Transfection Efficiency Improvement Of Human Ribosomal DNA Targeting Vector By NLS And DMSO

Non-viral vectors are attractive alternative to viral vectors due to security,versatility and convenience of preparation.However,the low transfection efficiency and transient expression of the therapeutic gene handicap its clinical application.The human ribosomal DNA targeting vector(hrDNA vector) pHrneo constructed by our group could integrate the therapeutic gene into the human ribosomal DNA loci at a high frequency(10^-5-10^-4) and express the therapeutic gene stably in cell lines such as HT1080 and HL7702.However,the low transfection efficiency of hrDNA vector severely hinders its clinical utilization,especially in primary human dermal fibroblast(HDF),one of the ideal targeting cell types employed in ex vivo gene therapy for its easy acquisition and culture.The result could be worse when the plasmid DNA are larger than 10kb(＞700KD) and sequestered in the cytoplasm for a longer time, leading to a dramatical decrease of its transfection efficiency.Therefore, sequestration of the cytoplasm and nuclear membrane might contribute to low transfection efficiency of the recombinant plasmids of pHrneo with the therapeutic genes,which sizes are around 10-14kb.Objective.To improve the transfection efficiency of pHrneo plasmids,two strategies were recruited to overcome the nuclear envelope barrier by facilitating the nuclear entry of plasmid DNA:1) The plasmid DNA was conjugated with the nuclear localization signal peptide to promote the translocation into nucleus before the nuclease degradation.2) The plasmid DNA entered into nucleus when the nuclear pore complex permeability was augmented by small amphipathic molecules.Method:1) The pHrneo plasmid carrying GFP were conjugated by SPB with SV 40 NLS peptide through electrostatic interaction,and the efficiency of conjugation was evaluated by agarose gel shift mobility assay.The protection of plasmid from nuclease in vivo was mimicked by DNaseâ… digestion.Polyethylenimine was employed to transfect the primary human dermal fibroblasts when the pHrnGFP was coupled with NLS at gradient molar ratio and the transfection efficiency was measured by flow cytometry 48 hours later.The subcellular location of cy3-labeled pHrnGFP were idetified with confocal microscopy 1 hour after transfection.2) Lipofectamine2000 was employed to transfect DMSO treated HDF and the transfection efficiency was assessed with flow cytometry 24 hours later.The cell cycle of DMSO treated HDF was also measured by flow cytometry.The subcellular location of cy3-labeled pHrnGFP was identified by confocal microscopy 30 minutes post transfection.The cytotoxicity of NLS and DMSO were determined by MTT assay.Result:1) The mobility of plasmid in agarose gel was decreased obviously when conjugated with NLS.The transfection efficiency was increased 4-5 fold when the coupling molar ratio of NLS and pHrnGFP was at 2×10⁴,at which the NLS also was protected from the degradation by DNaseâ… .The pHrnGFP was facilitated into nucleus within 1 hour under the view of confocal microscopy.2) The transfection efficiency was boosted 2-3 fold when the HDF cells were pretreated with 2% DMSO for 48 hours.The HDF cells were reversibly arrested at G1/S boundary in the presence of 2%DMSO.The Cy3-labeled plasmid was localized in nucleus 30 minutes after transfection under the view of confocal microscopy.Conclusion:NLS peptide improves the transfection efficiency of hrDNA vector via promoting its nuclear entry.The transfection efficiency of HDF cells was also improved when treated with 2%DMSO 48 hours prior to transfection,by which the hydrophobic Phe-rich repeat of nuclear pore complex was interfered.And the methods we developed could accelerate the pre-clinical research of hrDNA vector.