CRISPR/Cas9-mediated knock-out of AGXT1 in HepG2 cells as a new in vitro model of Primary Hyperoxaluria Type 1

Gatticchi, Leonardo; Grottelli, Silvia; Ambrosini, Giulia; Pampalone, Gioena; Gualtieri, Ottavia; Dando, Ilaria; Bellezza, Ilaria; Cellini, Barbara

doi:10.1016/j.biochi.2022.08.005

Cited by 7 publications

(3 citation statements)

References 62 publications

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“…While our CETSA assay provides a better indication of compound cellular efficacy than previously reported indirect assay in non-human cell lines, its current application in HEK293T cells could introduce non-physiological levels of HAO1 expression and differences in the cellular environment compared to hepatocytes, where therapeutic molecules in development need to engage with HAO1. Future work adapting SplitLuc CETSA for use in the immortalised hepatoma-derived cell line HepG2, which has been recently validated as a model cell line for primary hyperoxaluria 40 , would overcome this limitation. An additional avenue to explore in future would be the relationship between compound binding to HAO1…”

Section: Discussionmentioning

confidence: 99%

Luminescence-based complementation assay to assess target engagement and cell permeability of glycolate oxidase (HAO1) inhibitors

Mackinnon,

Zarganes-Tzitzikas,

Adams

et al. 2024

Preprint

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Glycolate oxidase (HAO1) catalyses the synthesis of glyoxylate, a common metabolic intermediate that causes renal failure if accumulated. HAO1 inhibition is an emerging treatment for primary hyperoxaluria, a rare disorder of glyoxylate metabolism. Here we report the first cell-based measurement of inhibitor uptake and engagement with HAO1, by adapting the cellular thermal shift assay (CETSA) based on Nano luciferase complementation and luminescence readout. By profiling the interaction between HAO1 and four well-characterised inhibitors in intact and lysed HEK293T cells, we showed that our CETSA method differentiates between low-permeability/high-engagement and high-permeability/low-engagement ligands and is able to rank HAO1 inhibitors in line with both recombinant protein methods and previously reported indirect cellular assays. Our methodology addresses the unmet need for a robust, sensitive, and scalable cellular assay to guide HAO1 inhibitor development and, in broader terms, can be rapidly adapted for other targets to simultaneously monitor compound affinity and cellular permeability.

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Section: Discussionmentioning

confidence: 99%

Luminescence-based complementation assay to assess target engagement and cell permeability of glycolate oxidase (HAO1) inhibitors

Mackinnon,

Zarganes-Tzitzikas,

Adams

et al. 2024

Preprint

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“…This situation has posed new challenges in the interpretation of missense variants, and has highlighted the use of functional studies to support pathogenicity assessment [ 57 ]. As for PH1, our group recently setup a new cellular model of disease based on HepG2 cells, a cell line of hepatic origin showing a conserved glyoxylate/oxalate metabolism [ 58 , 59 ]. The model was used to support diagnosis in a PH1 patient who was compound heterozygous for a validated pathogenic mutation and a novel mutation, p.Gly365Cys [ 60 ▪ ].…”

Section: Molecular Pathogenesis Of Primary Hyperoxaluria Type 1 (Ph1)mentioning

confidence: 99%

A molecular journey on the pathogenesis of primary hyperoxaluria

Cellini

2024

Current Opinion in Nephrology &Amp; Hypertension

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Purpose of review Primary hyperoxalurias (PHs) are rare disorders caused by the deficit of liver enzymes involved in glyoxylate metabolism. Their main hallmark is the increased excretion of oxalate leading to the deposition of calcium oxalate stones in the urinary tract. This review describes the molecular aspects of PHs and their relevance for the clinical management of patients. Recent findings Recently, the study of PHs pathogenesis has received great attention. The development of novel in vitro and in vivo models has allowed to elucidate how inherited mutations lead to enzyme deficit, as well as to confirm the pathogenicity of newly-identified mutations. In addition, a better knowledge of the metabolic consequences in disorders of liver glyoxylate detoxification has been crucial to identify the key players in liver oxalate production, thus leading to the identification and validation of new drug targets. Summary The research on PHs at basic, translational and clinical level has improved our knowledge on the critical factors that modulate disease severity and the response to the available treatments, leading to the development of new drugs, either in preclinical stage or, very recently, approved for patient treatment.

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“…43 percent of Pakistani children patients with kidney stones have undifferentiated hyperoxaluria [8]. Above 200 various pathogenic or likely pathogenic variants in the AGXT gene (NM_000030, located on 2q37.3) are discovered so far [6] and are reported in the ClinVardatabase (http://www.clinvar.com/) [21]. The progression of PH1 to renal failure is diverse, and its age of start is unpredictable.…”

Section: Introductionmentioning

confidence: 99%

Molecular Analysis of the AGXT Gene Detected a Missense and Pathogenic Variant Associated with Primary Hyperoxaluria Type 1; a Case Study

saba,

Khan,

Rehman

et al. 2023

Preprint

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Background Primary Hyperoxaluria Type 1 (PH1) is an autosomal recessive genetic disorder triggered by a mutation in the alanine glyoxylate aminotransferase (AGXT) gene. Early detection of PH1 is a pre-requisite as it causes End Stage Renal Disease (ESRD) in most patients in the early stages. An eleven years old girl with a history of kidney disease and stones and with phenotypic characteristics of PH1 was brought to the laboratory. A c.568G>A mutation in AGXT gene, which is responsible for PH1, is found in a homozygous condition. Further study revealed the detection of the mutation in heterozygous form in both the parents. This study provides insight to generate more reliable genetic markers for the early detection of PH1 in a family or a population. This can lead to better and earlier treatment strategies. Case Presentation This study aimed to detect the AGXT gene mutationswhich are responsible for primary hyperoxaluriain the patient.AGXT gene screening was done in her parents for identifying the root cause and zygosity of the mutation. The AGXT gene on chromosome2q37.3was amplified via polymerase chain reaction and sequenced by Sanger sequencing. Molecular modeling and genetic change analysis was performed by using in-silico parameters. Conclusion The sequence analysis revealed the presence of a missense and pathogenic variant in the homozygous condition in the AGXT gene exon 5;c.568G>A with protein change p. Gly190Arg in the patient. Parental screening showed that the patient received one allele from her father and the other from her mother. A liver transplant followed by a kidney transplant was carried out in the patient with 6 months difference. The study emphasized that as theb mutation p.Gly190Arg is reported as a cause of PH1, this mutation can be considered an early diagnostic marker for PH1.

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CRISPR/Cas9-mediated knock-out of AGXT1 in HepG2 cells as a new in vitro model of Primary Hyperoxaluria Type 1

Cited by 7 publications

References 62 publications

Luminescence-based complementation assay to assess target engagement and cell permeability of glycolate oxidase (HAO1) inhibitors

Luminescence-based complementation assay to assess target engagement and cell permeability of glycolate oxidase (HAO1) inhibitors

A molecular journey on the pathogenesis of primary hyperoxaluria

Molecular Analysis of the AGXT Gene Detected a Missense and Pathogenic Variant Associated with Primary Hyperoxaluria Type 1; a Case Study

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