Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system

Area of Science:

• Molecular Biology
• Biotechnology
• Vaccinology

Background:

• Antibiotic resistance markers in plasmid DNA vectors pose safety concerns.
• Regulatory agencies advocate for their removal from therapeutic and vaccine vectors.
• Current selection systems often rely on antibiotics, complicating manufacturing and safety.

Purpose of the Study:

• To develop and apply a novel antibiotic-free selection system for plasmid DNA vectors.
• To create safer and more potent DNA vaccines and therapeutics.
• To improve manufacturing yields and in vivo performance.

Main Methods:

• Development of vectors incorporating RNA-OUT antisense RNA.
• Repression of a chromosomally integrated counter-selectable marker (sacB) using RNA-OUT.
• Plasmid selection on sucrose instead of antibiotics.
• Evaluation of DNA vaccine performance in terms of yield, expression, and immune response.

Main Results:

• Successful development and application of an antibiotic-free selection system.
• Achieved highly productive fermentation yielding >1g/L plasmid DNA.
• Demonstrated improved in vivo expression of encoded proteins.
• Showcased increased immune responses to target antigens.

Conclusions:

• The novel antibiotic-free selection system offers a safer alternative for DNA therapy and vaccination.
• Sucrose-selectable DNA vaccine vectors combine safety with high manufacturing yields.
• These vectors provide enhanced potency for therapeutic and vaccine applications.