CGAS-STING Pathway Regulates Transgene Expression By Mediating OAS-RNaseL And Its Mechanism

ExogenousDNA istaken up by cellsthrough virusinfection or transfection,usually through endocytosis,trafficking,entering the nucleusin the cell.Once inside the nucleus,DNA encoded transgene can expressin the cell.However,the foreign invading DNA will be recognized by a variety of nucleic acid sensorsleading to the production and secretion of type I interferons(IFNs),inducing expression of a large array of immune response genescollectively called interferon stimulated genes(ISGs),which actsasone of the first linesof defense against foreign DNA invasion.In-depth mechanismsof what may affect transgene expression within the cell remain unclear.CyclicGMP-AMP synthase(cGAS)waspreviously identified asa major cytoplasmic DNA sensor.Upon binding to DNA,cGAS can use ATP and GTP assubstratesto produce2',3'-cyclicGMP-AMP(cGAMP),which actsasa second messenger that bindsand activatesSTING(stimulator of interferon genes).Once activated by cGAMP,STING wasrelocalized from the endoplasmic reticulum to the Golgi complex and stimulatesTBK1 and IKK kinase complexes,resulting in the phosphorylation and activation of transcription factorsIRF3 and NF-?B,and leading to the production of type I interferons(IFNs).Through the activation of the Januskinase(JAK)-signal transducer and activator of transcription(STAT)pathway,type I interferonsinduce expression of a large array of ISGs,which establishesan antiviral state to curb replication of invading microbessuch asDNA viruses.Recent studiesdemonstrate that several cytosolic DNA sensors,such asDAI(DNA-dependent activator of IFN-regulatory factors),p204(interferon activated gene 204protein,IFI16),DHX9(DEx H-Box Helicase 9),can bind plasmid DNA at an early stage.These studiesfound increased expression of variouscytosolic DNA sensors,such asDDX41(DEAD-Box Helicase 41),DAI and p204 after electroporation.Moreover,previousstudieshave suggested that foreign DNA including transfected plasmid may stimulate cellular responsesto restrain foreign gene expression within the target cells,however,very little isknown regarding what may affect transgene expression within the cell.The underlying mechanismsneed to be further explored.In the present study,we investigated the underlying mechanism that controlstransgene expression by exploring the role of cGAS-STING-mediated DNA sensing pathway.Chemical inhibitorsthat disrupt the signaling cascadesmediated by the DNA-sensing pathway effectively lifted cellular suppression on transgene expression in multiple cell linesand primary cellsincluding T cells.The resultsare described in asfollows:(1)Plasmid DNA,through cGAS-STING pathway,induced type I interferon and ISGs,reduced m RNA transcription level and finally suppressed transgene expression.To determine whether cGAS-STING-mediated signaling hasan effect on the expression of a foreign gene carried by a plasmid,we examined the DNA transfection efficiency,aswell asdownstream ISGsexpression changes,and m RNA and DNA levelsof exogenousDNA in WT and Cgas^-/-L929 cells,or murine embryonic fibroblasts(MEFs)derived from WT?Cgas^-/-?Sting^gt/gt C57BL/6 mice,or primary lung fibroblastsfrom wild-type,Cgas^-/-,and Sting^gt/gt C57BL/6 mice upon DNA transfection.Deletion of cGAS or STING suppressed expression of ISGssuch asCXCL10,IFIT1 or ISG15.Lossof either cGAS or STING led to>2-fold increase in GFP positive cellsasmeasured by fluorescence activated cell sorting(FACS).Quantitative polymerase chain reaction(PCR)indicated that the uptake of plasmid DNA waslargely unaffected by cGAS,however the m RNA level of EGFP waselevated in both L929 and primary Cgas^-/-cellscompared to their wild-type counterparts.Next,we asked if the adaptor protein MAVS,which actsimmediately in the downstream of RNA receptor(i.e RIG-I),played any role in the foreign gene expression upon transfection.The data showed that in contrast to Cgas^-/-cells,knocking out MAVS didn't affect GFP expression after transfection asindicated by FACS analysisof GFP positive cells.These resultsfurther support the specific role of the cGAS-STING pathway in suppressing the expression of a transgene.(2)Plasmid DNA-induced IFN response suppressesforeign gene expression by binding IFNAR receptor through an autocrine manner.We generated conditioned media from the cellsthat were transfected with HT-DNA or poly(I:C),then treated L929 cellswith these conditioned media and examined their effectson the expression of EGFP after plasmid DNA transfection.Pre-treatment with both conditioned media strongly inhibited EGFP protein expression.Next,we pre-treated cellswith recombinant IFNb,anti-IFNb antibody,or the combination of both.The expression levelsof EGFP from a transfected plasmid were inhibited by IFNb at 24 and 48 hoursand enhanced by IFNb neutralizing antibody.Finally,we pre-treated cellswith recombinant IFNb,anti-IFNAR1 antibody,or combination of both.Asexpected,the expression levelsof Renilla luciferase from a transfected plasmid were inhibited by IFNb and enhanced by IFNAR1 neutralizing antibody.Taken together,these resultssupport the hypothesisthat foreign gene expression isrestricted by cGAS-STING pathway that inducesIFN responsesthrough autocrine manner.(3)Degradation of m RNA and suppression of foreign gene expression derived from transgenesare mediated by the OAS-RNaseL system that activated by cGAS-STING pathway.We performed gene expression profile analysesusing the RNA-Seq technology.We hypothesized that OAS-RNaseL family may directly suppressexpression of transfected genes.We deleted the individual OAS genesaswell asRNaseL gene in BJ-5ta cells.When these cellswere transfected with two other plasmids,p EGFP-N1 or p LVX-m Cherry,the proportion of fluorescence-positive cellswere enhanced by 1.5?3-fold in OAS1^-/-,OAS3^-/-,and RNaseL^-/-,but not in OAS2^-/-cells.Asexpected,transfection of plasmid DNA caused induction of ISG15 in all cells.The m RNA level of EGFP waselevated in OAS1^-/-,OAS3^-/-,and RNaseL^-/-,but not in OAS2^-/-cells.These resultssuggest that the activation of OAS1,OAS3,and RNaseL leadsto degradation of m RNA and suppression of transgene expression.To directly detect RNaseL activity in cellsfollowing plasmid DNA delivery,we transfected a plasmid encoding RNaseL fused with a Flag tag at C-terminal(p CMV-RNaseL-Flag)into cells.The result showed RNaseL activity wasindeed triggered by DNA transfection in a cGAS-STING-dependent manner.RNaseL activity wasinhibited when deletion of cGAS or STING.Moreover,plasmid DNA-induced RNaseL activity wasabolished in BX795treated cells.These resultssupport the major role of OAS-RNaseL in suppressing transgene expression through secretion IFNsvia cGAS-STING pathway.(4)Small molecule inhibitorstargeting cGAS-STING signaling improve expression of transfected genes.We next tested small molecule inhibitorstargeting cGAS-induced signaling in L929cells,or BJ-5ta cells,or WT?Cgas^-/-and Sting^gt/gt MEF cells.FACS data showed that the pretreatment of L929,BJ-5ta or MEFswith STING inhibitorsC-176 and C-178,TBK1inhibitorsBX795,Amlexanox and MRT67307,JAK1/2 inhibitor Ruxolitinib,JAK3inhibitor Tofacitinib followed by transfection of p EGFP-N1 plasmid increased the percentage of GFP positive cells.These inhibitorscaused increased levelsof EGFP m RNA and ISGsexpression resulted from plasmid transfection,thereby suggesting their rolesin regulating transgene expression.(5)Chemical inhibitorsimprove transfection in primary T cellsPreviousdata indicated small molecule inhibitorstargeting cGAS-induced signaling improve expression of transfected genesin primary cells.Transfection of T cellsremainsa challenge in research and gene therapy practices.We then treated cultured T cellsor bulk PBMCscellswith inhibitorsor DMSO and delivered EGFP plasmid through electroporation.In cultured T cells,bulk PBMCs,or na(?)ve and rest T cells,small molecule inhibitorstargeting cGAS-STING signaling robustly suppressed ISGsexpression,exhibited significant improvement of transfection efficiency in these cells.These inhibitorsdidn't have detectable adverse effect on cell growth up to 18 daysfollowing electroporation.These resultsprovide evidence supporting that simple administration of small molecule inhibitorscan improve T cell transfection by overcoming the impediment caused by activation of cGAS-STING signaling.In summary,the cGAS-STING pathway playsa vital role in regulating transgene expression.Mobilization of OAS-RNaseL anti-viral system isthe predominant force that causesdegradation of m RNA and suppression of transgene expression.We investigated the underlying mechanism that controlstransgene gene expression by exploring the role of cGAS-STING-mediated DNA sensing pathway.We suggest that inhibition of innate immune response will significantly improve the DNA transfection efficiency,thereby may benefit CAR-T treatmentsand gene therapy,and DNA-mediated vaccination.