Hepatolenticular degeneration, also known as Wilson's disease (WD), is a rare autosomal recessive disorder of copper metabolism, and the pathogenic gene is ATP7B. The ATP7B gene, located on chromosome 13 (13Q14.3), encodes a 1411 amino acid Cu transporter P-type ATPASE. Mutation of ATP7B gene results in weakened or lost ATPASE function, resulting in decreased synthesis of ceruloplasmin (CP) in serum and impaired biliary duct Cu discharge, and deposition of Cu ions in liver, brain, kidney and cornea. It would causes progressive cirrhosis, extrapyramidal symptoms, psychiatric symptoms, kidney damage, and corneal pigmentation ring (Kayser-Fleischerring, K.F ring), resulting in Wilson's disease. The worldwide incidence of WD is 1/30 000 ~ 1/100 000, and the prevalence of WD is about 1/90. In Asia, the most common type is R778L, but the pathogenesis of this mutation remains unclear. This disease is more common in China, which is more common in young people and slightly more common in males than females. If not properly treated, WD can cause disability or even death. WD is also one of the few treatable neurogenetic diseases. The key is early detection, early diagnosis and early treatment.
The combination therapy of MT1X induction and Cu chelation provides an effective idea for the treatment of Wilson's disease.
Cu chelators and zinc salts are the two most important drugs used in the treatment of WD patients; However, the molecular mechanisms associated with ATP7B expression in these drugs have not been identified. A targeted knockout of ATP7B (KO) was established in the most widely used human hepatoma cell line, HepG2 for molecular studies of the pathogenesis and treatment of the disease. KO cells showed similar growth, Cu uptake, release, and gene expression as compared to parental cells. However, in the presence of Cu, morphological changes, oxidative stress, apoptosis, and loss of viability were observed. Induction of metallothionein (MT1X) after Cu exposure was significantly reduced in KO cells. Following zinc treatment, MT1X expression was strongly induced and a high percentage of KO cells could be rescued from Cu induced toxicity. D-penicillamine treatment had a minor effect on the viability of KO cells whereas the parental cell line showed a pronounced improvement. Combined treatment displayed a highly synergistic effect in KO cells. The data suggest that zinc has a previously unrecognized effect on the viability of hepatocytes that lack ATP7B due to a high induction of MT1X expression that compensates low gene expression after Cu exposure. A combination therapy that simultaneously targets at MT1X induction and Cu chelation improves the overall survival of hepatocytes for most efficient therapy of patients with WD. KO cells played a key role in this study, Ubigene now has nearly 2,000 types of KO cells in stock, covering thousands of genes from 8 popular signaling pathways, popular immune checkpoints and diseases (most are cancer disease).
Verify the effects of Cu on lipid homeostasis in intestinal cells with ATP7B KO cell line.
In another study, Guttmann et al. constructed ATP7B knockout cell lines in human intestinal cacO-2 cells to determine the relationship between Cu and lipid homeostasis and metabolism in intestinal cells. Caco-2 cells were transfected with plasmids containing endonuclease Cas9 and the gRNA target to exon 2 of human ATP7B. The results showed that KO cells had increased sensitivity to Cu, increased intracellular Cu storage, and induced genes regulating oxidative stress. The results showed that the sensitivity to Cu and intracellular Cu storage had increased, and induced genes regulating oxidative stress in KO cells. The chylomicron structural protein ApoB48 was significantly down-regulated by Cu in KO cells. Apolipoprotein ApoA1, ApoC3, and ApoE were induced by knockout of ATP7B. Cu enhances the formation of small lipid droplets and decreases the formation of large lipid droplets. At the same time, Cu decreased the storage and secretion of triglycerides (TG). The KO cells exposing in oleic acid (OA) leads to enhanced TG storage. In a word, The results suggest that Cu inhibits TG adipogenesis in the intestine, while ATP7B deficiency leads to TG storage induced by OA.
The mouse model of ATP7B gene R778L and P992L point mutation was constructed by CRISPR/Cas9 technique.
Dong et al. used CRISPR/Cas9 technology to target Arg778Leu and Pro992Leu, which are the common mutation sites of ATP7B gene in WD patients in China, and injected Cas9 mRNA into mouse single-cell embryos by microinjection. Finally, a mouse model simulating ATP7B gene R778L and P992L point mutations was successfully constructed. The clinical phenotypes of the two mutant mice were observed from the physiological and pathological perspectives, and their phenotypes were verified to conform to the clinical symptoms of WD patients. It was found that the serum ceruloplasmin level of R778L point mutant mice was significantly lower than that of wild-type mice, while the serum non-ceruloplasmin binding Cu content of R778L and P992L mice was significantly higher than that of wild-type mice. At the same time, these mice showed pathological liver damage, while R778L mice showed behavioral abnormalities. This study accurately constructed a point mutation mouse model of WD in Chinese population, providing an ideal tool for the pathogenesis and gene therapy research of WD.
"Make gene editing easier!" has always been Ubigene aspiration. Ubigene developed the CRISPR-U™ technology, which is greatly improve the efficiency of homologous recombination. With CRISPR-U™ , Ubigene has successfully generated over 5000 KO cell lines for the researchers all over the world. In addition, Ubigene now has nearly 2,000 types of KO cells in stock. For more details, please feel free to contact us!
ursimran C , Nadine S , Vanessa S , et al. The Effect of Zinc and D-Penicillamine in a Stable Human Hepatoma ATP7B Knockout Cell Line[J]. Plos One, 2014, 9(6):e98809.
Guttmann S , Nadzemova O , I Grünewald, et al. ATP7B knockout disturbs copper and lipid metabolism in Caco-2 cells[J]. PLoS ONE, 2020, 15(3):e0230025.
Jianjian D. Construction of Wilson's disease mouse model using CRISPR/Cas9 technique and study on gene therapy[D].University of Science and Technology of China, 2021.DOI:10.27517/d.cnki.gzkju.2021.000522.