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

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 eukaryoti...

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Autores principales: Pastor, Marie, Quiviger, Mickäel, Pailloux, Julie, Scherman, Daniel, Marie, Corinne
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270507/
https://www.ncbi.nlm.nih.gov/pubmed/32505001
http://dx.doi.org/10.1016/j.omtn.2020.05.014
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author Pastor, Marie
Quiviger, Mickäel
Pailloux, Julie
Scherman, Daniel
Marie, Corinne
author_facet Pastor, Marie
Quiviger, Mickäel
Pailloux, Julie
Scherman, Daniel
Marie, Corinne
author_sort Pastor, Marie
collection PubMed
description 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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spelling pubmed-72705072020-06-08 Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver Pastor, Marie Quiviger, Mickäel Pailloux, Julie Scherman, Daniel Marie, Corinne Mol Ther Nucleic Acids Article 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. American Society of Gene & Cell Therapy 2020-05-20 /pmc/articles/PMC7270507/ /pubmed/32505001 http://dx.doi.org/10.1016/j.omtn.2020.05.014 Text en © 2020 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Article
Pastor, Marie
Quiviger, Mickäel
Pailloux, Julie
Scherman, Daniel
Marie, Corinne
Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver
title Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver
title_full Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver
title_fullStr Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver
title_full_unstemmed Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver
title_short Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver
title_sort reduced heterochromatin formation on the pfar4 miniplasmid allows sustained transgene expression in the mouse liver
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270507/
https://www.ncbi.nlm.nih.gov/pubmed/32505001
http://dx.doi.org/10.1016/j.omtn.2020.05.014
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