Gene Expression
Nebulas
Gene Expression NebulasSummary: The mRNA-based therapeutics such as COVID-19 vaccines are rapidly progressing into the clinic with a tremendous potential of benefiting millions of people worldwide. Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive aspect, however until recently, it has remained poorly explored. In this study, we examined for the first time therapeutic utility of mRNA delivery in liver fibrosis and cirrhosis, which contributes to millions of deaths, annually. Here, demonstrated the therapeutic efficacy of the human transcription factor hepatic nuclear factor alpha (HNF4A) encoding mRNA in murine chronically injured liver leading to fibrosis and cirrhosis. We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNP) encapsulating HNF4A mRNA. To identify potential mechanism, we performed microarray-based gene expression profiling, single cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for further functional validation. Expression of HNF4A encoding mRNA led to restoration of metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of HNF4A mRNA encapsulated-LNP induced a robust inhibition of fibrogenesis in four independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells. Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver.
Overall Design: Single cell RNA-seq analysis of control and HNF4A treated mice. The Control data is split in 12 fastq files. The HNF4A data is split into 12 fastq files.
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| Growth Protocol: | 8-12 weeks old male BALB/c mice for the experiments were obtained from the central animal facility of Hannover Medical School. The experiments were performed according to the regulations of the Institutional Animal Care and Use Committee of the Hannover Medical School, Germany (Protocol number: TVA 17/2658). Liver fibrosis mouse models were established by either injecting 4µl/g 10% CCl4/olive oil into BALB/c mice, twice a week for 8 weeks (fibrosis) and 16 weeks (cirrhosis), or fed with the DDC diet consistently for six weeks. 12-16 week old multidrug resistance 2 knockout mice (Mdr2-/-), a mouse ortholog of human MDR3 (also known as ATP binding cassette subfamily B member 4) on FVB background were used as a model of progressive familial intrahepatic cholestasis. Mouse primary hepatocytes were isolated via liver perfusion with Liberase (Roche) and subsequently purified by Percoll density gradient centrifugation. 0.5 million-mouse hepatocytes were seeded per well of collagen-coated 12-well plates. Four hours after seeding, the DMEM medium supplemented with 10% FBS was changed to hepatocyte culture medium (Lonza). Primary human hepatocytes were purchased from Primacyt Cell Culture Technology Gmbh (Schwerin, Germany). |
| Treatment Protocol: | Liver biopsies were performed percutaneously under local anesthesia. An approximately 5 mm portion of the needle biopsy was immediately preserved in Allprotect Tissue Reagent (QIAGEN) and then stored at -80° C. The remaining cylinder was fixed in 4% neutral buffered formalin and embedded in paraffin wax. RNA was isolated from cryo-conserved liver biopsy fragments as described recently. |
| Extract Protocol: | After liver perfusion and Percoll-density gradient centrifugation, alive cells were enriched using dead cell removal kit (Miltenyi Biotec). Then the cell viability was confirmed by sort-lab of Hannover Medical School. |
| Library Construction Protocol: | Library preparation for single cell mRNA-Seq analysis was performed according to the Chromium NextGEM Single Cell 3 Reagent Kits v3.1 User Guide (Manual Part Number CG000204 Rev B, 10x Genomics). A twofold excess of cells was loaded to the 10x controller in the specified volume in order to reach a target number of 2500 cells (N2030-N2031) or 7500 cells (N2032-N2033) per sample. Fragment length distribution of generated cDNAs and libraries was monitored using Bioanalyzer High Sensitivity DNA Assay (5067-4626, Agilent Technologies). Quantification of libraries was performed by use of the ‘Qubit® dsDNA HS Assay Kit’ (Q32854, ThermoFisher Scientific). |
| Molecule Type: | polyA(+) RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 550 |
| Strand-Specific: | - |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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