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A novel high‐throughput cell‐based method for integrated quantification of type I interferons and in vitro screening of immunostimulatory RNA drug delivery
Author(s) -
Nguyen David N.,
Kim Phillip,
MartínezSobrido Luis,
Beitzel Brett,
GarcíaSastre Adolfo,
Langer Robert,
Anderson Daniel G.
Publication year - 2009
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22312
Subject(s) - interferon , innate immune system , rna , effector , in vitro , biology , transfection , hek 293 cells , immune system , microbiology and biotechnology , computational biology , high throughput screening , cell , receptor , gene , biochemistry , virology , immunology
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‐α and IFN‐β 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. Biotechnol. Bioeng. 2009;103: 664–675. © 2009 Wiley Periodicals, Inc.