DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery
Identifieur interne : 000A68 ( Main/Exploration ); précédent : 000A67; suivant : 000A69DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery
Auteurs : Ethlinn V. B. Van Gaal [Pays-Bas] ; Ronald S. Oosting [Pays-Bas] ; Roel Van Eijk [Pays-Bas] ; Marta Bakowska [Pays-Bas] ; Dries Feyen [Pays-Bas] ; Robbert Jan Kok [Pays-Bas] ; Wim E. Hennink [Pays-Bas] ; Daan J. A. Crommelin [Pays-Bas] ; Enrico Mastrobattista [Pays-Bas]Source :
- Pharmaceutical Research [ 0724-8741 ] ; 2011.
Abstract
To evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA.
A set of DTS was identified and cloned into EGFP-reporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry.
Neither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNF-α, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.
No beneficial effects of DTS on gene expression or nuclear uptake were observed in this study.
Url:
DOI: 10.1007/s11095-011-0407-8
PubMed: 21424159
PubMed Central: 3109246
Affiliations:
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Hennink</name><affiliation wicri:level="4"><nlm:aff id="Aff1">Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht</wicri:regionArea><orgName type="university">Université d'Utrecht</orgName><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName></affiliation></author><author><name sortKey="Crommelin, Daan J A" sort="Crommelin, Daan J A" uniqKey="Crommelin D" first="Daan J. A." last="Crommelin">Daan J. A. Crommelin</name><affiliation wicri:level="4"><nlm:aff id="Aff1">Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht</wicri:regionArea><orgName type="university">Université d'Utrecht</orgName><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName></affiliation><affiliation wicri:level="3"><nlm:aff id="Aff3">Dutch Top Institute Pharma, Leiden, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Dutch Top Institute Pharma, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Mastrobattista, Enrico" sort="Mastrobattista, Enrico" uniqKey="Mastrobattista E" first="Enrico" last="Mastrobattista">Enrico Mastrobattista</name><affiliation wicri:level="4"><nlm:aff id="Aff1">Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht</wicri:regionArea><orgName type="university">Université d'Utrecht</orgName><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">21424159</idno><idno type="pmc">3109246</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109246</idno><idno type="RBID">PMC:3109246</idno><idno type="doi">10.1007/s11095-011-0407-8</idno><date when="2011">2011</date><idno type="wicri:Area/Pmc/Corpus">000177</idno><idno type="wicri:Area/Pmc/Curation">000177</idno><idno type="wicri:Area/Pmc/Checkpoint">000370</idno><idno type="wicri:Area/Ncbi/Merge">000090</idno><idno type="wicri:Area/Ncbi/Curation">000090</idno><idno type="wicri:Area/Ncbi/Checkpoint">000090</idno><idno type="wicri:doubleKey">0724-8741:2011:Van Gaal E:dna:nuclear:targeting</idno><idno type="wicri:Area/Main/Merge">000A70</idno><idno type="wicri:Area/Main/Curation">000A68</idno><idno type="wicri:Area/Main/Exploration">000A68</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery</title><author><name sortKey="Van Gaal, Ethlinn V B" sort="Van Gaal, Ethlinn V B" uniqKey="Van Gaal E" first="Ethlinn V. B." last="Van Gaal">Ethlinn V. B. Van Gaal</name><affiliation wicri:level="4"><nlm:aff id="Aff1">Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht</wicri:regionArea><orgName type="university">Université d'Utrecht</orgName><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName></affiliation></author><author><name sortKey="Oosting, Ronald S" sort="Oosting, Ronald S" uniqKey="Oosting R" first="Ronald S." last="Oosting">Ronald S. Oosting</name><affiliation wicri:level="4"><nlm:aff id="Aff2">Department of Pharmacology, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Pharmacology, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht</wicri:regionArea><orgName type="university">Université d'Utrecht</orgName><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName></affiliation></author><author><name sortKey="Van Eijk, Roel" sort="Van Eijk, Roel" uniqKey="Van Eijk R" first="Roel" last="Van Eijk">Roel Van Eijk</name><affiliation wicri:level="4"><nlm:aff id="Aff1">Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht</wicri:regionArea><orgName type="university">Université d'Utrecht</orgName><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName></affiliation></author><author><name sortKey="Bakowska, Marta" sort="Bakowska, Marta" uniqKey="Bakowska M" first="Marta" last="Bakowska">Marta Bakowska</name><affiliation wicri:level="4"><nlm:aff id="Aff1">Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht</wicri:regionArea><orgName type="university">Université d'Utrecht</orgName><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName></affiliation></author><author><name sortKey="Feyen, Dries" sort="Feyen, Dries" uniqKey="Feyen D" first="Dries" last="Feyen">Dries Feyen</name><affiliation wicri:level="4"><nlm:aff id="Aff1">Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht</wicri:regionArea><orgName type="university">Université d'Utrecht</orgName><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName></affiliation></author><author><name sortKey="Kok, Robbert Jan" sort="Kok, Robbert Jan" uniqKey="Kok R" first="Robbert Jan" last="Kok">Robbert Jan Kok</name><affiliation wicri:level="4"><nlm:aff id="Aff1">Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht</wicri:regionArea><orgName type="university">Université d'Utrecht</orgName><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName></affiliation></author><author><name sortKey="Hennink, Wim E" sort="Hennink, Wim E" uniqKey="Hennink W" first="Wim E." last="Hennink">Wim E. Hennink</name><affiliation wicri:level="4"><nlm:aff id="Aff1">Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht</wicri:regionArea><orgName type="university">Université d'Utrecht</orgName><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName></affiliation></author><author><name sortKey="Crommelin, Daan J A" sort="Crommelin, Daan J A" uniqKey="Crommelin D" first="Daan J. A." last="Crommelin">Daan J. A. Crommelin</name><affiliation wicri:level="4"><nlm:aff id="Aff1">Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht</wicri:regionArea><orgName type="university">Université d'Utrecht</orgName><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName></affiliation><affiliation wicri:level="3"><nlm:aff id="Aff3">Dutch Top Institute Pharma, Leiden, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Dutch Top Institute Pharma, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Mastrobattista, Enrico" sort="Mastrobattista, Enrico" uniqKey="Mastrobattista E" first="Enrico" last="Mastrobattista">Enrico Mastrobattista</name><affiliation wicri:level="4"><nlm:aff id="Aff1">Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands</nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht</wicri:regionArea><orgName type="university">Université d'Utrecht</orgName><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName></affiliation></author></analytic><series><title level="j">Pharmaceutical Research</title><idno type="ISSN">0724-8741</idno><idno type="eISSN">1573-904X</idno><imprint><date when="2011">2011</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>ABSTRACT</title><sec><title>Purpose</title><p>To evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA.</p></sec><sec><title>Methods</title><p>A set of DTS was identified and cloned into EGFP-reporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry.</p></sec><sec><title>Results</title><p>Neither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNF-α, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.</p></sec><sec><title>Conclusion</title><p>No beneficial effects of DTS on gene expression or nuclear uptake were observed in this study.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Forrest, Ml" uniqKey="Forrest M">ML Forrest</name></author><author><name sortKey="Pack, Dw" uniqKey="Pack D">DW Pack</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Funhoff, Am" uniqKey="Funhoff A">AM Funhoff</name></author><author><name sortKey="Nostrum, Cf" uniqKey="Nostrum C">CF Nostrum</name></author><author><name sortKey="Koning, Ga" uniqKey="Koning G">GA Koning</name></author><author><name sortKey="Schuurmans Nieuwenbroek, Nm" uniqKey="Schuurmans Nieuwenbroek N">NM Schuurmans-Nieuwenbroek</name></author><author><name sortKey="Crommelin, Dj" 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uniqKey="Ito R">R Ito</name></author><author><name sortKey="Mizuguchi, H" uniqKey="Mizuguchi H">H Mizuguchi</name></author><author><name sortKey="Hayakawa, T" uniqKey="Hayakawa T">T Hayakawa</name></author><author><name sortKey="Harashima, H" uniqKey="Harashima H">H Harashima</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Foecking, Mk" uniqKey="Foecking M">MK Foecking</name></author><author><name sortKey="Hofstetter, H" uniqKey="Hofstetter H">H Hofstetter</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Thanaketpaisarn, O" uniqKey="Thanaketpaisarn O">O Thanaketpaisarn</name></author><author><name sortKey="Nishikawa, M" uniqKey="Nishikawa M">M Nishikawa</name></author><author><name sortKey="Okabe, T" uniqKey="Okabe T">T Okabe</name></author><author><name sortKey="Yamashita, F" uniqKey="Yamashita F">F Yamashita</name></author><author><name sortKey="Hashida, M" uniqKey="Hashida M">M Hashida</name></author></analytic></biblStruct></listBibl></div1></back></TEI><affiliations><list><country><li>Pays-Bas</li></country><region><li>Hollande-Méridionale</li><li>Utrecht (province)</li></region><settlement><li>Leyde</li><li>Utrecht</li></settlement><orgName><li>Université d'Utrecht</li></orgName></list><tree><country name="Pays-Bas"><region name="Utrecht (province)"><name sortKey="Van Gaal, Ethlinn V B" sort="Van Gaal, Ethlinn V B" uniqKey="Van Gaal E" first="Ethlinn V. B." last="Van Gaal">Ethlinn V. B. Van Gaal</name></region><name sortKey="Bakowska, Marta" sort="Bakowska, Marta" uniqKey="Bakowska M" first="Marta" last="Bakowska">Marta Bakowska</name><name sortKey="Crommelin, Daan J A" sort="Crommelin, Daan J A" uniqKey="Crommelin D" first="Daan J. A." last="Crommelin">Daan J. A. Crommelin</name><name sortKey="Crommelin, Daan J A" sort="Crommelin, Daan J A" uniqKey="Crommelin D" first="Daan J. A." last="Crommelin">Daan J. A. Crommelin</name><name sortKey="Feyen, Dries" sort="Feyen, Dries" uniqKey="Feyen D" first="Dries" last="Feyen">Dries Feyen</name><name sortKey="Hennink, Wim E" sort="Hennink, Wim E" uniqKey="Hennink W" first="Wim E." last="Hennink">Wim E. Hennink</name><name sortKey="Kok, Robbert Jan" sort="Kok, Robbert Jan" uniqKey="Kok R" first="Robbert Jan" last="Kok">Robbert Jan Kok</name><name sortKey="Mastrobattista, Enrico" sort="Mastrobattista, Enrico" uniqKey="Mastrobattista E" first="Enrico" last="Mastrobattista">Enrico Mastrobattista</name><name sortKey="Oosting, Ronald S" sort="Oosting, Ronald S" uniqKey="Oosting R" first="Ronald S." last="Oosting">Ronald S. Oosting</name><name sortKey="Van Eijk, Roel" sort="Van Eijk, Roel" uniqKey="Van Eijk R" first="Roel" last="Van Eijk">Roel Van Eijk</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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