Combinative workflow for mRNA vaccine development

Recently, mRNA has gained a lot of attention in the field of vaccines, gene therapy, and protein replacement therapies. Herein, we are demonstrating a comprehensive approach to designing, cloning, and characterizing an antigenic cassette for the development of mRNA vaccine for COVID-19. The gene encoding the antigenic spike protein of the SARS-CoV-2 Omicron variant (B.1.1.529) was designed using the databases, characterized by in-silico tools, and assembled using overlapping oligonucleotide-based assembly by PCR. Next, the gene was cloned, mRNA was synthesized, and characterized using orthogonal approaches (Capillary electrophoresis, Sanger DNA sequencing, Next-generation sequencing, HPLC, qPCR, etc.). Furthermore, the antigen expression was monitored in-vitro using an animal cell model by western blot, flow cytometer, and surface plasmon resonance. The demonstrated approach has also been followed for developing the mRNA vaccines for various other indications such as Malaria, Herpes, Dengue, HPV, etc. •Self-amplifying mRNA-based vaccines are the emerging field that has received considerable attention during COVID-19.•A comprehensive workflow to support mRNA vaccine development has been demonstrated.•The workflow includes designing, cloning, and characterizing the final product as mRNA.•The workflow uses in-silico tools, molecular biology techniques, and in-vitro approaches to confirm the correctness of the gene and conformationally active form of the encoded protein.•The scope of the demonstrated workflow is not only limited to the vaccines but other mRNA modalities including cell/gene therapy and protein replacement therapies.