Inhibition of cGAS-Mediated Interferon Response Facilitates Transgene Expression.

Yajuan Fu,Baoting Huang,Liqun Zheng,Suxing Chen,Lijun Sun,Shan Xu,Wei Bao,Hanze Wang,Qi Chen,Zhang Lin,Tingting Yu,Lei Yang,Hao Hu,Yijun Fang

doi:10.1016/j.isci.2020.101026

Abstract

SummaryDNA transfection is often the bottleneck of research and gene therapy practices. To explore the mechanism regulating transgene expression, we investigated the role of the cGAS-STING signaling pathway, which induces type-I interferons in response to DNA. We confirmed that deletion of cGAS enhances transgene expression at the protein level by ~2- to 3-fold. This enhancement is inversely correlated with the expression of interferons and interferon stimulated genes (ISGs), which suppress expression of transfected genes at the mRNA level. Mechanistically, DNA transfection activates the cGAS-STING pathway and induces the expression of the OAS family proteins, leading to the activation of RNaseL and degradation of mRNA derived from transgenes. Administration of chemical inhibitors that block cGAS-mediated signaling cascades improves the expression of transgenes by ~1.5- to 3-fold in multiple cell lines and primary cells, including T cells. These data suggest that targeting the cGAS-STING pathway can improve transgene expression, and this strategy may be applied to gene therapy.

Highlights

DNA transfection is a common approach to deliver the genes of interest into cells (Kim and Eberwine, 2010) and has been widely used in the study of gene function, producing recombinant proteins including monoclonal antibodies for therapeutic purpose (Jager et al, 2013; Wurm, 2004), and treatment of genetic diseases (Cavazzana-Calvo et al, 2000)
To explore the mechanism regulating transgene expression, we investigated the role of the Cyclic GMP-AMP synthase (cGAS)-STING signaling pathway, which induces type-I interferons in response to DNA
DNA transfection activates the cGAS-STING pathway and induces the expression of the oligoadenylate synthetase (OAS) family proteins, leading to the activation of RNaseL and degradation of mRNA derived from transgenes

Summary

Introduction

DNA transfection is a common approach to deliver the genes of interest into cells (Kim and Eberwine, 2010) and has been widely used in the study of gene function, producing recombinant proteins including monoclonal antibodies for therapeutic purpose (Jager et al, 2013; Wurm, 2004), and treatment of genetic diseases (Cavazzana-Calvo et al, 2000). A variety of transfection methods have been developed, including DEAE-dextran, branched polyethylenimine (PEI) (Fischer et al, 1999), calcium phosphate coprecipitation, cationic lipid vehicles (such as Lipofectamine), electroporation (Chu et al, 1987), or nucleofection (Kim and Eberwine, 2010; Lai et al, 2003; Niidome and Huang, 2002). All of these methods are focused on improving the delivery of foreign DNA into target cells. Previous studies have suggested that foreign DNA including transfected plasmid may stimulate cellular responses to restrain foreign gene expression within the target cells (Bosnjak et al, 2017; Langereis et al, 2015; Sellins et al, 2005); the in-depth mechanisms remain unclear

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Conclusion