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Systemic Delivery of scAAV8-Encoded MiR-29a Ameliorates Hepatic Fibrosis in Carbon Tetrachloride-Treated Mice.

Knabel MK, Ramachandran K, Karhadkar S, Hwang HW, Creamer TJ, Chivukula RR, Sheikh F, Clark KR, Torbenson M, Montgomery RA, Cameron AM, Mendell JT, Warren DS - PLoS ONE (2015)

Bottom Line: Members of the miR-29 family, which include miR-29a, miR-29b and miR-29c, are strong inhibitors of ECM synthesis and fibrosis-associated decreases in miR-29 have been reported in multiple organs.The observed therapeutic benefits were associated with AAV transduction of hepatocytes but not hepatic stellate cells, which are the main ECM producing cells in fibroproliferative liver diseases.Our data therefore demonstrate that delivery of miR-29 to the hepatic parenchyma using a clinically relevant gene delivery platform protects injured livers against fibrosis and, given the consistent fibrosis-associated downregulation of miR-29, suggests AAV-miR-29 based therapies may be effective in treating a variety of fibroproliferative disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; The McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Fibrosis refers to the accumulation of excess extracellular matrix (ECM) components and represents a key feature of many chronic inflammatory diseases. Unfortunately, no currently available treatments specifically target this important pathogenic mechanism. MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally repress target gene expression and the development of miRNA-based therapeutics is being actively pursued for a diverse array of diseases. Because a single miRNA can target multiple genes, often within the same pathway, variations in the level of individual miRNAs can potently influence disease phenotypes. Members of the miR-29 family, which include miR-29a, miR-29b and miR-29c, are strong inhibitors of ECM synthesis and fibrosis-associated decreases in miR-29 have been reported in multiple organs. We observed downregulation of miR-29a/b/c in fibrotic livers of carbon tetrachloride (CCl4) treated mice as well as in isolated human hepatocytes exposed to the pro-fibrotic cytokine TGF-β. Importantly, we demonstrate that a single systemic injection of a miR-29a expressing adeno-associated virus (AAV) can prevent and even reverse histologic and biochemical evidence of fibrosis despite continued exposure to CCl4. The observed therapeutic benefits were associated with AAV transduction of hepatocytes but not hepatic stellate cells, which are the main ECM producing cells in fibroproliferative liver diseases. Our data therefore demonstrate that delivery of miR-29 to the hepatic parenchyma using a clinically relevant gene delivery platform protects injured livers against fibrosis and, given the consistent fibrosis-associated downregulation of miR-29, suggests AAV-miR-29 based therapies may be effective in treating a variety of fibroproliferative disorders.

No MeSH data available.


Related in: MedlinePlus

Primary and mature miR-29 expression levels in murine liver and isolated human hepatocytes.(a) Carbon tetrachloride-mediated liver fibrosis. Trichrome-stained liver sections demonstrating progressive fibrosis during 8 weeks of CCl4 exposure. Scale bar = 100μm (b) Heat map of miRNA expression levels after 1, 4, and 8 weeks of CCl4 exposure (compared to normal liver). The top 70 miRNAs with the largest average fold change are shown and sorted by fold change at 8 weeks. miR-29 family members indicated with arrows. (c) Hepatic expression levels of primary and mature miR-29a,b,c in CCl4-treated mice. Average fold changes for 1 week (n = 4), 4 week (n = 3) and 8 week (n = 2) treatment groups were calculated using normal liver (no CCl4) as a reference (n = 3). Error bars represent +/- one standard deviation. (* = p<0.05 compared to normal liver). (d) TGF-β represses miR-29 expression in human hepatocytes. Average fold change of primary and mature miR-29a/b/c as well as miR-122 and miR-130a in TGF-β treated hepatocytes were calculated using control media-treated cells as a reference. Error bars represent +/- one standard deviation. (* = p<0.05 compared to control media).
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pone.0124411.g001: Primary and mature miR-29 expression levels in murine liver and isolated human hepatocytes.(a) Carbon tetrachloride-mediated liver fibrosis. Trichrome-stained liver sections demonstrating progressive fibrosis during 8 weeks of CCl4 exposure. Scale bar = 100μm (b) Heat map of miRNA expression levels after 1, 4, and 8 weeks of CCl4 exposure (compared to normal liver). The top 70 miRNAs with the largest average fold change are shown and sorted by fold change at 8 weeks. miR-29 family members indicated with arrows. (c) Hepatic expression levels of primary and mature miR-29a,b,c in CCl4-treated mice. Average fold changes for 1 week (n = 4), 4 week (n = 3) and 8 week (n = 2) treatment groups were calculated using normal liver (no CCl4) as a reference (n = 3). Error bars represent +/- one standard deviation. (* = p<0.05 compared to normal liver). (d) TGF-β represses miR-29 expression in human hepatocytes. Average fold change of primary and mature miR-29a/b/c as well as miR-122 and miR-130a in TGF-β treated hepatocytes were calculated using control media-treated cells as a reference. Error bars represent +/- one standard deviation. (* = p<0.05 compared to control media).

Mentions: C57/BL6 mice received intraperitoneal injections of 1 ml/kg carbon tetrachloride (Sigma-Aldrich) diluted 1:7 in corn oil twice a week for up to 12 weeks. AAV was administered at a dose of 2x1011 viral genomes (vg) per animal (Figs 1, 2, 3 and 4) or 1x1012 vg/animal (high dose; S2 Fig only) via tail vein injection with a 30g needle. The Animal Care and Use Committee of the Johns Hopkins University School of Medicine reviewed and approved this study (Protocol MO13M227) and all housing and procedures were carried out in strict accordance with their policies and recommendations.


Systemic Delivery of scAAV8-Encoded MiR-29a Ameliorates Hepatic Fibrosis in Carbon Tetrachloride-Treated Mice.

Knabel MK, Ramachandran K, Karhadkar S, Hwang HW, Creamer TJ, Chivukula RR, Sheikh F, Clark KR, Torbenson M, Montgomery RA, Cameron AM, Mendell JT, Warren DS - PLoS ONE (2015)

Primary and mature miR-29 expression levels in murine liver and isolated human hepatocytes.(a) Carbon tetrachloride-mediated liver fibrosis. Trichrome-stained liver sections demonstrating progressive fibrosis during 8 weeks of CCl4 exposure. Scale bar = 100μm (b) Heat map of miRNA expression levels after 1, 4, and 8 weeks of CCl4 exposure (compared to normal liver). The top 70 miRNAs with the largest average fold change are shown and sorted by fold change at 8 weeks. miR-29 family members indicated with arrows. (c) Hepatic expression levels of primary and mature miR-29a,b,c in CCl4-treated mice. Average fold changes for 1 week (n = 4), 4 week (n = 3) and 8 week (n = 2) treatment groups were calculated using normal liver (no CCl4) as a reference (n = 3). Error bars represent +/- one standard deviation. (* = p<0.05 compared to normal liver). (d) TGF-β represses miR-29 expression in human hepatocytes. Average fold change of primary and mature miR-29a/b/c as well as miR-122 and miR-130a in TGF-β treated hepatocytes were calculated using control media-treated cells as a reference. Error bars represent +/- one standard deviation. (* = p<0.05 compared to control media).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4414421&req=5

pone.0124411.g001: Primary and mature miR-29 expression levels in murine liver and isolated human hepatocytes.(a) Carbon tetrachloride-mediated liver fibrosis. Trichrome-stained liver sections demonstrating progressive fibrosis during 8 weeks of CCl4 exposure. Scale bar = 100μm (b) Heat map of miRNA expression levels after 1, 4, and 8 weeks of CCl4 exposure (compared to normal liver). The top 70 miRNAs with the largest average fold change are shown and sorted by fold change at 8 weeks. miR-29 family members indicated with arrows. (c) Hepatic expression levels of primary and mature miR-29a,b,c in CCl4-treated mice. Average fold changes for 1 week (n = 4), 4 week (n = 3) and 8 week (n = 2) treatment groups were calculated using normal liver (no CCl4) as a reference (n = 3). Error bars represent +/- one standard deviation. (* = p<0.05 compared to normal liver). (d) TGF-β represses miR-29 expression in human hepatocytes. Average fold change of primary and mature miR-29a/b/c as well as miR-122 and miR-130a in TGF-β treated hepatocytes were calculated using control media-treated cells as a reference. Error bars represent +/- one standard deviation. (* = p<0.05 compared to control media).
Mentions: C57/BL6 mice received intraperitoneal injections of 1 ml/kg carbon tetrachloride (Sigma-Aldrich) diluted 1:7 in corn oil twice a week for up to 12 weeks. AAV was administered at a dose of 2x1011 viral genomes (vg) per animal (Figs 1, 2, 3 and 4) or 1x1012 vg/animal (high dose; S2 Fig only) via tail vein injection with a 30g needle. The Animal Care and Use Committee of the Johns Hopkins University School of Medicine reviewed and approved this study (Protocol MO13M227) and all housing and procedures were carried out in strict accordance with their policies and recommendations.

Bottom Line: Members of the miR-29 family, which include miR-29a, miR-29b and miR-29c, are strong inhibitors of ECM synthesis and fibrosis-associated decreases in miR-29 have been reported in multiple organs.The observed therapeutic benefits were associated with AAV transduction of hepatocytes but not hepatic stellate cells, which are the main ECM producing cells in fibroproliferative liver diseases.Our data therefore demonstrate that delivery of miR-29 to the hepatic parenchyma using a clinically relevant gene delivery platform protects injured livers against fibrosis and, given the consistent fibrosis-associated downregulation of miR-29, suggests AAV-miR-29 based therapies may be effective in treating a variety of fibroproliferative disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; The McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Fibrosis refers to the accumulation of excess extracellular matrix (ECM) components and represents a key feature of many chronic inflammatory diseases. Unfortunately, no currently available treatments specifically target this important pathogenic mechanism. MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally repress target gene expression and the development of miRNA-based therapeutics is being actively pursued for a diverse array of diseases. Because a single miRNA can target multiple genes, often within the same pathway, variations in the level of individual miRNAs can potently influence disease phenotypes. Members of the miR-29 family, which include miR-29a, miR-29b and miR-29c, are strong inhibitors of ECM synthesis and fibrosis-associated decreases in miR-29 have been reported in multiple organs. We observed downregulation of miR-29a/b/c in fibrotic livers of carbon tetrachloride (CCl4) treated mice as well as in isolated human hepatocytes exposed to the pro-fibrotic cytokine TGF-β. Importantly, we demonstrate that a single systemic injection of a miR-29a expressing adeno-associated virus (AAV) can prevent and even reverse histologic and biochemical evidence of fibrosis despite continued exposure to CCl4. The observed therapeutic benefits were associated with AAV transduction of hepatocytes but not hepatic stellate cells, which are the main ECM producing cells in fibroproliferative liver diseases. Our data therefore demonstrate that delivery of miR-29 to the hepatic parenchyma using a clinically relevant gene delivery platform protects injured livers against fibrosis and, given the consistent fibrosis-associated downregulation of miR-29, suggests AAV-miR-29 based therapies may be effective in treating a variety of fibroproliferative disorders.

No MeSH data available.


Related in: MedlinePlus