Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons
Identifieur interne : 001668 ( Main/Exploration ); précédent : 001667; suivant : 001669Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons
Auteurs : Paul J. Farrow ; Lee B. Barrett [États-Unis] ; Mark Stevenson ; Kerry D. Fisher ; Jonathan Finn [Canada] ; Rachel Spice [Royaume-Uni] ; Michael A. Allan [Royaume-Uni] ; Martin Berry [Royaume-Uni] ; Ann Logan [Royaume-Uni] ; Leonard W. Seymour ; Martin L. Read [Royaume-Uni]Source :
- Nucleic Acids Research [ 0305-1048 ] ; 2006.
Abstract
Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague–Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection.
Url:
DOI: 10.1093/nar/gkl442
Affiliations:
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Acids Res.</title><idno type="ISSN">0305-1048</idno><idno type="eISSN">1362-4962</idno><imprint><publisher>Oxford University Press</publisher><date type="published" when="2006">2006</date><biblScope unit="volume">34</biblScope><biblScope unit="issue">11</biblScope><biblScope unit="page" from="80">e80</biblScope><biblScope unit="page" to="80">e80</biblScope></imprint><idno type="ISSN">0305-1048</idno></series><idno type="istex">88BF51A4E996E20C7395DE27F2DB19BDB4AE88D1</idno><idno type="DOI">10.1093/nar/gkl442</idno><idno type="local">gkl442</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0305-1048</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague–Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection.</div></front></TEI><affiliations><list><country><li>Canada</li><li>Royaume-Uni</li><li>États-Unis</li></country><region><li>Massachusetts</li></region></list><tree><noCountry><name sortKey="Farrow, Paul J" sort="Farrow, Paul J" uniqKey="Farrow P" first="Paul J." last="Farrow">Paul J. Farrow</name><name sortKey="Fisher, Kerry D" sort="Fisher, Kerry D" uniqKey="Fisher K" first="Kerry D." last="Fisher">Kerry D. Fisher</name><name sortKey="Seymour, Leonard W" sort="Seymour, Leonard W" uniqKey="Seymour L" first="Leonard W." last="Seymour">Leonard W. Seymour</name><name sortKey="Stevenson, Mark" sort="Stevenson, Mark" uniqKey="Stevenson M" first="Mark" last="Stevenson">Mark Stevenson</name></noCountry><country name="États-Unis"><region name="Massachusetts"><name sortKey="Barrett, Lee B" sort="Barrett, Lee B" uniqKey="Barrett L" first="Lee B." last="Barrett">Lee B. Barrett</name></region></country><country name="Canada"><noRegion><name sortKey="Finn, Jonathan" sort="Finn, Jonathan" uniqKey="Finn J" first="Jonathan" last="Finn">Jonathan Finn</name></noRegion></country><country name="Royaume-Uni"><noRegion><name sortKey="Spice, Rachel" sort="Spice, Rachel" uniqKey="Spice R" first="Rachel" last="Spice">Rachel Spice</name></noRegion><name sortKey="Allan, Michael A" sort="Allan, Michael A" uniqKey="Allan M" first="Michael A." last="Allan">Michael A. Allan</name><name sortKey="Berry, Martin" sort="Berry, Martin" uniqKey="Berry M" first="Martin" last="Berry">Martin Berry</name><name sortKey="Logan, Ann" sort="Logan, Ann" uniqKey="Logan A" first="Ann" last="Logan">Ann Logan</name><name sortKey="Read, Martin L" sort="Read, Martin L" uniqKey="Read M" first="Martin L." last="Read">Martin L. Read</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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