Project description:To study monocyte and macrophage activation in ANCA-associtated vasculitis (AAV), we performed bulk RNA sequencing of bead-selected monocytes and in vitro cultured monocyte-derived macrophages from AAV patients and healthy controls. Overview patients included for sequencing monocytes: - AAV active disease, n=4, MPO-AAV=4 - AAV remission, n=10, PR3-AAV=5, MPO-AAV=5 - Healthy controls, n=6 Overview patients included for sequencing monocyte-derived macrophages: - AAV active, n=1, PR3-AAV=1 - AAV remission, n=3, PR3-AAV=3 - Healthy controls, n=3
Project description:Recombinant AAV vectors have the unique ability to promote targeted integration of transgenes via homologous integration at specified genomic sites reaching frequencies of 0.1-1%. We studied genomic parameters that influence targeting efficiencies on a large scale. To do this, we generated more than 1000 engineered, doxycycline-inducible target sites in the human HAP1 cell line and infected this polyclonal population with a library of AAV-DJ targeting vectors each carrying a unique barcode. The heterogeneity of barcode integration at each target site provided an assessment of the targeting efficiency at that locus. We compared targeting efficiency with and without target site transcription for identical chromosomal positions, finding that targeting efficiency was enhanced by target site transcription. Chromatin states associated with active transcription were also predictive of higher targeting efficiency. Furthermore, there was an effect on the amenability of a site to targeting due to other factors such as the level of transcription from intersecting genes. These results define important parameters that may not only assist in designing optimal targeting vectors for genome editing, but also provide new insights into the mechanism of AAV-mediated homologous recombination.
Project description:This is an ATAC sequencing experiment to explore chromatin accessibility change in mouse skeletal muscle treated with either AAV-GFP or AAV-CAAHR (a constitutively active mutant aryl hydrocarbon receptor). Mice received intramuscular injection of the AAV 5 months before the harvest of muscle.
Project description:Deciphering patterns of connectivity between neurons in the mammalian brain is a critical step toward understanding brain function. Conventional imaging based neuroanatomical tracing methods identify area-to-area or sparse neuron-to-neuron connectivity patterns, but with extremely limited throughput. Recently developed barcode-based connectomics methods can efficiently map large numbers of single-neuron projections, but linking these data to single-cell transcriptomics remains a challenge. Here, we established a retro-AAV barcode-based multiplexed tracing method called MERGE-seq (Multiplexed projection neuRons retroGrade barcodE sequencing), which is capable of simultaneously characterizing the projectome and transcriptome at the single neuron level. We uncovered dedicated and collateral projection patterns of ventromedial prefrontal cortex (vmPFC) neurons to five downstream targets (AI, DMS, BLA, MD and LH). We found that projection-defined vmPFC neurons are molecularly heterogeneous, which are composed of different neuronal subtypes. We further identified transcriptional signatures of various dedicated and bifurcated vmPFC neurons, and verified Pou3f1 as the marker gene of neurons sending collateral axons to DMS and LH. Finally, we fitted our single-neuron connectome/transcriptome data into a machine learning-based model and revealed groups of genes that were predictive of certain projection pattern. In summary, we have developed a new multiplexed technique whose paired connectome and gene expression data can help reveal organizational principles that form neural circuits and process information.
Project description:Deciphering patterns of connectivity between neurons in the mammalian brain is a critical step toward understanding brain function. Conventional imaging based neuroanatomical tracing methods identify area-to-area or sparse neuron-to-neuron connectivity patterns, but with extremely limited throughput. Recently developed barcode-based connectomics methods can efficiently map large numbers of single-neuron projections, but linking these data to single-cell transcriptomics remains a challenge. Here, we established a retro-AAV barcode-based multiplexed tracing method called MERGE-seq (Multiplexed projection neuRons retroGrade barcodE sequencing), which is capable of simultaneously characterizing the projectome and transcriptome at the single neuron level. We uncovered dedicated and collateral projection patterns of ventromedial prefrontal cortex (vmPFC) neurons to five downstream targets (AI, DMS, BLA, MD and LH). We found that projection-defined vmPFC neurons are molecularly heterogeneous, which are composed of different neuronal subtypes. We further identified transcriptional signatures of various dedicated and bifurcated vmPFC neurons, and verified Pou3f1 as the marker gene of neurons sending collateral axons to DMS and LH. Finally, we fitted our single-neuron connectome/transcriptome data into a machine learning-based model and revealed groups of genes that were predictive of certain projection pattern. In summary, we have developed a new multiplexed technique whose paired connectome and gene expression data can help reveal organizational principles that form neural circuits and process information.
Project description:To explore how Mier1 influence the genome H3K27ac levels during liver regeneration, we performed H3K27ac chromatin immunoprecipitation followed by sequencing in control and liver-specific Mier1 ko liver tissues at 0 h and 24 h after 70% partial hepatectomy (PHx). The mice we used were knocked in a Cre-induced Cas9 expression cassette. Through tail vein injection, we delivered the AAV expressing Cre-recombinase under TBG promoter, and sgRNA targeting Mier1 (AAV-Mier1 sgRNA), into the adult Cas9 knockin mice to knock out the Mier1 gene in liver. AAV vectors with no sgRNA inserted (AAV-Cre) were used in control animals.Then we performed 70% partial hepatectomy, 3 weeks after AAV injection. Consistent with the increased expression of cell cycle genes during liver regeneration, we observed increased signals of H3K27ac at 24 h after hepatectomy, especially near some cell cycle genes, after MIER1 depletion in liver tissue.
Project description:AAV gene therapy has recently been approved for clinical use and shown to be efficacious and safe in a growing number of clinical trials. However, the safety of AAV as a gene therapy has been challenged by a few studies that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. The association between AAV and HCC has been difficult to reconcile and is the subject of intense debate because numerous AAV studies have not reported toxicity. Here, we report a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the Rian locus and the over-expression of a proximal gene, Rtl1, were associated with HCC. In addition, we identify a number of genes with differential expression that maybe useful in the study, diagnosis and treatment of HCC. We demonstrate that AAV vector dose, enhancer-promoter selection, and the timing of gene delivery are the defining factors in AAV-mediated insertional mutagenesis. Our results help explain the AAV-mediated genotoxicity previously observed and have important implications for the design of both safer AAV vectors and gene therapy studies. To investigate the possibility that insertional mutagenesis by AAV contributed to the development of HCC, we collected normal and tumor tissues from adult mouse livers that received AAV injection at a neonatal stage.
Project description:AAV gene therapy has recently been approved for clinical use and shown to be efficacious and safe in a growing number of clinical trials. However, the safety of AAV as a gene therapy has been challenged by a few studies that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. The association between AAV and HCC has been difficult to reconcile and is the subject of intense debate because numerous AAV studies have not reported toxicity. Here, we report a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the Rian locus and the over-expression of a proximal gene, Rtl1, were associated with HCC. In addition, we identify a number of genes with differential expression that maybe useful in the study, diagnosis and treatment of HCC. We demonstrate that AAV vector dose, enhancer-promoter selection, and the timing of gene delivery are the defining factors in AAV-mediated insertional mutagenesis. Our results help explain the AAV-mediated genotoxicity previously observed and have important implications for the design of both safer AAV vectors and gene therapy studies. To investigate the possibility that insertional mutagenesis by AAV contributed to the development of HCC, we collected normal and tumor tissues from adult mouse livers that received AAV injection at a neonatal stage.
Project description:To explore the effect of Bicd2 in Con A-induced acute autoimmune hepatitis, we conducted single cell RNA sequencing of AAV-scramble or AAV-shBicd2 infected mice livers in response to Con A injection.