title: A novel high-throughput cell-based method for integrated quantification of type I interferons and in vitro screening of immunostimulatory RNA drug delivery.
abstract: A hallmark of immune activation by certain RNA sequences is the generation of interferon responses. However, the study of immunostimulatory RNA (isRNA) has been hindered by costly and slow methods, particularly in vitro. We have developed a cell-based assay to detect human type I interferon (IFN) that reliably senses both IFN-alpha and IFN-beta simultaneously. The human 293T cell line was stably transfected with a fusion gene of monomeric red fluorescent protein (mRFP) under the transcriptional control of an interferon-stimulated response element (ISRE). High levels of mRFP are expressed following activation of the type I IFN receptor (IFNAR). Using this method, detection limits for IFN similar to that of ELISA can be achieved but with a greater dynamic range and in a high-throughput manner. As a proof of concept, we utilized this method to screen a library of cationic lipid-like materials that form nanoparticle complexes with RNA for induction of innate immune responses in vitro. We expect the screening and detection methods described herein may provide a useful tool in elucidating mechanisms that govern the delivery of RNA molecules to effector cells and receptors of the innate immune system. We apply this tool to investigate isRNA drug delivery, but it may also find use in other applications for which high-throughput detection of type 1 IFN is desired.
authors.affiliation: Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA.
authors.forename: David N
authors.initials: DN
authors.lastname: Nguyen
authors.forename: Phillip
authors.initials: P
authors.lastname: Kim
authors.forename: Luis
authors.initials: L
authors.lastname: Martínez-Sobrido
authors.forename: Brett
authors.initials: B
authors.lastname: Beitzel
authors.forename: Adolfo
authors.initials: A
authors.lastname: García-Sastre
authors.forename: Robert
authors.initials: R
authors.lastname: Langer
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Anderson