title: Model-driven engineering of gene expression from RNA replicons.
abstract: RNA replicons are an emerging platform for engineering synthetic biological systems. Replicons self-amplify, can provide persistent high-level expression of proteins even from a small initial dose, and, unlike DNA vectors, pose minimal risk of chromosomal integration. However, no quantitative model sufficient for engineering levels of protein expression from such replicon systems currently exists. Here, we aim to enable the engineering of multigene expression from more than one species of replicon by creating a computational model based on our experimental observations of the expression dynamics in single- and multireplicon systems. To this end, we studied fluorescent protein expression in baby hamster kidney (BHK-21) cells using a replicon derived from Sindbis virus (SINV). We characterized expression dynamics for this platform based on the dose-response of a single species of replicon over 50 h and on a titration of two cotransfected replicons expressing different fluorescent proteins. From this data, we derive a quantitative model of multireplicon expression and validate it by designing a variety of three-replicon systems, with profiles that match desired expression levels. We achieved a mean error of 1.7-fold on a 1000-fold range, thus demonstrating how our model can be applied to precisely control expression levels of each Sindbis replicon species in a system.
authors.affiliation: Raytheon BBN Technologies, Cambridge, Massachusetts United States.
authors.forename: Jacob
authors.initials: J
authors.lastname: Beal
authors.forename: Tyler E
authors.initials: TE
authors.lastname: Wagner
authors.forename: Tasuku
authors.initials: T
authors.lastname: Kitada
authors.forename: Odisse
authors.initials: O
authors.lastname: Azizgolshani
authors.forename: Jordan Moberg
authors.initials: JM
authors.lastname: Parker
authors.forename: Douglas
authors.initials: D
authors.lastname: Densmore
authors.forename: Ron
authors.initials: R
authors.lastname: Weiss
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics; D008957:Models, Genetic; D012367:RNA, Viral / Q000235:genetics; D012093:Replicon; D012845:Sindbis Virus / Q000235:genetics; D058615:Synthetic Biology; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D012367:RNA, Viral
keywords: Sindbis; TASBE characterization; alphavirus; circuit prediction; expression control; flow cytometry; quantitative modeling; replicon