BACKGROUND: Efficient gene delivery by synthetic vectors is a major challenge in gene therapy. However, inefficient nuclear delivery of cDNA is thought to be a major limiting step in gene transfer using non-viral vectors. It is commonly thought that, in the cytosol, cDNA has to be released from its vector before importation to the nucleus. The stability of naked cDNA in the cytoplasm is not well established. METHODS: cDNA plasmids, either free or complexed with poly(ethyleneimine) (PEI), were microinjected into the cytoplasm of mammalian cells and their turnover was assessed by fluorescence in situ hybridization (FISH). Incubations of cDNA plasmids in cytosolic extracts were also performed. RESULTS: FISH experiments showed that naked cDNA rapidly fade with time when injected into the cytosol. Fading was not observed when naked cDNA plasmids were injected into the nucleus. Incubation of naked cDNA in a cytosolic fraction isolated from mammalian cells reproduced cDNA degradation as observed in microinjection experiments. Nuclease inhibitors, including aurin tricarboxylic acid or Zn2+, prevented in vitro cDNA degradation. The cytosolic nuclease activity was optimal at physiological pH and physiological Ca2+ concentration. By contrast, it was insensitive to Mg2+ or Na+ concentrations. Finally, cDNA complexation with PEI or addition of oligonucleotides prevented in vitro cDNA degradation. CONCLUSION: Altogether, these experiments suggest that cDNA digestion by cytosolic nucleases occur when the decomplexed transgene is present in the cytosol. We propose that the inefficient transfer of cDNA into the nucleus during transfection with synthetic vectors may result from rapid digestion of naked cDNA by a Ca2+-sensitive cytosolic nuclease.
BACKGROUND: Efficient gene delivery by synthetic vectors is a major challenge in gene therapy. However, inefficient nuclear delivery of cDNA is thought to be a major limiting step in gene transfer using non-viral vectors. It is commonly thought that, in the cytosol, cDNA has to be released from its vector before importation to the nucleus. The stability of naked cDNA in the cytoplasm is not well established. METHODS: cDNA plasmids, either free or complexed with poly(ethyleneimine) (PEI), were microinjected into the cytoplasm of mammalian cells and their turnover was assessed by fluorescence in situ hybridization (FISH). Incubations of cDNA plasmids in cytosolic extracts were also performed. RESULTS: FISH experiments showed that naked cDNA rapidly fade with time when injected into the cytosol. Fading was not observed when naked cDNA plasmids were injected into the nucleus. Incubation of naked cDNA in a cytosolic fraction isolated from mammalian cells reproduced cDNA degradation as observed in microinjection experiments. Nuclease inhibitors, including aurin tricarboxylic acid or Zn2+, prevented in vitro cDNA degradation. The cytosolic nuclease activity was optimal at physiological pH and physiological Ca2+ concentration. By contrast, it was insensitive to Mg2+ or Na+ concentrations. Finally, cDNA complexation with PEI or addition of oligonucleotides prevented in vitro cDNA degradation. CONCLUSION: Altogether, these experiments suggest that cDNA digestion by cytosolic nucleases occur when the decomplexed transgene is present in the cytosol. We propose that the inefficient transfer of cDNA into the nucleus during transfection with synthetic vectors may result from rapid digestion of naked cDNA by a Ca2+-sensitive cytosolic nuclease.
Authors: Rahul Rattan; Sriram Vaidyanathan; Gordon S-H Wu; Anisha Shakya; Bradford G Orr; Mark M Banaszak Holl Journal: Mol Pharm Date: 2013-07-24 Impact factor: 4.939
Authors: Richard N Cohen; Marieke A E M van der Aa; Nichole Macaraeg; Ai Ping Lee; Francis C Szoka Journal: J Control Release Date: 2009-01-12 Impact factor: 9.776