Targeted Correction Of Point Mutations In Low Density Lipoprotein Receptor Gene Mediated By Modified Single Stranded Oligonucleotides In Mouse Liver In Vivo

ObjectiveThe aim of this study is to determine whether the point mutation in low density lipoprotein receptor(LDLR)gene in mouse liver in vivo could be corrected by antisense modified Single strand oligonucleotide (A-MSO).Method1 Targeted correction of nonsense mutation in LDLR in HepG2 cell1.1 Construction and identification of LDLR-luc fusion protein vectorWild type(WT)LDLR gene or 660-LDLR(with nonsense mutation) was linked to 5' end of luciferase gene(luc)for construction of pWT-LDLR-luc or p660-LDLR-luc.660-LDLR carries a C to A transversion,creating a nonsense mutation and a Hinf I site.These plasmids were verified by direct sequencing.1.2 Preparation and identification of HepG2 cell model with 660-LDLR-luc gene p660-LDLR-luc was transfected into HepG2 cell and the luciferase activity was assayed for the identification of nonsense mutation model 48 h after transfection.1.3 Targeted correction of nonsense mutation in LDLR mediated by MSO in HepG2 cellA-MSO or S-MSO(sense MSO)results in sequence-specific alterations leading to correction of mutations.Seventy two hours after co-transfecting HepG2 cell with p660-LDLR-luc and A-MSO or S-MSO together,the luciferase activity was determined to verify the correction of nonsense mutation and to bolt the better MSO.2 Targeted correction of nonsense mutation in LDLR mediated by MSO in mouse liver in vivo2.1 Confirmation of liver-specific transfection by hydrodynamic gene transfer(HGT)pWT-LDLR-luc was transferred to mouse liver by HGT.To determine whether HGT leads to liver-specific transfection of objective DNA,the FLAs present in liver,heart,lung,kidney and spleen were examined.2.2 Preparation and identification of mouse model with 660-LDLR-luc gene in liver in vivop660-LDLR-luc was transferred to mouse liver by HGT.Both assay of FLA present in liver and PCR coupled with restriction fragment length polymorphism(PCR-RFLP)were performed to verify nonsense mutation model.2.3 Targeted correction of nonsense mutation in LDLR mediated by MSO in mouse liver in vivoTwelve hours after transfer p660-LDLR-luc to mouse liver by HGT, A-MSO was delivered by polyethylenimine(PEI)coupled with HGT to liver.Sixty hours after administration of A-MSO,the FLA was determined to verify the correction of nonsense mutation.2.4 Confirming correction of nonsense mutation in DNA levelPCR-RFLP and DNA sequencing were preformed to verify the correction of nonsense mutation.Result1 The results of experiments with HepG2 cell1.1 DNA sequencing of WT-LDLR and 660-LDLRp660-LDLR-luc carries a C to A transversion(TGA),but pWT-LDLR-luc does not.1.2 Expression of luciferase in HepG2 cellAssay of FLA present in the HepG2 cell transfected with various plasmids showed that FLA of p660-LDLR-luc group is higher than that of pWT-LDLR-EGFP group(p＜0.01)but lower than that of pWT-LDLR-luc group(p＜0.01),indicating that expression of luciferase could not be completely stopped by nonsense mutation. 1.3 p660-LDLR-luc expression of luciferase was elevated by A-MSO and S-MSO in HepG2 cellComparing the FLA present in the HepG2 cell,co-transfected with p660-LDLR-luc and various MSO together,showed that the luciferase expression of p660-LDLR-luc was elevated by A-MSO or S-MSO and A-MSO was better than S-MSO,p＜0.01.2 The results of experiments with mouse2.1 HGT leaded to liver-specific gene transferSeventy two hours after transferring pWT-LDLR-luc to mouse liver, FLA assay showed that FLAs present in heart,lung,spleen and kidney lysates were significantly lower than that present in liver(p＜0.01),which clearly indicates that HGT resulted in a highly liver-specific gene transfer.2.2 Expression of luciferase and the results of PCR-RFLPComparing FLA present in liver transfected with various plasmids respectively indicated that FLA of p660-LDLR-luc group was lower than that of pWT-LDLR-luc group but higher than that of pEGFP-N1(p＜0.01), suggesting that expression of luciferase could not be completely stopped by nonsense mutation in liver in vivo.PCR-RFLP showed that nonsense mutation has not been auto-corrected in liver in vivo.2.3 p660-LDLR-luc expression of luciferase was elevated by A-MSO in mouse liver in vivo Comparing the FLA present in liver co-transfected with p660-LDLR-luc and A-MSO or C-MSO(control MSO)together,showed that FLA of A-MSO group was higher than that of C-MSO group(p＜0.01).2.4 A to C transversion in LDLR genePCR-RFLP and DNA sequencing confirmed that A was transfered into C in LDLR gene.ConclusionMouse transfected with p660-LDLR-luc by HGT can be used as an animal model of point mutation in LDLR gene in liver in vivo.MSO can be delivered to mouse liver by PEI coupled with HGT.The point mutation in LDLR gene in liver in vivo can be corrected by A-MSO.