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

<div><p>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.</p></div

Intervention with scAAV8.miR29a.eGFP reverses histologic evidence of CCl₄-mediated hepatic fibrosis.

<p>(a) Timeline of AAV delivery and CCl₄ treatment. (b) Estimate of viral genomes/cell in livers of scAAV-transduced mice. A portion of the viral genome (GFP) and a non-repetitive locus in the mouse genome (DGCR8) were separately amplified from whole liver genomic DNA. Standard curves of known amounts of AAV8.eGFP plasmid DNA and whole liver genomic DNA were used to determine the number of viral and cellular genomes in each sample, respectively. (c) Hepatic expression of primary and mature miR-29a in CCl₄-treated scAAV8.eGFP (n = 4; one of the five scAAV8 injected mice died during CCl₄ treatment) and scAAV8.miR29a.eGFP transduced animals (n = 5). In parallel, three additional animals were sacrificed after 4 weeks of CCl₄ treatment to establish the level of fibrosis present at the time of viral delivery (pre-treatment). Average fold change for each treatment group was calculated using normal (no CCl₄) scAAV8.eGFP treated mice as a reference (n = 3). Error bars represent +/- one standard deviation. (d) Trichrome staining reveals reduced collagen deposition (blue) in scAAV8.miR29a.eGFP transduced livers compared to either pre-treatment (4 weeks CCl₄) or scAAV8.eGFP treatment (12 weeks CCl₄). Scale bar = 100μm (e) The degree of fibrosis was scored on a scale of 0–4 by a trained pathologist (blinded to treatment condition) and the score for each individual animal is shown. (f) Quantitative determination of hepatic collagen levels in transduced animals. Error bars represent +/- one standard deviation. (* = p<0.05).</p

Primary and mature miR-29 expression levels in murine liver and isolated human hepatocytes.

<p>(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).</p

scAAV8 transduction and miR-29 expression levels in murine liver.

<p>(a) Schematic representation of scAAV vectors depicting locations of inverted terminal repeats (ITRs), elongation factor 1-alpha promoter (EF1α), miRNA (shown in hairpin form), and enhanced green fluorescent protein (eGFP) open reading frame. (b) Transduction with scAAV8 does not disrupt normal liver architecture. Trichrome stained liver sections from AAV-transduced animals demonstrating normal histology. Scale bar = 100μm (c) Viral genomic DNA (gDNA) and mRNA from the EF1α transcription unit (cDNA) are readily detectable in mouse liver following transduction with 2x10¹¹ vg of scAAV8.eGFP (n = 3) or scAAV8.miR29a.eGFP (n = 3). The presence of the hairpin accounts for the increased size of the scAAV8.miR29a.eGFP gDNA amplicon. (d) Hepatic expression of primary and mature miR-29a in scAAV8 transduced mice. Average fold change for each treatment group was calculated using scAAV8.eGFP treated mice as a reference (n = 3). Error bars represent +/- one standard deviation. (e-i) Localization of AAV-mediated GFP expression in transduced mouse liver. Sections of transduced livers were co-immunostained for GFP (e and g-i; green) and markers of hepatocytes (Albumin f and g; red) or stellate cells (Desmin h; Vimentin i; red). Open arrowheads indicate GFP+ hepatocyte and filled arrowheads indicate desmin+ or vimentin+ stellate cells. All sections were counterstained with Hoechst (blue). Confocal images were captured with a 40x objective and are shown at 2x zoom. Scale bar = 20μm.</p

Pre-treatment with scAAV8.miR29a.eGFP prevents CCl₄-mediated hepatic fibrosis.

<p>(a) Timeline of AAV delivery and CCl₄ treatment. (b) Estimate of viral genomes/cell in livers of scAAV-transduced mice. A portion of the viral genome (GFP) and a non-repetitive locus in the mouse genome (DGCR8) were separately amplified from whole liver genomic DNA. Standard curves of known amounts of AAV8.eGFP plasmid DNA and whole liver genomic DNA were used to determine the number of viral and cellular genomes in each sample, respectively. (c) Hepatic expression of primary and mature miR-29a in the livers of CCl₄-treated scAAV8.eGFP (n = 5) and scAAV8.miR29a.eGFP transduced animals (n = 5). Average fold change for each treatment group was calculated using normal (no CCl₄) scAAV8.eGFP treated mice as a reference (n = 3). Error bars represent +/- one standard deviation. (d) Trichrome staining reveals reduced collagen deposition (blue) in scAAV8.miR29a.eGFP transduced livers. Scale bar = 100μm (e) The degree of fibrosis was scored on a scale of 0–4 by a trained pathologist (blinded to treatment condition) and the score for each individual animal is shown. (f) Quantitative determination of hepatic collagen levels in transduced animals. Error bars represent +/- one standard deviation. (* = p<0.05).</p