Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10382428_1.fastq.gz | Fastqsanger.gz | |||
| SRR10382428_2.fastq.gz | Fastqsanger.gz | |||
| SRR10382429_1.fastq.gz | Fastqsanger.gz | |||
| SRR10382429_2.fastq.gz | Fastqsanger.gz | |||
| SRR10382430_1.fastq.gz | Fastqsanger.gz |
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Project description:Primary Hyperoxaluria Type 1 (PH1) is a rare inherited metabolic disorder characterized by oxalate overproduction in the liver, resulting in renal damage. It is caused by mutations in the AGXT gene. Combined liver and kidney transplantation is currently the only permanent curative treatment. We combined locus-specific gene correction and hepatic direct cell reprogramming to generate autologous healthy induced hepatocytes (iHeps) from PH1 patient-derived fibroblasts. First, site-specific AGXT corrected cells were obtained by homology directed repair (HDR) assisted by CRISPR/Cas9, following two different strategies: accurate point mutation (c.853T>C) correction or knock-in of an enhanced version of AGXT cDNA. Then, iHeps were generated, by overexpression of hepatic transcription factors. Generated AGXT-corrected iHeps showed hepatic gene expression profile and exhibited in vitro reversion of oxalate accumulation compared to non-edited PH1-derived iHeps. This strategy set up a potential alternative cellular source for liver cell replacement therapy and a personalized PH1 in vitro disease model.
2024-03-27 | GSE226019 | GEO
Project description:Misfolding loss-of-function diseases are a huge burden for people and states. Primary hyperoxaluria type 1 (PH1) is a rare genetic disorder caused by mutations in the alanine:glyoxylate aminotransferase 1 (AGT) enzyme. The underlying molecular mechanisms causing PH1 are associated with protein misfolding (enhanced aggregation and mitochondrial mistargeting). The main therapeutic approach to increase patients lifespan and quality of life is a double transplantation of kidney and liver. Alternative treatments are currently under study, such as gene and enzyme replacement therapies and pharmacological chaperones as treatments, but other alternatives are necessary. In this work, we developed and characterize a novel biotechnological approach using six single domain nanobodies (NB-AGT-1 to -6) as potential therapeutics for PH1 misfolding. We show that NB-AGTs are very stable proteins and bind to pathogenic and non-pathogenic variants of AGT with extreme affinities (with Kd values from low nM to low pM). Structural studies showed that NB-AGTs bind to different epitopes of AGT with selectivity for different AGT variants. Experiments in cellular PH1 models showed that internalization of engineered NB-AGT-3 enhanced the specific activity of disease-associated variants and retargeted the protein to peroxisomes. Overall, we show that NBs are a novel and promising approach to treat PH1 as well as other loss-of-function misfolding diseases.
2026-01-06 | PXD060227 | Pride
Project description:The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. Additionally, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.
2023-11-10 | GSE245791 | GEO
Project description:This study aims to investigate a wheat recombination hotspot (H1) in comparison with a “regular” recombination site (Rec7) on the sequence and epigenetic level in conditions with functional and non-functional Ph1 locus.
2022-12-14 | GSE220598 | GEO
Project description:Hepatocytes-like cells (HLC) derived from human induced pluripotent stem cells show great promise for cell-based liver therapies and disease modelling. However, their application is currently hindered by the low production yields of existing protocols. We aim to develop a bioprocess able to generate high numbers of HLC. We used stirred-tank bioreactors with a rational control of dissolved oxygen concentration (DO) for the optimization of HLC production as 3D aggregates. We evaluated the impact of controlling DO at physiological levels (4%O2) during hepatic progenitors’ stage on cell proliferation and differentiation efficiency. Whole transcriptome analysis and biochemical assays were performed to provide a detailed characterization of HLC quality attributes. When DO was controlled at 4%O2 during the hepatic progenitors’ stage, cells presented an upregulation of genes associated with hypoxia-inducible factor pathway and a downregulation of oxidative stress genes. This condition promoted higher HLC production (maximum cell concentration: 2×106 cell/mL) and improved differentiation efficiencies (80% Albumin-positive cells) when compared to the bioreactor operated under atmospheric oxygen levels (21%O2, 0.6×106 cell/mL, 43% Albumin positive cells). These HLC exhibited functional characteristics of hepatocytes: capacity to metabolize drugs, ability to synthesize hepatic metabolites, and inducible cytochrome P450 activity. Bioprocess robustness was confirmed with HLC derived from different donors, including a primary hyperoxaluria type 1 (PH1) patient. The generated PH1.HLC showed metabolic features of PH1 disease with higher secretion of oxalate compared with HLC generated from healthy individuals. This work reports a reproducible bioprocess, that shows the importance of controlling DO at physiological levels to increase HLC production, and the HLC capability to display PH1 disease features.
2024-10-23 | GSE267210 | GEO
Project description:Primary hyperoxaluria type I (PH1) is a genetic disease caused by a deficiency in the peroxisomal alanine:glyoxylate aminotransferase (AGT) activity. Mutations in AGT mostly cause protein mistargeting and enhanced aggregation, although the molecular and structural basis of these mechanisms are unknown. In this work, we use hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) to provide novel insight into these pathogenic mechanisms. We characterize the wild-type (WT) protein, the LM variant (containing the mutations P11L and I340M, a haplotype more frequent in PH1 patients) and the LM G170R (the most common genotype in PH1, introducing the G170R mutation on the LM background). Our study provides the first experimental analysis of the local stability and dynamics of AGT, showing that stability is heterogeneous in the native state and providing a blueprint for frustrated regions with potentially functional relevance. The LM and LM G170R variants destabilize locally the structure. Enzymatic transamination of the PLP bound to AGT hardly affects stability. Our study thus supports that AGT misfolding is not caused by dramatic effects on the stability and dynamics of the holo-protein.
2024-01-25 | PXD046749 | Pride
Project description:In this study, a whole-genome CRISPRi library was developed in Y. lipolytica and applied for enforcing the tolerance to furfural and acetic acid. Several novel gene targets were discovered through NGS before and after screening. And to analyze the tolerance mechanism caused by transcriptional repression of the most prominent gene targets, the transcriptome and proteomics were performed.
2024-03-31 | PXD051116 |