한빛사 논문
Amal Senevirathne1, Ji-Young Park1, Chamith Hewawaduge, Kirthika Perumalraja, John Hwa Lee*
College of Veterinary Medicine, Jeonbuk National University, Iksan Campus, 54596, Iksan, Republic of Korea
*Corresponding author.
1Equally contributed authors.
Abstract
This study describes an efficient eukaryotic expression system (pJHL204) built into the Salmonella delivery system to enhance the essential efficacy and effectiveness of conventional DNA therapy. The expression system utilizes RNA-dependent RNA polymerase activity (RdRp) of Semiliki Forest Virus attributing to dramatic antigen expression by cytoplasmic mRNA amplification. Functional characterization of the pJHL204 by in vitro and in vivo transfection studies revealed the improved expression of mRNA at least 150 folds than the RdRp mutant plasmid under in vitro conditions. Using green fluorescence protein (GFP) and mCherry as bait proteins this system was extensively characterized for plasmid delivery capacity, antigen expression, and safety using in vivo and in vitro models by employing flow cytometry, fluorescence microscopy, and immunohistochemical staining. Employment of Salmonella as a carrier significantly extends plasmid in vivo survivability and prolongs the effective duration until the elimination of the Salmonella carrier strain in the host. The strategy can be easily adapted for P2A connected multiple antigen delivery in a single vector system due to the significantly high cargo capacity of Salmonella. A mouse challenge study was carried out utilizing P2A connected H1N1 hemagglutinin (HA) and neuraminidase (NA) via the Salmonella carrier strain JOL2500 significantly reduced viral activity and protected mice against the H1N1 challenge and demonstrates potential to redefine in vivo DNA therapy as a reliable and safe system to treat human diseases using useful microbes like Salmonella.
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