Study On Targeted Correction Of Point Mutation In Low Density Lipoprotein Receptor Gene In Situ Of Chromosome And Regulatable Expression

ObjectiveFamilial hypercholesterolemia (FH) results from a mutation (usually point mutation) in the low density lipoprotein receptor (LDLR) that normally removes LDL from plasma and has been considered as one of the best candidate for gene therapy. Experimental evidences indicated that targeted DNA repair employing synthetic modified single-stranded DNA oligonucleotides (MSO) promised great potentials. To test the ability of MSO in repairing point mutation of LDLR gene in vitro, a cell model of LDLR gene point mutation was created by stably transfection. Our results showed that transfection with specific MSOs successfully repaired the point mutation. The present study reported for first time that targeted gene repair of LDLR gene in vitro can be achieved following MSO delivery, providing guidance for designing effective MSO as tools for treating FH and also opening the possibility of developing MSO as a therapeutic approach for treatment of human hereditary diseases caused by point mutation.MethodEstablishment of a targeted LDLR gene correction systemTo construct plasmid pIRES-Hyg-WT-LDLR-EGFP, pIRES-Hyg-556-LDLR-EGFP, pIRES-Hyg-660-LDLR-EGFP, Fulllength cDNA sequence of LDLR was insert into the multiple clone site of the plasmid pIRES-Hyg. In above plasmids, The WT-LDLR, 556-LDLR and 660-LDLR represent wild type or mutants of LDLR respectively. The 556 missense mutation is a G to C transversion at cDNA position 1730 in exon 12, causing the tryptophane to serine substitution at 556 position. The 660 mutation is a C to A transversion at position 2043 in exon 14, creating a premature termination codon at 660 position. pIRES-Hyg-WT-LDLR-EGFP, pIRES-Hyg-556-LDLR-EGFP and pIRES-Hyg-660-LDLR-EGFP were linearized and transfected into cultured HepG2, CHO, Vero cells respectively, which were then subjected to hygromycin selection, resistant cell clones were verified by fluorescence microscopy, PCR-restriction fragment length polymorphisms (RFLP) and sequencing, DiI labeled LDL was prepared and used to test the function of the exogenous LDLR. Finally, the expression of the LDLR-EGFP protein was confirmed by western blot.Targeted correction of point mutation in low density lipoprotein receptor gene by modified single-stranded oligonucleotides in vitroAn EGFP-based reporter system containing wild type or C660X mutant LDLR gene was constructed and used for generating stable Vero-WT and Vero-660 cell lines to test the ability of MSO in repairing point mutation of LDLR gene. Since the translation of LDLR gene was disrupted by the C to A mutation creating a premature termination codon, no EGFP blotting signal was detected for Vero-660 cells. MSOs in various lengths (15-45 nt) correspond in sequences to either coding strand (antisense strand ) or template strand (sense strand) of LDLR gene with phosphorothioate (PTO) linkages were synthesized and purified by HPLC, After the transfection of MSO, FACS analysis was carried out to evaluate the putatively repair efficiency, Then the cell populations were sorted according to morphology and EGFP positive fluorescence by flow cytometry. The repaired LDLR-EGFP gene of sorted cells was verified by pryosequencing, western bloting and Dil-LDLs uptake.Establishment of stable and regulable cell lines expressing LDLR geneFor constructing plasmid pIRES-TetR-Hyg, Tet R fragment was amplified from plasmid DNA of pcDNA6/TR by PCR, which was inserting into the multiple clone site of the plasmid pIRES-Hyg. The second plasmid pCDNA4/TO-WT-LDLR-EGFP was constracted simultaneouly by inserting full length cDNA sequence of wild type LDLR into the multiple clone sites of the plasmid pCDNA4/T0.Recombinant plasmid pCDNA4/TO-WT-LDLR-EGFP and pIRES-TetR-Hyg were cotransfected into cultured HepG2, CHO cells respectively by Lipofectamine 2000, which were then subjected to hygromycin and zeocin selection, the selected resistant clones was tested by laser confocal microscope and Western blot analysis respectively after induced by tetracycline. DiI labeled LDL was used to test the function of the exogenous LDLR. The expression level at different inducing times of Tet-Regulated cell lines was examined.ResultThe results of this study clearly showed that the cell lines with stably integrated wild LDLR gene, 660 and 556 mutational LDLR gene were successfully established. The hygromycin-resistant stable clones integrated with pIRES-Hyg-WT-LDLR-EGFP pIRES-Hyg-556-LDLR-EGFP and pIRES-Hyg-660-LDLR-EGFP were designated HepG2-WT, CHO-WT, Vero-WT, HepG2-556, CHO-556, Vero-556 and HepG2-660, CHO-660, Vero-660 respectively. Cell clones were verified by fluorescence microscopy, PCR-restriction fragment length polymorphisms (RFLP), Western blot and function of LDLR analysis.We chosed Vero-660 as the target cell to gene repair, the reason for choosing Vero-660 cells is because it shows much lower background of green fluorescence than other cell lines such as HepG2 and CHO cells. Vero-660 cells were transfected with MSOs, The 35A- MSOs showed better repair efficiency, The putatively repair efficiencies (with green fluorescence) were～5 % detected by flow cytometry. The repair efficiencies of sorted EGFP positive cells were～0.5 % after being rectified by pryosequencing.The results also showed that the Tet-Regulated cell lines, with stably integrated wild LDLR-EGFP gene were successfully established. Two Tet-Regulated cell lines were designated HepG2-TetR-WT and CHO-TetR -WT respectively. Laser confocal microscope views and Western blot results showed that LDLR-EGFP gene can be induced by the tetracycline. The function of LDLR was verified by Dil-LDL uptake. ConclusionIn conclusion, we established a point mutation model of LDLR gene and the mutation was corrected with an efficiency of～0.5% by MSO in vitro. The results are encouraging, suggesting that the targeted gene repair is potentially a method of choice, but it is still far from achieving clinical success. More effective structures of oligonucleotide, efficient delivery systems, and gene correction efficiency should be pursued. Development of these strategies holds great potentials for treatment of genetic defects including FH.Two stable cell lines for regulatable expression of LDLR gene were established, which provides good cell model for studying LDLR function and its molecular mechanism.