Evaluation Of Doggybone DNA As Template For In Vitro Transcription

By Lauren Hinkel, Susannah Chilton, Olivia McPhee, Eli Henderson, Trang Tong, Max Mashrick, Ben Longenbach, Calleigh Herren, Andrew Voter, Christian Cobaugh, Vernal Biosciences

Linear DNA is used as the template to in vitro transcribe (IVT) synthetic mRNA. Common sources for templates are pDNA linearized with a restriction enzyme and PCR amplicons. Enzymatically produced Doggybone™ DNA (dbDNA), is a novel IVT template that may improve the stability of DNA-templated poly(A) regions. Further, the highly scalable, cell-free process used to manufacture dbDNA does not produce endotoxin or bioburden.

In this study, we evaluated the performance of two dbDNA IVT templates, each encoding the same mRNA (firefly luciferase or Fluc). The first template was compatible with baseline process conditions (BSLN), while the second template was compatible with Vernal’s optimized process conditions (VRN). We evaluated the mRNA purity and in vivo potency of the mRNA IVT’d from each template.