Ligand substitution of receptor targeted DNA complexes affects gene transfer into hepatoma cells
We have targeted the serpin enzyme complex receptor for gene transfer in human hepatoma cell lines using peptides < 30 amino acids in length which contain the five amino acid recognition sequence for this receptor, coupled to poly K of average chain length 100 K, using the heterobifunctional coup...
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Veröffentlicht in: | Gene therapy 1998-12, Vol.5 (12), p.1685-1697 |
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description | We have targeted the serpin enzyme complex receptor for gene transfer in human hepatoma cell lines using peptides < 30 amino acids in length which contain the five amino acid recognition sequence for this receptor, coupled to poly K of average chain length 100 K, using the heterobifunctional coupling reagent sulfo-LC SPDP. The number of sulfo-LC SPDP modified poly-L-lysine residues, as well as the degree of peptide substitution was assessed by nuclear magnetic resonance spectroscopy. Conjugates were prepared in which 3.5%, 7.8% or 26% of the lysine residues contained the sulfo-LC SPDP moiety. Each of these conjugates was then coupled with ligand peptides so that one in 370, one in 1039, or one in 5882 lysines were substituted with receptor ligand. Electron microscopy and atomic force microscopy were used to assess complex structure and size. HuH7 human hepatoma cells were transfected with complexes of these conjugates with the plasmid pGL3 and luciferase expression measured 2 to 16 days after treatment. All the protein conjugates in which 26% of the K residues were modified with sulfo-LC SPDP were poor gene transfer reagents. Complexes containing less substituted poly K, averaged 17 +/- 0.5 nm in diameter and gave peak transgene expression of 3-4 x 10(6) ILU/mg which persisted (> 7 x 10(5) ILU) at 16 days. Of these, more substituted polymers condensed DNA into complexes averaging 20 +/- 0.7 nm in diameter and gave five-fold less luciferase than complexes containing less substituted conjugates. As few as eight to 11 ligands per complex are optimal for DNA delivery via the SEC receptor. The extent of substitution of receptor-mediated gene transfer complexes affects the size of the complexes, as well as the intensity and duration of transgene expression. These observations may permit tailoring of complex construction for the usage required. |
doi_str_mv | 10.1038/sj.gt.3300777 |
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V ; PERLMUTTER, D. H ; DAVIS, P. B</creator><creatorcontrib>ZIADY, A.-G ; FERKOL, T ; GERKEN, T ; DAWSON, D. V ; PERLMUTTER, D. H ; DAVIS, P. B</creatorcontrib><description>We have targeted the serpin enzyme complex receptor for gene transfer in human hepatoma cell lines using peptides < 30 amino acids in length which contain the five amino acid recognition sequence for this receptor, coupled to poly K of average chain length 100 K, using the heterobifunctional coupling reagent sulfo-LC SPDP. The number of sulfo-LC SPDP modified poly-L-lysine residues, as well as the degree of peptide substitution was assessed by nuclear magnetic resonance spectroscopy. Conjugates were prepared in which 3.5%, 7.8% or 26% of the lysine residues contained the sulfo-LC SPDP moiety. Each of these conjugates was then coupled with ligand peptides so that one in 370, one in 1039, or one in 5882 lysines were substituted with receptor ligand. Electron microscopy and atomic force microscopy were used to assess complex structure and size. HuH7 human hepatoma cells were transfected with complexes of these conjugates with the plasmid pGL3 and luciferase expression measured 2 to 16 days after treatment. All the protein conjugates in which 26% of the K residues were modified with sulfo-LC SPDP were poor gene transfer reagents. Complexes containing less substituted poly K, averaged 17 +/- 0.5 nm in diameter and gave peak transgene expression of 3-4 x 10(6) ILU/mg which persisted (> 7 x 10(5) ILU) at 16 days. Of these, more substituted polymers condensed DNA into complexes averaging 20 +/- 0.7 nm in diameter and gave five-fold less luciferase than complexes containing less substituted conjugates. As few as eight to 11 ligands per complex are optimal for DNA delivery via the SEC receptor. The extent of substitution of receptor-mediated gene transfer complexes affects the size of the complexes, as well as the intensity and duration of transgene expression. These observations may permit tailoring of complex construction for the usage required.</description><identifier>ISSN: 0969-7128</identifier><identifier>EISSN: 1476-5462</identifier><identifier>DOI: 10.1038/sj.gt.3300777</identifier><identifier>PMID: 10023448</identifier><language>eng</language><publisher>Basingstoke: Nature Publishing Group</publisher><subject>Amino acid sequence ; Animals ; Atomic force microscopy ; Biological and medical sciences ; Biotechnology ; Cell lines ; Cell size ; Deoxyribonucleic acid ; DNA ; Electron microscopy ; Fundamental and applied biological sciences. Psychology ; Gene Expression ; Gene therapy ; Gene transfer ; Genetic Therapy - methods ; Genetic Vectors - chemistry ; Genetic Vectors - metabolism ; Health. Pharmaceutical industry ; Hepatoma ; Humans ; Industrial applications and implications. Economical aspects ; Ligands ; Liver cancer ; Liver Neoplasms, Experimental - therapy ; Luciferases - genetics ; Lysine ; Magnetic resonance spectroscopy ; Microscopy ; Microscopy, Atomic Force ; Microscopy, Electron ; NMR ; Nuclear magnetic resonance ; Nuclear Magnetic Resonance, Biomolecular ; Peptides ; Poly-L-lysine ; Polylysine ; Receptors, Cell Surface - genetics ; Structure-Activity Relationship ; Time Factors ; Transfection - methods</subject><ispartof>Gene therapy, 1998-12, Vol.5 (12), p.1685-1697</ispartof><rights>1999 INIST-CNRS</rights><rights>Macmillan Publishers Limited 1998.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c377t-84aff546d5c37212dbad4449305bf2826e27e9844f8219b7bf0483062c2f23cf3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1640322$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10023448$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>ZIADY, A.-G</creatorcontrib><creatorcontrib>FERKOL, T</creatorcontrib><creatorcontrib>GERKEN, T</creatorcontrib><creatorcontrib>DAWSON, D. V</creatorcontrib><creatorcontrib>PERLMUTTER, D. H</creatorcontrib><creatorcontrib>DAVIS, P. B</creatorcontrib><title>Ligand substitution of receptor targeted DNA complexes affects gene transfer into hepatoma cells</title><title>Gene therapy</title><addtitle>Gene Ther</addtitle><description>We have targeted the serpin enzyme complex receptor for gene transfer in human hepatoma cell lines using peptides < 30 amino acids in length which contain the five amino acid recognition sequence for this receptor, coupled to poly K of average chain length 100 K, using the heterobifunctional coupling reagent sulfo-LC SPDP. The number of sulfo-LC SPDP modified poly-L-lysine residues, as well as the degree of peptide substitution was assessed by nuclear magnetic resonance spectroscopy. Conjugates were prepared in which 3.5%, 7.8% or 26% of the lysine residues contained the sulfo-LC SPDP moiety. Each of these conjugates was then coupled with ligand peptides so that one in 370, one in 1039, or one in 5882 lysines were substituted with receptor ligand. Electron microscopy and atomic force microscopy were used to assess complex structure and size. HuH7 human hepatoma cells were transfected with complexes of these conjugates with the plasmid pGL3 and luciferase expression measured 2 to 16 days after treatment. All the protein conjugates in which 26% of the K residues were modified with sulfo-LC SPDP were poor gene transfer reagents. Complexes containing less substituted poly K, averaged 17 +/- 0.5 nm in diameter and gave peak transgene expression of 3-4 x 10(6) ILU/mg which persisted (> 7 x 10(5) ILU) at 16 days. Of these, more substituted polymers condensed DNA into complexes averaging 20 +/- 0.7 nm in diameter and gave five-fold less luciferase than complexes containing less substituted conjugates. As few as eight to 11 ligands per complex are optimal for DNA delivery via the SEC receptor. The extent of substitution of receptor-mediated gene transfer complexes affects the size of the complexes, as well as the intensity and duration of transgene expression. These observations may permit tailoring of complex construction for the usage required.</description><subject>Amino acid sequence</subject><subject>Animals</subject><subject>Atomic force microscopy</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Cell lines</subject><subject>Cell size</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>Electron microscopy</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression</subject><subject>Gene therapy</subject><subject>Gene transfer</subject><subject>Genetic Therapy - methods</subject><subject>Genetic Vectors - chemistry</subject><subject>Genetic Vectors - metabolism</subject><subject>Health. Pharmaceutical industry</subject><subject>Hepatoma</subject><subject>Humans</subject><subject>Industrial applications and implications. Economical aspects</subject><subject>Ligands</subject><subject>Liver cancer</subject><subject>Liver Neoplasms, Experimental - therapy</subject><subject>Luciferases - genetics</subject><subject>Lysine</subject><subject>Magnetic resonance spectroscopy</subject><subject>Microscopy</subject><subject>Microscopy, Atomic Force</subject><subject>Microscopy, Electron</subject><subject>NMR</subject><subject>Nuclear magnetic resonance</subject><subject>Nuclear Magnetic Resonance, Biomolecular</subject><subject>Peptides</subject><subject>Poly-L-lysine</subject><subject>Polylysine</subject><subject>Receptors, Cell Surface - genetics</subject><subject>Structure-Activity Relationship</subject><subject>Time Factors</subject><subject>Transfection - methods</subject><issn>0969-7128</issn><issn>1476-5462</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1998</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0U2L1TAUBuAginMdXbqVgOKu13w1SZfDjF9wmdnouqbpSe2lbWpOCs6_nwz3guLGVTjw8HJyXkJec7bnTNoPeNwPeS8lY8aYJ2THldFVrbR4Snas0U1luLAX5AXikTGmjBXPyQVnTEil7I78OIyDW3qKW4d5zFse40JjoAk8rDkmml0aIENPb26vqI_zOsFvQOpCAJ-RDrAAzcktGCDRccmR_oTV5Tg76mGa8CV5FtyE8Or8XpLvnz5-u_5SHe4-f72-OlReGpMrq0piWbuvyyy46DvXK6UayeouCCs0CAONVSpYwZvOdIEpK5kWXgQhfZCX5P0pd03x1waY23nExw3cAnHDVje8XILV_4WF8do2TYFv_4HHuKWlfKIVWinNjVKiqOqkfIqICUK7pnF26b7lrH1sqMVjO-T23FDxb86pWzdD_5c-VVLAuzNw6N0Uym39iH-cVkwKIR8ADBGYoA</recordid><startdate>19981201</startdate><enddate>19981201</enddate><creator>ZIADY, A.-G</creator><creator>FERKOL, T</creator><creator>GERKEN, T</creator><creator>DAWSON, D. V</creator><creator>PERLMUTTER, D. H</creator><creator>DAVIS, P. B</creator><general>Nature Publishing Group</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QP</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>P64</scope><scope>RC3</scope><scope>7QO</scope><scope>7X8</scope></search><sort><creationdate>19981201</creationdate><title>Ligand substitution of receptor targeted DNA complexes affects gene transfer into hepatoma cells</title><author>ZIADY, A.-G ; FERKOL, T ; GERKEN, T ; DAWSON, D. V ; PERLMUTTER, D. H ; DAVIS, P. B</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c377t-84aff546d5c37212dbad4449305bf2826e27e9844f8219b7bf0483062c2f23cf3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1998</creationdate><topic>Amino acid sequence</topic><topic>Animals</topic><topic>Atomic force microscopy</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Cell lines</topic><topic>Cell size</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>Electron microscopy</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Expression</topic><topic>Gene therapy</topic><topic>Gene transfer</topic><topic>Genetic Therapy - methods</topic><topic>Genetic Vectors - chemistry</topic><topic>Genetic Vectors - metabolism</topic><topic>Health. Pharmaceutical industry</topic><topic>Hepatoma</topic><topic>Humans</topic><topic>Industrial applications and implications. Economical aspects</topic><topic>Ligands</topic><topic>Liver cancer</topic><topic>Liver Neoplasms, Experimental - therapy</topic><topic>Luciferases - genetics</topic><topic>Lysine</topic><topic>Magnetic resonance spectroscopy</topic><topic>Microscopy</topic><topic>Microscopy, Atomic Force</topic><topic>Microscopy, Electron</topic><topic>NMR</topic><topic>Nuclear magnetic resonance</topic><topic>Nuclear Magnetic Resonance, Biomolecular</topic><topic>Peptides</topic><topic>Poly-L-lysine</topic><topic>Polylysine</topic><topic>Receptors, Cell Surface - genetics</topic><topic>Structure-Activity Relationship</topic><topic>Time Factors</topic><topic>Transfection - methods</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>ZIADY, A.-G</creatorcontrib><creatorcontrib>FERKOL, T</creatorcontrib><creatorcontrib>GERKEN, T</creatorcontrib><creatorcontrib>DAWSON, D. V</creatorcontrib><creatorcontrib>PERLMUTTER, D. H</creatorcontrib><creatorcontrib>DAVIS, P. B</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Gene therapy</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>ZIADY, A.-G</au><au>FERKOL, T</au><au>GERKEN, T</au><au>DAWSON, D. V</au><au>PERLMUTTER, D. H</au><au>DAVIS, P. B</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Ligand substitution of receptor targeted DNA complexes affects gene transfer into hepatoma cells</atitle><jtitle>Gene therapy</jtitle><addtitle>Gene Ther</addtitle><date>1998-12-01</date><risdate>1998</risdate><volume>5</volume><issue>12</issue><spage>1685</spage><epage>1697</epage><pages>1685-1697</pages><issn>0969-7128</issn><eissn>1476-5462</eissn><abstract>We have targeted the serpin enzyme complex receptor for gene transfer in human hepatoma cell lines using peptides < 30 amino acids in length which contain the five amino acid recognition sequence for this receptor, coupled to poly K of average chain length 100 K, using the heterobifunctional coupling reagent sulfo-LC SPDP. The number of sulfo-LC SPDP modified poly-L-lysine residues, as well as the degree of peptide substitution was assessed by nuclear magnetic resonance spectroscopy. Conjugates were prepared in which 3.5%, 7.8% or 26% of the lysine residues contained the sulfo-LC SPDP moiety. Each of these conjugates was then coupled with ligand peptides so that one in 370, one in 1039, or one in 5882 lysines were substituted with receptor ligand. Electron microscopy and atomic force microscopy were used to assess complex structure and size. HuH7 human hepatoma cells were transfected with complexes of these conjugates with the plasmid pGL3 and luciferase expression measured 2 to 16 days after treatment. All the protein conjugates in which 26% of the K residues were modified with sulfo-LC SPDP were poor gene transfer reagents. Complexes containing less substituted poly K, averaged 17 +/- 0.5 nm in diameter and gave peak transgene expression of 3-4 x 10(6) ILU/mg which persisted (> 7 x 10(5) ILU) at 16 days. Of these, more substituted polymers condensed DNA into complexes averaging 20 +/- 0.7 nm in diameter and gave five-fold less luciferase than complexes containing less substituted conjugates. As few as eight to 11 ligands per complex are optimal for DNA delivery via the SEC receptor. The extent of substitution of receptor-mediated gene transfer complexes affects the size of the complexes, as well as the intensity and duration of transgene expression. These observations may permit tailoring of complex construction for the usage required.</abstract><cop>Basingstoke</cop><pub>Nature Publishing Group</pub><pmid>10023448</pmid><doi>10.1038/sj.gt.3300777</doi><tpages>13</tpages></addata></record> |
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subjects | Amino acid sequence Animals Atomic force microscopy Biological and medical sciences Biotechnology Cell lines Cell size Deoxyribonucleic acid DNA Electron microscopy Fundamental and applied biological sciences. Psychology Gene Expression Gene therapy Gene transfer Genetic Therapy - methods Genetic Vectors - chemistry Genetic Vectors - metabolism Health. Pharmaceutical industry Hepatoma Humans Industrial applications and implications. Economical aspects Ligands Liver cancer Liver Neoplasms, Experimental - therapy Luciferases - genetics Lysine Magnetic resonance spectroscopy Microscopy Microscopy, Atomic Force Microscopy, Electron NMR Nuclear magnetic resonance Nuclear Magnetic Resonance, Biomolecular Peptides Poly-L-lysine Polylysine Receptors, Cell Surface - genetics Structure-Activity Relationship Time Factors Transfection - methods |
title | Ligand substitution of receptor targeted DNA complexes affects gene transfer into hepatoma cells |
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