Vectors based on reducible polycations facilitate intracellular release of nucleic acids
Identifieur interne : 000C04 ( Istex/Corpus ); précédent : 000C03; suivant : 000C05Vectors based on reducible polycations facilitate intracellular release of nucleic acids
Auteurs : Martin L. Read ; K. Helen Bremner ; David Oupick ; Nicola K. Green ; Peter F. Searle ; Leonard W. SeymourSource :
- The Journal of Gene Medicine [ 1099-498X ] ; 2003-03.
English descriptors
- Teeft :
- Assay, Bioconjug chem, Cancer cells, Cancer gene, Cancer gene therapy, Cationic, Cationic lipid, Cationic lipids, Cell lines, Cells transfected, Cellular uptake, Chem, Chloroquine, Copyright, Cytometry analysis, Cytosolic nucleases, Dotap, Endosomal, Ethidium, Ethidium bromide, Expression plasmid, Fresh media, Gene, Gene delivery, Gene expression, Gene therapy, Gene transfer, Grey bars, Hela, Hela cells, Hepes buffer, High levels, Higher level, Imagequanttm software, Initial experiments, Intracellular, Intracellular activation, Intracellular delivery, Intracellular release, John wiley sons, Lipid, Lipoplexes, Lncap, Lncap cells, Luciferase, Luciferase activity, Molecular weight, Mrna, Nacl, Nitroreductase, Nuclease, Nuclease degradation, Nucleic, Nucleic acids, Oxidative polycondensation, Pegfpn1, Peptide, Photon correlation spectroscopy, Physiological conditions, Plasmid, Polycation, Polycations, Polyplexes, Prodrug, Reducible, Reducible polycations, Reporter gene expression, Similar levels, Standard deviation, Surface charge, Synthetic vectors, Toxicity, Transfected, Transfected cells, Transfection, Transfection activity, Uorescence, White bars.
Abstract
Background: Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L‐lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids. Methods: RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys‐Lys10‐Cys and used to condense nucleic acids. The stability of RPC‐based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techniques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC‐3 and B16‐F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presence of chloroquine or the cationic lipid (N‐(1‐(2,3‐dioleoyloxy)propyl)‐N, N, N‐trimethylammonium chloride) (DOTAP). A CMV‐driven plasmid expressing the nitroreductase (ntr) gene was used to evaluate the therapeutic efficacy of RPC‐based delivery vectors. Results: A 187‐fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non‐reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 ± 7.9%), LNCaP (55.2 ± 6.7%) and PC‐3 (66.1 ± 3.7%) cells. Transfection with the ntr gene and treatment with the prodrug CB1954 resulted in significant cell killing, achieving IC50 values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20–75% of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (<5% of cells). Conclusions: These results demonstrate that lipid‐mediated activation of RPC‐based polyplexes is a useful strategy to enhance intracellular delivery of nucleic acids and potentiate therapeutic activity. Copyright © 2002 John Wiley & Sons, Ltd.
Url:
DOI: 10.1002/jgm.331
Links to Exploration step
ISTEX:5B64D673FF4FBAD3BE0BB46200FF07964DD46B8CLe document en format XML
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Helen Bremner</name><affiliation><mods:affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</mods:affiliation></affiliation></author><author><name sortKey="Oupick, David" sort="Oupick, David" uniqKey="Oupick D" first="David" last="Oupick">David Oupick</name><affiliation><mods:affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</mods:affiliation></affiliation></author><author><name sortKey="Green, Nicola K" sort="Green, Nicola K" uniqKey="Green N" first="Nicola K." last="Green">Nicola K. Green</name><affiliation><mods:affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</mods:affiliation></affiliation></author><author><name sortKey="Searle, Peter F" sort="Searle, Peter F" uniqKey="Searle P" first="Peter F." last="Searle">Peter F. Searle</name><affiliation><mods:affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</mods:affiliation></affiliation></author><author><name sortKey="Seymour, Leonard W" sort="Seymour, Leonard W" uniqKey="Seymour L" first="Leonard W." last="Seymour">Leonard W. 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Read</name><affiliation><mods:affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: mread@globalnet.co.uk</mods:affiliation></affiliation><affiliation><mods:affiliation>Correspondence address: Department of Clinical Pharmacology, University of Oxford, Oxford OX2 6HE, UK.</mods:affiliation></affiliation></author><author><name sortKey="Bremner, K Helen" sort="Bremner, K Helen" uniqKey="Bremner K" first="K. Helen" last="Bremner">K. Helen Bremner</name><affiliation><mods:affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</mods:affiliation></affiliation></author><author><name sortKey="Oupick, David" sort="Oupick, David" uniqKey="Oupick D" first="David" last="Oupick">David Oupick</name><affiliation><mods:affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</mods:affiliation></affiliation></author><author><name sortKey="Green, Nicola K" sort="Green, Nicola K" uniqKey="Green N" first="Nicola K." last="Green">Nicola K. Green</name><affiliation><mods:affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</mods:affiliation></affiliation></author><author><name sortKey="Searle, Peter F" sort="Searle, Peter F" uniqKey="Searle P" first="Peter F." last="Searle">Peter F. Searle</name><affiliation><mods:affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</mods:affiliation></affiliation></author><author><name sortKey="Seymour, Leonard W" sort="Seymour, Leonard W" uniqKey="Seymour L" first="Leonard W." last="Seymour">Leonard W. Seymour</name><affiliation><mods:affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">The Journal of Gene Medicine</title><title level="j" type="alt">JOURNAL OF GENE MEDICINE, THE</title><idno type="ISSN">1099-498X</idno><idno type="eISSN">1521-2254</idno><imprint><biblScope unit="vol">5</biblScope><biblScope unit="issue">3</biblScope><biblScope unit="page" from="232">232</biblScope><biblScope unit="page" to="245">245</biblScope><biblScope unit="page-count">14</biblScope><publisher>John Wiley & Sons, Ltd.</publisher><pubPlace>Chichester, UK</pubPlace><date type="published" when="2003-03">2003-03</date></imprint><idno type="ISSN">1099-498X</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1099-498X</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Assay</term><term>Bioconjug chem</term><term>Cancer cells</term><term>Cancer gene</term><term>Cancer gene therapy</term><term>Cationic</term><term>Cationic lipid</term><term>Cationic lipids</term><term>Cell lines</term><term>Cells transfected</term><term>Cellular uptake</term><term>Chem</term><term>Chloroquine</term><term>Copyright</term><term>Cytometry analysis</term><term>Cytosolic nucleases</term><term>Dotap</term><term>Endosomal</term><term>Ethidium</term><term>Ethidium bromide</term><term>Expression plasmid</term><term>Fresh media</term><term>Gene</term><term>Gene delivery</term><term>Gene expression</term><term>Gene therapy</term><term>Gene transfer</term><term>Grey bars</term><term>Hela</term><term>Hela cells</term><term>Hepes buffer</term><term>High levels</term><term>Higher level</term><term>Imagequanttm software</term><term>Initial experiments</term><term>Intracellular</term><term>Intracellular activation</term><term>Intracellular delivery</term><term>Intracellular release</term><term>John wiley sons</term><term>Lipid</term><term>Lipoplexes</term><term>Lncap</term><term>Lncap cells</term><term>Luciferase</term><term>Luciferase activity</term><term>Molecular weight</term><term>Mrna</term><term>Nacl</term><term>Nitroreductase</term><term>Nuclease</term><term>Nuclease degradation</term><term>Nucleic</term><term>Nucleic acids</term><term>Oxidative polycondensation</term><term>Pegfpn1</term><term>Peptide</term><term>Photon correlation spectroscopy</term><term>Physiological conditions</term><term>Plasmid</term><term>Polycation</term><term>Polycations</term><term>Polyplexes</term><term>Prodrug</term><term>Reducible</term><term>Reducible polycations</term><term>Reporter gene expression</term><term>Similar levels</term><term>Standard deviation</term><term>Surface charge</term><term>Synthetic vectors</term><term>Toxicity</term><term>Transfected</term><term>Transfected cells</term><term>Transfection</term><term>Transfection activity</term><term>Uorescence</term><term>White bars</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Background: Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L‐lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids. Methods: RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys‐Lys10‐Cys and used to condense nucleic acids. The stability of RPC‐based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techniques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC‐3 and B16‐F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presence of chloroquine or the cationic lipid (N‐(1‐(2,3‐dioleoyloxy)propyl)‐N, N, N‐trimethylammonium chloride) (DOTAP). A CMV‐driven plasmid expressing the nitroreductase (ntr) gene was used to evaluate the therapeutic efficacy of RPC‐based delivery vectors. Results: A 187‐fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non‐reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 ± 7.9%), LNCaP (55.2 ± 6.7%) and PC‐3 (66.1 ± 3.7%) cells. Transfection with the ntr gene and treatment with the prodrug CB1954 resulted in significant cell killing, achieving IC50 values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20–75% of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (<5% of cells). Conclusions: These results demonstrate that lipid‐mediated activation of RPC‐based polyplexes is a useful strategy to enhance intracellular delivery of nucleic acids and potentiate therapeutic activity. Copyright © 2002 John Wiley & Sons, Ltd.</div></front></TEI><istex><corpusName>wiley</corpusName><keywords><teeft><json:string>polyplexes</json:string><json:string>dotap</json:string><json:string>transfection</json:string><json:string>intracellular</json:string><json:string>hela</json:string><json:string>gene expression</json:string><json:string>cationic</json:string><json:string>polycations</json:string><json:string>transfected</json:string><json:string>lipid</json:string><json:string>uorescence</json:string><json:string>mrna</json:string><json:string>hela cells</json:string><json:string>prodrug</json:string><json:string>copyright</json:string><json:string>john wiley sons</json:string><json:string>lncap</json:string><json:string>polycation</json:string><json:string>chloroquine</json:string><json:string>gene transfer</json:string><json:string>plasmid</json:string><json:string>nucleic acids</json:string><json:string>peptide</json:string><json:string>toxicity</json:string><json:string>reducible</json:string><json:string>standard deviation</json:string><json:string>transfection activity</json:string><json:string>ethidium</json:string><json:string>chem</json:string><json:string>intracellular activation</json:string><json:string>gene</json:string><json:string>nucleic</json:string><json:string>cationic lipids</json:string><json:string>lipoplexes</json:string><json:string>luciferase</json:string><json:string>nuclease</json:string><json:string>nacl</json:string><json:string>nitroreductase</json:string><json:string>pegfpn1</json:string><json:string>endosomal</json:string><json:string>synthetic vectors</json:string><json:string>cell lines</json:string><json:string>intracellular delivery</json:string><json:string>white bars</json:string><json:string>cytometry analysis</json:string><json:string>oxidative polycondensation</json:string><json:string>gene therapy</json:string><json:string>higher level</json:string><json:string>gene delivery</json:string><json:string>cells transfected</json:string><json:string>reducible polycations</json:string><json:string>ethidium bromide</json:string><json:string>bioconjug chem</json:string><json:string>fresh media</json:string><json:string>cationic lipid</json:string><json:string>cancer gene therapy</json:string><json:string>assay</json:string><json:string>cancer cells</json:string><json:string>grey bars</json:string><json:string>imagequanttm software</json:string><json:string>initial experiments</json:string><json:string>hepes buffer</json:string><json:string>luciferase activity</json:string><json:string>expression plasmid</json:string><json:string>intracellular release</json:string><json:string>molecular weight</json:string><json:string>similar levels</json:string><json:string>photon correlation spectroscopy</json:string><json:string>lncap cells</json:string><json:string>surface charge</json:string><json:string>high levels</json:string><json:string>physiological conditions</json:string><json:string>transfected cells</json:string><json:string>cytosolic nucleases</json:string><json:string>nuclease degradation</json:string><json:string>cancer gene</json:string><json:string>reporter gene expression</json:string><json:string>cellular uptake</json:string></teeft></keywords><author><json:item><name>Martin L. Read</name><affiliations><json:string>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</json:string><json:string>E-mail: mread@globalnet.co.uk</json:string><json:string>Correspondence address: Department of Clinical Pharmacology, University of Oxford, Oxford OX2 6HE, UK.</json:string></affiliations></json:item><json:item><name>K. Helen Bremner</name><affiliations><json:string>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</json:string></affiliations></json:item><json:item><name>David Oupický</name><affiliations><json:string>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</json:string></affiliations></json:item><json:item><name>Nicola K. Green</name><affiliations><json:string>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</json:string></affiliations></json:item><json:item><name>Peter F. Searle</name><affiliations><json:string>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</json:string></affiliations></json:item><json:item><name>Leonard W. Seymour</name><affiliations><json:string>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>cancer</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>DNA</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>gene therapy</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>lipid</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>nitroreductase</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>poly(L‐lysine)</value></json:item></subject><articleId><json:string>JGM331</json:string></articleId><arkIstex>ark:/67375/WNG-PHJ5C7S9-J</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>article</json:string></originalGenre><abstract>Background: Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L‐lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids. Methods: RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys‐Lys10‐Cys and used to condense nucleic acids. The stability of RPC‐based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techniques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC‐3 and B16‐F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presence of chloroquine or the cationic lipid (N‐(1‐(2,3‐dioleoyloxy)propyl)‐N, N, N‐trimethylammonium chloride) (DOTAP). A CMV‐driven plasmid expressing the nitroreductase (ntr) gene was used to evaluate the therapeutic efficacy of RPC‐based delivery vectors. Results: A 187‐fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non‐reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 ± 7.9%), LNCaP (55.2 ± 6.7%) and PC‐3 (66.1 ± 3.7%) cells. Transfection with the ntr gene and treatment with the prodrug CB1954 resulted in significant cell killing, achieving IC50 values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20–75% of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (>5% of cells). Conclusions: These results demonstrate that lipid‐mediated activation of RPC‐based polyplexes is a useful strategy to enhance intracellular delivery of nucleic acids and potentiate therapeutic activity. Copyright © 2002 John Wiley & Sons, Ltd.</abstract><qualityIndicators><score>10</score><pdfWordCount>8791</pdfWordCount><pdfCharCount>53381</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>14</pdfPageCount><pdfPageSize>595 x 859 pts</pdfPageSize><pdfWordsPerPage>628</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>285</abstractWordCount><abstractCharCount>2009</abstractCharCount><keywordCount>6</keywordCount></qualityIndicators><title>Vectors based on reducible polycations facilitate intracellular release of nucleic acids</title><pmid><json:string>12666189</json:string></pmid><genre><json:string>article</json:string></genre><host><title>The Journal of Gene Medicine</title><language><json:string>unknown</json:string></language><doi><json:string>10.1002/(ISSN)1521-2254</json:string></doi><issn><json:string>1099-498X</json:string></issn><eissn><json:string>1521-2254</json:string></eissn><publisherId><json:string>JGM</json:string></publisherId><volume>5</volume><issue>3</issue><pages><first>232</first><last>245</last><total>14</total></pages><genre><json:string>journal</json:string></genre><subject><json:item><value>Research Article</value></json:item><json:item><value>Research Articles</value></json:item></subject></host><namedEntities><unitex><date><json:string>2.3 and 2/4.6</json:string><json:string>2003</json:string></date><geogName></geogName><orgName><json:string>Malvern Instruments</json:string><json:string>Sigma</json:string><json:string>Berthold Instruments, UK</json:string><json:string>Promega</json:string><json:string>Sons, Ltd.</json:string><json:string>Clontech, Oxford, UK</json:string><json:string>Department of Clinical Pharmacology, University of Oxford, Oxford OX</json:string><json:string>Sons, Ltd</json:string><json:string>Seymour Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B</json:string><json:string>University of Birmingham, UK</json:string></orgName><orgName_funder></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Peter Whittle</json:string><json:string>Paul Farrow</json:string><json:string>John Wiley</json:string><json:string>David Oupick</json:string><json:string>Martin L. Read</json:string><json:string>Jane Grove</json:string><json:string>The</json:string><json:string>Richard Dales</json:string><json:string>Alan Parker</json:string><json:string>Manfred Ogris</json:string><json:string>Libuse Oupicka</json:string><json:string>K. Helen</json:string></persName><placeName><json:string>Carlisle</json:string><json:string>Southampton</json:string><json:string>Germany</json:string><json:string>Munich</json:string><json:string>UK</json:string><json:string>Poole</json:string><json:string>Northampton</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>[24,31]</json:string><json:string>Adami et al.</json:string><json:string>[50]</json:string><json:string>[34]</json:string><json:string>[15,16]</json:string><json:string>[39,40]</json:string><json:string>[29]</json:string><json:string>[44]</json:string><json:string>[42,43]</json:string><json:string>[17]</json:string><json:string>Pollard et al.</json:string><json:string>[54]</json:string><json:string>[8,9]</json:string><json:string>[38]</json:string><json:string>Wattiaux et al.</json:string><json:string>[33,49]</json:string><json:string>[53]</json:string><json:string>[3,4]</json:string><json:string>[48]</json:string><json:string>[51,52]</json:string><json:string>Sorgi et al.</json:string><json:string>[20,26]</json:string><json:string>[41]</json:string><json:string>M. 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Read et al.</json:string><json:string>[16,55,56]</json:string><json:string>[47]</json:string><json:string>[1,2]</json:string><json:string>[33,34]</json:string><json:string>Coll et al.</json:string><json:string>[46]</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/WNG-PHJ5C7S9-J</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - medicine, research & experimental</json:string><json:string>2 - genetics & heredity</json:string><json:string>2 - biotechnology & applied microbiology</json:string></wos><scienceMetrix><json:string>1 - applied sciences</json:string><json:string>2 - enabling & strategic technologies</json:string><json:string>3 - biotechnology</json:string></scienceMetrix><scopus><json:string>1 - Health Sciences</json:string><json:string>2 - Medicine</json:string><json:string>3 - Genetics(clinical)</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Pharmacology, Toxicology and Pharmaceutics</json:string><json:string>3 - Drug Discovery</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Genetics</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Molecular Biology</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Molecular Medicine</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences medicales</json:string><json:string>4 - pharmacologie. traitements medicamenteux</json:string></inist></categories><publicationDate>2003</publicationDate><copyrightDate>2003</copyrightDate><doi><json:string>10.1002/jgm.331</json:string></doi><id>5B64D673FF4FBAD3BE0BB46200FF07964DD46B8C</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/WNG-PHJ5C7S9-J/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/WNG-PHJ5C7S9-J/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/WNG-PHJ5C7S9-J/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Vectors based on reducible polycations facilitate intracellular release of nucleic acids</title><title level="a" type="short" xml:lang="en">Intracellular Activation of RPC‐ Based Gene Therapy Vectors</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>John Wiley & Sons, Ltd.</publisher><pubPlace>Chichester, UK</pubPlace><availability><licence>Copyright © 2003 John Wiley & Sons, Ltd.</licence></availability><date type="published" when="2003-03"></date></publicationStmt><notesStmt><note type="content-type" subtype="article" source="article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-6N5SZHKN-D">article</note><note type="publication-type" subtype="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><analytic><title level="a" type="main">Vectors based on reducible polycations facilitate intracellular release of nucleic acids</title><title level="a" type="short" xml:lang="en">Intracellular Activation of RPC‐ Based Gene Therapy Vectors</title><author xml:id="author-0000" role="corresp"><persName><forename type="first">Martin L.</forename><surname>Read</surname></persName><email>mread@globalnet.co.uk</email><affiliation><orgName type="institution">Cancer Research UK Institute for Cancer Studies</orgName><orgName type="institution">University of Birmingham</orgName><address><addrLine>Birmingham B15 2TT</addrLine><addrLine>UK</addrLine><country key="GB" xml:lang="en">UNITED KINGDOM</country></address></affiliation></author><author xml:id="author-0001"><persName><forename type="first">K. Helen</forename><surname>Bremner</surname></persName><affiliation><orgName type="institution">Cancer Research UK Institute for Cancer Studies</orgName><orgName type="institution">University of Birmingham</orgName><address><addrLine>Birmingham B15 2TT</addrLine><addrLine>UK</addrLine><country key="GB" xml:lang="en">UNITED KINGDOM</country></address></affiliation></author><author xml:id="author-0002"><persName><forename type="first">David</forename><surname>Oupický</surname></persName><affiliation><orgName type="institution">Cancer Research UK Institute for Cancer Studies</orgName><orgName type="institution">University of Birmingham</orgName><address><addrLine>Birmingham B15 2TT</addrLine><addrLine>UK</addrLine><country key="GB" xml:lang="en">UNITED KINGDOM</country></address></affiliation></author><author xml:id="author-0003"><persName><forename type="first">Nicola K.</forename><surname>Green</surname></persName><affiliation><orgName type="institution">Cancer Research UK Institute for Cancer Studies</orgName><orgName type="institution">University of Birmingham</orgName><address><addrLine>Birmingham B15 2TT</addrLine><addrLine>UK</addrLine><country key="GB" xml:lang="en">UNITED KINGDOM</country></address></affiliation></author><author xml:id="author-0004"><persName><forename type="first">Peter F.</forename><surname>Searle</surname></persName><affiliation><orgName type="institution">Cancer Research UK Institute for Cancer Studies</orgName><orgName type="institution">University of Birmingham</orgName><address><addrLine>Birmingham B15 2TT</addrLine><addrLine>UK</addrLine><country key="GB" xml:lang="en">UNITED KINGDOM</country></address></affiliation></author><author xml:id="author-0005"><persName><forename type="first">Leonard W.</forename><surname>Seymour</surname></persName><affiliation><orgName type="institution">Cancer Research UK Institute for Cancer Studies</orgName><orgName type="institution">University of Birmingham</orgName><address><addrLine>Birmingham B15 2TT</addrLine><addrLine>UK</addrLine><country key="GB" xml:lang="en">UNITED KINGDOM</country></address></affiliation></author><idno type="istex">5B64D673FF4FBAD3BE0BB46200FF07964DD46B8C</idno><idno type="ark">ark:/67375/WNG-PHJ5C7S9-J</idno><idno type="DOI">10.1002/jgm.331</idno><idno type="unit">JGM331</idno><idno type="toTypesetVersion">file:JGM.JGM331.pdf</idno></analytic><monogr><title level="j" type="main">The Journal of Gene Medicine</title><title level="j" type="alt">JOURNAL OF GENE MEDICINE, THE</title><idno type="pISSN">1099-498X</idno><idno type="eISSN">1521-2254</idno><idno type="book-DOI">10.1002/(ISSN)1521-2254</idno><idno type="book-part-DOI">10.1002/jgm.v5:3</idno><idno type="product">JGM</idno><imprint><biblScope unit="vol">5</biblScope><biblScope unit="issue">3</biblScope><biblScope unit="page" from="232">232</biblScope><biblScope unit="page" to="245">245</biblScope><biblScope unit="page-count">14</biblScope><publisher>John Wiley & Sons, Ltd.</publisher><pubPlace>Chichester, UK</pubPlace><date type="published" when="2003-03"></date></imprint></monogr></biblStruct></sourceDesc></fileDesc><encodingDesc><schemaRef type="ODD" url="https://xml-schema.delivery.istex.fr/tei-istex.odd"></schemaRef><appInfo><application ident="pub2tei" version="1.0.10" when="2019-12-20"><label>pub2TEI-ISTEX</label><desc>A set of style sheets for converting XML documents encoded in various scientific publisher formats into a common TEI format.
<ref target="http://www.tei-c.org/">We use TEI</ref></desc></application></appInfo></encodingDesc><profileDesc><abstract xml:lang="en" style="main"><head>Abstract</head><head>Background</head><p>Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L‐lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids.</p><head>Methods</head><p>RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys‐Lys<hi rend="subscript">10</hi>‐Cys and used to condense nucleic acids. The stability of RPC‐based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techniques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC‐3 and B16‐F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presence of chloroquine or the cationic lipid (<hi rend="italic">N</hi>‐(1‐(2,3‐dioleoyloxy)propyl)‐<hi rend="italic">N</hi>, <hi rend="italic">N</hi>, <hi rend="italic">N</hi>‐trimethylammonium chloride) (DOTAP). A CMV‐driven plasmid expressing the nitroreductase (<hi rend="italic">ntr</hi>) gene was used to evaluate the therapeutic efficacy of RPC‐based delivery vectors.</p><head>Results</head><p>A 187‐fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non‐reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 ± 7.9%), LNCaP (55.2 ± 6.7%) and PC‐3 (66.1 ± 3.7%) cells. Transfection with the <hi rend="italic">ntr</hi> gene and treatment with the prodrug CB1954 resulted in significant cell killing, achieving IC<hi rend="subscript">50</hi> values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20–75% of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (<5% of cells).</p><head>Conclusions</head><p>These results demonstrate that lipid‐mediated activation of RPC‐based polyplexes is a useful strategy to enhance intracellular delivery of nucleic acids and potentiate therapeutic activity. Copyright © 2002 John Wiley & Sons, Ltd.</p></abstract><textClass><keywords xml:lang="en"><term xml:id="kwd1">cancer</term><term xml:id="kwd2">DNA</term><term xml:id="kwd3">gene therapy</term><term xml:id="kwd4">lipid</term><term xml:id="kwd5">nitroreductase</term><term xml:id="kwd6">poly(L‐lysine)</term></keywords><keywords rend="articleCategory"><term>Research Article</term></keywords><keywords rend="tocHeading1"><term>Research Articles</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2019-12-20" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/WNG-PHJ5C7S9-J/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Wiley, elements deleted: body"><istex:xmlDeclaration>version="1.0" encoding="UTF-8" standalone="yes"</istex:xmlDeclaration><istex:document><component version="2.0" type="serialArticle" xml:lang="en"><header><publicationMeta level="product"><publisherInfo><publisherName>John Wiley & Sons, Ltd.</publisherName><publisherLoc>Chichester, UK</publisherLoc></publisherInfo><doi registered="yes">10.1002/(ISSN)1521-2254</doi><issn type="print">1099-498X</issn><issn type="electronic">1521-2254</issn><idGroup><id type="product" value="JGM"></id></idGroup><titleGroup><title type="main" xml:lang="en" sort="JOURNAL OF GENE MEDICINE, THE">The Journal of Gene Medicine</title><title type="subtitle">A cross‐disciplinary journal for research on the science of gene transfer and its clinical applications</title><title type="short">J. Gene Med.</title></titleGroup></publicationMeta><publicationMeta level="part" position="30"><doi origin="wiley" registered="yes">10.1002/jgm.v5:3</doi><numberingGroup><numbering type="journalVolume" number="5">5</numbering><numbering type="journalIssue">3</numbering></numberingGroup><coverDate startDate="2003-03">March 2003</coverDate></publicationMeta><publicationMeta level="unit" type="article" position="8" status="forIssue"><doi origin="wiley" registered="yes">10.1002/jgm.331</doi><idGroup><id type="unit" value="JGM331"></id></idGroup><countGroup><count type="pageTotal" number="14"></count></countGroup><titleGroup><title type="articleCategory">Research Article</title><title type="tocHeading1">Research Articles</title></titleGroup><copyright ownership="publisher">Copyright © 2003 John Wiley & Sons, Ltd.</copyright><eventGroup><event type="manuscriptReceived" date="2002-05-21"></event><event type="manuscriptRevised" date="2002-08-05"></event><event type="manuscriptAccepted" date="2002-08-08"></event><event type="publishedOnlineEarlyUnpaginated" date="2003-01-06"></event><event type="firstOnline" date="2003-01-06"></event><event type="publishedOnlineFinalForm" date="2003-03-20"></event><event type="xmlConverted" agent="Converter:JWSART34_TO_WML3G version:2.3.2 mode:FullText source:FullText result:FullText" date="2010-03-04"></event><event type="xmlConverted" agent="Converter:WILEY_ML3G_TO_WILEY_ML3GV2 version:3.8.8" date="2014-01-30"></event><event type="xmlConverted" agent="Converter:WML3G_To_WML3G version:4.1.7 mode:FullText,remove_FC" date="2014-11-04"></event></eventGroup><numberingGroup><numbering type="pageFirst">232</numbering><numbering type="pageLast">245</numbering></numberingGroup><correspondenceTo>Department of Clinical Pharmacology, University of Oxford, Oxford OX2 6HE, UK.</correspondenceTo><linkGroup><link type="toTypesetVersion" href="file:JGM.JGM331.pdf"></link></linkGroup></publicationMeta><contentMeta><countGroup><count type="figureTotal" number="10"></count><count type="tableTotal" number="1"></count><count type="referenceTotal" number="56"></count></countGroup><titleGroup><title type="main" xml:lang="en">Vectors based on reducible polycations facilitate intracellular release of nucleic acids</title><title type="short" xml:lang="en">Intracellular Activation of RPC‐ Based Gene Therapy Vectors</title></titleGroup><creators><creator xml:id="au1" creatorRole="author" affiliationRef="#af1" corresponding="yes"><personName><givenNames>Martin L.</givenNames><familyName>Read</familyName></personName><contactDetails><email>mread@globalnet.co.uk</email></contactDetails></creator><creator xml:id="au2" creatorRole="author" affiliationRef="#af1"><personName><givenNames>K. Helen</givenNames><familyName>Bremner</familyName></personName></creator><creator xml:id="au3" creatorRole="author" affiliationRef="#af1"><personName><givenNames>David</givenNames><familyName>Oupický</familyName></personName></creator><creator xml:id="au4" creatorRole="author" affiliationRef="#af1"><personName><givenNames>Nicola K.</givenNames><familyName>Green</familyName></personName></creator><creator xml:id="au5" creatorRole="author" affiliationRef="#af1"><personName><givenNames>Peter F.</givenNames><familyName>Searle</familyName></personName></creator><creator xml:id="au6" creatorRole="author" affiliationRef="#af1"><personName><givenNames>Leonard W.</givenNames><familyName>Seymour</familyName></personName></creator></creators><affiliationGroup><affiliation xml:id="af1" countryCode="GB" type="organization"><unparsedAffiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</unparsedAffiliation></affiliation></affiliationGroup><keywordGroup xml:lang="en" type="author"><keyword xml:id="kwd1">cancer</keyword><keyword xml:id="kwd2">DNA</keyword><keyword xml:id="kwd3">gene therapy</keyword><keyword xml:id="kwd4">lipid</keyword><keyword xml:id="kwd5">nitroreductase</keyword><keyword xml:id="kwd6">poly(L‐lysine)</keyword></keywordGroup><abstractGroup><abstract type="main" xml:lang="en"><title type="main">Abstract</title><section xml:id="abs1-1"><title type="main">Background</title><p>Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L‐lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids.</p></section><section xml:id="abs1-2"><title type="main">Methods</title><p>RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys‐Lys<sub>10</sub>‐Cys and used to condense nucleic acids. The stability of RPC‐based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techniques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC‐3 and B16‐F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presence of chloroquine or the cationic lipid (<i>N</i>‐(1‐(2,3‐dioleoyloxy)propyl)‐<i>N</i>, <i>N</i>, <i>N</i>‐trimethylammonium chloride) (DOTAP). A CMV‐driven plasmid expressing the nitroreductase (<i>ntr</i>) gene was used to evaluate the therapeutic efficacy of RPC‐based delivery vectors.</p></section><section xml:id="abs1-3"><title type="main">Results</title><p>A 187‐fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non‐reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 ± 7.9%), LNCaP (55.2 ± 6.7%) and PC‐3 (66.1 ± 3.7%) cells. Transfection with the <i>ntr</i> gene and treatment with the prodrug CB1954 resulted in significant cell killing, achieving IC<sub>50</sub> values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20–75% of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (<5% of cells).</p></section><section xml:id="abs1-4"><title type="main">Conclusions</title><p>These results demonstrate that lipid‐mediated activation of RPC‐based polyplexes is a useful strategy to enhance intracellular delivery of nucleic acids and potentiate therapeutic activity. Copyright © 2002 John Wiley & Sons, Ltd.</p></section></abstract></abstractGroup></contentMeta></header></component></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Vectors based on reducible polycations facilitate intracellular release of nucleic acids</title></titleInfo><titleInfo type="abbreviated" lang="en"><title>Intracellular Activation of RPC‐ Based Gene Therapy Vectors</title></titleInfo><titleInfo type="alternative" contentType="CDATA" lang="en"><title>Vectors based on reducible polycations facilitate intracellular release of nucleic acids</title></titleInfo><name type="personal"><namePart type="given">Martin L.</namePart><namePart type="family">Read</namePart><affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</affiliation><affiliation>E-mail: mread@globalnet.co.uk</affiliation><affiliation>Correspondence address: Department of Clinical Pharmacology, University of Oxford, Oxford OX2 6HE, UK.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">K. Helen</namePart><namePart type="family">Bremner</namePart><affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">David</namePart><namePart type="family">Oupický</namePart><affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Nicola K.</namePart><namePart type="family">Green</namePart><affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Peter F.</namePart><namePart type="family">Searle</namePart><affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Leonard W.</namePart><namePart type="family">Seymour</namePart><affiliation>Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, UK</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="article" displayLabel="article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-6N5SZHKN-D">article</genre><originInfo><publisher>John Wiley & Sons, Ltd.</publisher><place><placeTerm type="text">Chichester, UK</placeTerm></place><dateIssued encoding="w3cdtf">2003-03</dateIssued><dateCaptured encoding="w3cdtf">2002-05-21</dateCaptured><dateValid encoding="w3cdtf">2002-08-08</dateValid><copyrightDate encoding="w3cdtf">2003</copyrightDate></originInfo><language><languageTerm type="code" authority="rfc3066">en</languageTerm><languageTerm type="code" authority="iso639-2b">eng</languageTerm></language><physicalDescription><extent unit="figures">10</extent><extent unit="tables">1</extent><extent unit="references">56</extent></physicalDescription><abstract lang="en">Background: Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L‐lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids. Methods: RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys‐Lys10‐Cys and used to condense nucleic acids. The stability of RPC‐based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techniques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC‐3 and B16‐F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presence of chloroquine or the cationic lipid (N‐(1‐(2,3‐dioleoyloxy)propyl)‐N, N, N‐trimethylammonium chloride) (DOTAP). A CMV‐driven plasmid expressing the nitroreductase (ntr) gene was used to evaluate the therapeutic efficacy of RPC‐based delivery vectors. Results: A 187‐fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non‐reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 ± 7.9%), LNCaP (55.2 ± 6.7%) and PC‐3 (66.1 ± 3.7%) cells. Transfection with the ntr gene and treatment with the prodrug CB1954 resulted in significant cell killing, achieving IC50 values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20–75% of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (<5% of cells). Conclusions: These results demonstrate that lipid‐mediated activation of RPC‐based polyplexes is a useful strategy to enhance intracellular delivery of nucleic acids and potentiate therapeutic activity. Copyright © 2002 John Wiley & Sons, Ltd.</abstract><subject lang="en"><genre>keywords</genre><topic>cancer</topic><topic>DNA</topic><topic>gene therapy</topic><topic>lipid</topic><topic>nitroreductase</topic><topic>poly(L‐lysine)</topic></subject><relatedItem type="host"><titleInfo><title>The Journal of Gene Medicine</title><subTitle>A cross‐disciplinary journal for research on the science of gene transfer and its clinical applications</subTitle></titleInfo><titleInfo type="abbreviated"><title>J. Gene Med.</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><subject><genre>article-category</genre><topic>Research Article</topic><topic>Research Articles</topic></subject><identifier type="ISSN">1099-498X</identifier><identifier type="eISSN">1521-2254</identifier><identifier type="DOI">10.1002/(ISSN)1521-2254</identifier><identifier type="PublisherID">JGM</identifier><part><date>2003</date><detail type="volume"><caption>vol.</caption><number>5</number></detail><detail type="issue"><caption>no.</caption><number>3</number></detail><extent unit="pages"><start>232</start><end>245</end><total>14</total></extent></part></relatedItem><relatedItem type="references" displayLabel="cit1"><titleInfo><title>Antisense strategies and non‐viral gene therapy for cancer</title></titleInfo><name type="personal"><namePart type="given">ML</namePart><namePart type="family">Read</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">KH</namePart><namePart type="family">Bremner</namePart><role><roleTerm type="text">author</roleTerm></role></name><genre>journal-article</genre><note type="citation/reference">Read ML, Bremner KH. 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