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Updated: May 10, 2025
Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes
Published on: June 2, 2022
Investigation on ABCC6-Deficient Human Hepatocytes Generated by CRISPR-Cas9 Genome Editing
Ricarda Plümers1, Svenja Jelinek1, Christopher Lindenkamp1
1Herz- und Diabeteszentrum Nordrhein-Westfalen, Institut für Laboratoriums- und Transfusionsmedizin, Universitätsklinik der Ruhr-Universität Bochum, Medizinische Fakultät OWL (Universität Bielefeld), Georgstraße 11, 32545 Bad Oeynhausen, Germany.
Abstract:
Patients affected by the rare disease pseudoxanthoma elasticum (PXE) exhibit the calcification of elastic fibers in ocular, dermal, and vascular tissues. These symptoms are triggered by mutations in the ATP-binding cassette transporter subfamily C member 6 (ABCC6), whose substrate remains unknown. Interestingly, ABCC6 is predominantly expressed in the liver tissue, leading to the hypothesis that PXE is a metabolic disorder. We developed a genome-editing system targeting ABCC6 in human immortalized hepatocytes (HepIms) for further investigations. The HepIms were transfected with an ABCC6-specific clustered regulatory interspaced short palindromic repeat (CRISPR-Cas9) genome-editing plasmid, resulting in the identification of a heterozygous (htHepIm) and a compound heterozygous (chtHepIm) clone. These clones were analyzed for key markers associated with the PXE pathobiochemistry. Hints of impaired lipid trafficking, defects in the extracellular matrix remodeling, the induction of calcification inhibitor expression, and the down regulation of senescence and inflammatory markers in ABCC6-deficienct HepIms were found. Our ABCC6 knock-out model of HepIms provides a valuable tool for studying the metabolic characteristics of PXE in vitro. The initial analysis of the clones mirrors various features of the PXE pathobiochemistry and provides an outlook on future research approaches.
Insights
Researchers created a new model for studying pseudoxanthoma elasticum (PXE), a rare genetic disorder. This model uses gene editing in liver cells to investigate the role of ATP-binding cassette transporter subfamily C member 6 (ABCC6) in PXE.
Area of Science:
- Genetics and Molecular Biology
- Rare Disease Research
- Cellular Metabolism
Background:
- Pseudoxanthoma elasticum (PXE) is a rare genetic disorder characterized by calcification of elastic fibers in various tissues.
- Mutations in the ATP-binding cassette transporter subfamily C member 6 (ABCC6) gene are linked to PXE, but its substrate and PXE's exact nature as a metabolic disorder remain unclear.
- ABCC6's predominant expression in the liver suggests a potential metabolic basis for PXE.
Purpose of the Study:
- To develop a genome-editing system targeting ABCC6 in human hepatocytes to create a model for studying PXE.
- To investigate the pathobiochemical characteristics of PXE in ABCC6-deficient liver cells.
- To provide an in vitro tool for understanding PXE's metabolic aspects.
Main Methods:
- Developed a clustered regulatory interspaced short palindromic repeat (CRISPR-Cas9) genome-editing system targeting ABCC6 in human immortalized hepatocytes (HepIms).
- Generated heterozygous (htHepIm) and compound heterozygous (chtHepIm) ABCC6-deficient cell clones.
- Analyzed these clones for key markers related to PXE pathobiochemistry, including lipid trafficking, extracellular matrix remodeling, and inflammatory markers.
Main Results:
- Identified ABCC6-deficient HepIm clones exhibiting potential defects in lipid trafficking and extracellular matrix remodeling.
- Observed induction of calcification inhibitor expression and downregulation of senescence and inflammatory markers in the deficient cells.
- The developed model recapitulates several features of PXE pathobiochemistry.
Conclusions:
- The ABCC6-deficient HepIm model offers a valuable in vitro tool for investigating the metabolic underpinnings of PXE.
- Initial findings suggest ABCC6 deficiency impacts cellular processes relevant to PXE pathogenesis.
- This model facilitates future research into PXE's metabolic characteristics and potential therapeutic strategies.
