accessible target for the surveillance of gene doping. The methods developed here to test gene doping also provide the basis for detecting other WADA-prohibited gene-doping targets.…”

Section: E+07mentioning

confidence: 99%

Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping

Guiner

Gernoux

et al. 2011

Gene Ther

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Legitimate uses of gene transfer technology can benefit from sensitive detection methods to determine vector biodistribution in pre-clinical studies and in human clinical trials, and similar methods can detect illegitimate gene transfer to provide sports-governing bodies with the ability to maintain fairness. Real-time PCR assays were developed to detect a performance-enhancing transgene (erythropoietin, EPO) and backbone sequences in the presence of endogenous cellular sequences. In addition to developing real-time PCR assays, the steps involved in DNA extraction, storage and transport were investigated. By real-time PCR, the vector transgene is distinguishable from the genomic DNA sequence because of the absence of introns, and the vector backbone can be identified by heterologous gene expression control elements. After performance of the assays was optimized, cynomolgus macaques received a single dose by intramuscular (IM) injection of plasmid DNA, a recombinant adeno-associated viral vector serotype 1 (rAAV1) or a rAAV8 vector expressing cynomolgus macaque EPO. Macaques received a high plasmid dose intended to achieve a significant, but not life-threatening, increase in hematocrit. rAAV vectors were used at low doses to achieve a small increase in hematocrit and to determine the limit of sensitivity for detecting rAAV sequences by single-step PCR. DNA extracted from white blood cells (WBCs) was tested to determine whether WBCs can be collaterally transfected by plasmid or transduced by rAAV vectors in this context, and can be used as a surrogate marker for gene doping. We demonstrate that IM injection of a conventional plasmid and rAAV vectors results in the presence of DNA that can be detected at high levels in blood before rapid elimination, and that rAAV genomes can persist for several months in WBCs.

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“…Briefly, both mCs and MNPs can be economically produced at a large scale at high purity with the development of novel technologies such as affinity-based chromatographic purification and flame spray method [60,61]. Additionally, the robust one-step protocol used here requires minimal training and basic laboratory equipment/containment level.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Part II: Functional delivery of a neurotherapeutic gene to neural stem cells using minicircle DNA and nanoparticles: Translational advantages for regenerative neurology

Fernandes

Chari

2016

Journal of Controlled Release

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Both neurotrophin-based therapy and neural stem cell (NSC)-based strategies have progressed to clinical trials for treatment of neurological diseases and injuries. Brain-derived neurotrophic factor (BDNF) in particular can confer neuroprotective and neuro-regenerative effects in preclinical studies, complementing the cell replacement benefits of NSCs.Therefore, combining both approaches by genetically-engineering NSCs to express BDNF is an attractive approach to achieve combinatorial therapy for complex neural injuries. Current genetic engineering approaches almost exclusively employ viral vectors for gene delivery to NSCs though safety and scalability pose major concerns for clinical translation and applicability. Magnetofection, a non-viral gene transfer approach deploying magnetic nanoparticles and DNA with magnetic fields offers a safe alternative but significant improvements are required to enhance its clinical application for delivery of large sized therapeutic plasmids. Here, we demonstrate for the first time the feasibility of using minicircles with magnetofection technology to safely engineer NSCs to overexpress BDNF.Primary mouse NSCs overexpressing BDNF generated increased daughter neuronal cell numbers post-differentiation, with accelerated maturation over a four-week period. Based on our findings we highlight the clinical potential of minicircle/magnetofection technology for therapeutic delivery of key neurotrophic agents.

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Minicircle‐DNA production by site specific recombination and protein–DNA interaction chromatography

Cited by 91 publications

References 59 publications

Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy

Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy

Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping

Part II: Functional delivery of a neurotherapeutic gene to neural stem cells using minicircle DNA and nanoparticles: Translational advantages for regenerative neurology

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