PCR-generated DNA templates enable efficient, rapid, and cost-effective mRNA synthesis

Area of Science:

• Molecular Biology
• Biotechnology
• Vaccinology

Background:

• In vitro transcription (IVT) is crucial for mRNA synthesis, vaccine, and therapy development.
• IVT efficiency relies heavily on the quality of linear DNA templates.
• Conventional plasmid-based DNA template preparation is time-consuming and expensive.

Purpose of the Study:

• To develop and evaluate a cell-free, PCR-based method for generating linear DNA templates for IVT.
• To compare the PCR-based method with the conventional plasmid-based method for mRNA synthesis and vaccine efficacy.

Main Methods:

• A novel cell-free, PCR-based DNA template generation method was developed.
• IVT efficiency, mRNA yield, and quality were assessed for both PCR- and plasmid-derived templates.
• Immunogenicity of mRNA-lipid nanoparticle (LNP) vaccines produced using both template types was evaluated in mice.

Main Results:

• The PCR-based method produced higher yields of DNA templates and transcribed mRNA compared to the plasmid-based method.
• mRNA quality and integrity were maintained with PCR-generated templates.
• mRNA-LNP vaccines from both methods induced comparable, robust immune responses in mice.

Conclusions:

• PCR-generated DNA templates offer a rapid, efficient, and cost-effective alternative for mRNA synthesis.
• This method has broad applicability in the development of mRNA-based vaccines and therapeutics.
• The PCR-based approach streamlines DNA template preparation for IVT applications.