Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver - CNRS - Centre national de la recherche scientifique Accéder directement au contenu
Article Dans Une Revue Molecular Therapy - Nucleic Acids Année : 2020

Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver

Résumé

Non-viral gene delivery into the liver generally mediates a transient transgene expression. A comparative analysis was performed using two gene vectors, pFAR4 and pKAR4, which differ by the absence or presence of an antibiotic resistance marker, respectively. Both plasmids carried the same eukaryotic expression cassette composed of a sulfamidase (Sgsh) cDNA expressed from the human alpha antitrypsin liver-specific promoter. Hydrodynamic injection of the pFAR4 construct resulted in prolonged sulfamidase secretion from the liver, whereas delivery of the pKAR4 construct led to a sharp decrease in circulating enzyme. After induction of hepatocyte division, a rapid decline of sulfamidase expression occurred, indicating that the pFAR4 derivative was mostly episomal. Quantification analyses revealed that both plasmids were present at similar copy numbers, whereas Sgsh transcript levels remained high only in mice infused with the pFAR4 construct. Using a chromatin immunoprecipitation assay, it was established that the 5' end of the expression cassette carried by pKAR4 exhibited a 7.9-fold higher heterochromatin-to-euchromatin ratio than the pFAR4 construct, whereas a bisulfite treatment did not highlight any obvious differences in the methylation status of the two plasmids. Thus, by preventing transgene expression silencing, the pFAR4 gene vector allows a sustained transgene product secretion from the liver.
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hal-03295373 , version 1 (10-09-2021)

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Marie Pastor, Mickael Quiviger, Julie Pailloux, Daniel Scherman, Corinne Marie. Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver. Molecular Therapy - Nucleic Acids, 2020, 21, pp.28-36. ⟨10.1016/j.omtn.2020.05.014⟩. ⟨hal-03295373⟩
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