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 antibiotic-free selection with highly productive fermentation manufacturing (>1 gm/L plasmid DNA yields), while improving in vivo expression of encoded proteins and increasing immune responses to target antigens. These vectors are safer, more potent, alternatives for DNA therapy or vaccination. Keywords DNA vaccine; plasmid; antibiotic-free 1) IntroductionPlasmid based DNA vaccines and therapeutics are in development for a variety of human, animal, bird and fish applications. Antibiotic resistance markers, typically kanamycin resistance (kanR), allow selective retention of plasmid DNA during bacterial fermentation and are the most commonly utilized selectable markers. The presence of an antibiotic resistance gene in the plasmid backbone is considered undesirable by regulatory agencies, due to: 1) the potential transfer of antibiotic resistance to endogenous microbial fauna; and 2) the potential activation and transcription of the genes from mammalian promoters after cellular incorporation into the genome [Reviewed in 1,2 ]. For example, a regulatory guidance with regard to DNA vaccine plasmids states: "The use of certain selection markers, such as resistance to antibiotics, which may adversely impact on other clinical therapies in the target population, should be avoided" [ 3 ]. Further, the use of antibiotics in fermentation culture requires expensive process validation of antibiotic removal during plasmid purification, to prevent contamination of the final product with residual antibiotics. Ideally, the plasmid would not contain any protein coding regions other than the gene of interest, since these could *Corresponding Author James A Williams, Nature Technology Corporation., 4701 Innovation Drive Lincoln Nebraska, 68521, Telephone: (402) ., jim@natx.com. Conflict of Interest Statement JL, AEC, CPH and JAW have an equity interest in Nature Technology CorporationPublisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptVaccine. Author manuscript; available in PMC 2010 October 30. potentially be expressed in mammalian cells. Alternative select...

Antibiotic-Free Selection for Bio-Production: Moving Towards a New "Gold Standard"

“…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.…”

“…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). …”

“…The antibiotic-free selection is based on RNA selectable marker described by Luke et al, 2009 and the autolytic plasmid DNA extraction method uses integrated bacteriophage endolysin gene (λR), encoding a peptidoglycan hydrolase (lysozyme) enzyme to permeabilize the bacterial cell wall, and to selectively extract the plasmid DNA from the cells in an acetate buffer. Authors found that their autolytic strain allowed efficient plasmid DNA recovery, similar to alkaline lysis plasmid DNA purification.…”

Designing Lentiviral Gene Vectors

“…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.…”

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

“…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 .…”