2009
DOI: 10.1016/j.vaccine.2009.06.017
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Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system

Abstract: To ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors. Here, we describe the development and application of a novel antibiotic-free selection system. Vectors incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a chromosomally integrated constitutively expressed counter-selectable marker (sacB), allowing plasmid selection on sucrose. Sucrose selectable DNA vaccine vectors combine antibioti… Show more

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Cited by 98 publications
(99 citation statements)
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“…In a recent paper, RNA based selectable marker, not restricted to ColE1 containing vectors is described (Luke et al, 2009). Briefly, a counter-selectable marker (sacB) levansucrase from Bacillus subtilis, under control of the RNA-IN promoter is integrated into the bacterial chromosome induces cell death in presence of sucrose.…”
Section: Rna-based Antibiotic-free Selection Systemsmentioning
confidence: 99%
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“…In a recent paper, RNA based selectable marker, not restricted to ColE1 containing vectors is described (Luke et al, 2009). Briefly, a counter-selectable marker (sacB) levansucrase from Bacillus subtilis, under control of the RNA-IN promoter is integrated into the bacterial chromosome induces cell death in presence of sucrose.…”
Section: Rna-based Antibiotic-free Selection Systemsmentioning
confidence: 99%
“…Among systems described in paragraph 5, some have been developed especially for DNA vaccine production such as pCOR (Soubrier et al, 1989), RNA/RNA interference (Pfaffenzeller et al, 2006) and RNA out (Luke et al, 2009). …”
Section: Dna Immunization and Gene Therapymentioning
confidence: 99%
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“…Large scale preparations of plasmid DNA with antibiotic resistance genes might lead to the undesirable escape of these genes into environment. Thus, genetic systems, which rely on a short RNA-expressing gene as a plasmid selection marker, can be contrived (Luke, Carnes et al 2009). Regrettably, it is relatively common for lentiviral vector backbone plasmids to suffer from structural and maintenance instability in bacteria.…”
Section: Bacterial Plasmid Segmentmentioning
confidence: 99%
“…To further optimize our manufacturing methods we collaborated with Nature Technology who generated minimalized, antibiotic-free (FDA-compliant) plasmids for CTL stimulation 13,14 . Using this strategy we consistently achieve nucleofection efficiencies of >35% while maintaining high cell viability (data not shown) 3 .…”
Section: Representative Resultsmentioning
confidence: 99%