Sequence simplification of antigen coding IVT mRNA allows accelerated synthetic DNA template generation and epitope...

Area of Science:

• Molecular Biology
• Immunology
• Biotechnology

Background:

• Therapeutic messenger RNA (mRNA) technology has advanced significantly, particularly for vaccine development.
• Key mRNA structural elements, including 5' and 3' untranslated regions (UTRs) and the poly-A tail, are crucial for stability and reduced immunogenicity.

Purpose of the Study:

• To investigate the impact of individual structural components on antigen-encoding mRNA protein expression.
• To evaluate the necessity of stabilizing structures (5' and 3' UTRs, poly-A tail) for antigen presentation.
• To develop an efficient method for synthetic DNA template generation for accelerated mRNA production.

Main Methods:

• Investigated mRNA structural components' contribution to protein expression in vitro and ex vivo.
• Utilized electroporation for delivering antigen-encoding mRNA into antigen-presenting cells.
• Performed structural deconstruction of antigen-coding mRNA by removing UTRs and poly-A tail.

Main Results:

• Sequence and chemical modifications impact mRNA translation and protein expression.
• Antigen presentation was preserved even after removing stabilizing structures like 5' and 3' UTRs and the poly-A tail.
• A streamlined method for generating synthetic DNA templates for in vitro transcription was established, accelerating mRNA production.

Conclusions:

• Stabilizing structures (UTRs, poly-A tail) are not essential for maintaining antigen presentation in deconstructed mRNA.
• The findings facilitate high-throughput screening and evaluation of antigen immunogenicity.
• This work simplifies and accelerates the generation of mRNA for therapeutic applications.