CRISPR/Cas9-mediated correction of mutated copper transporter ATP7B

Abstract

Wilson's disease (WD) is a monogenetic liver disease that is based on a mutation of the ATP7B gene and leads to a functional deterioration in copper (Cu) excretion in the liver. The excess Cu accumulates in various organs such as the liver and brain. WD patients show clinical heterogeneity, which can range from acute or chronic liver failure to neurological symptoms. The course of the disease can be improved by a lifelong treatment with zinc or chelators such as D-penicillamine in a majority of patients, but serious side effects have been observed in a significant portion of patients, e.g. neurological deterioration and nephrotoxicity, so that a liver transplant would be inevitable. An alternative therapy option would be the genetic correction of the ATP7B gene. The novel gene therapy method CRISPR/Cas9, which has recently been used in the clinic, may represent a suitable therapeutic opportunity. In this study, we first initiated an artificial ATP7B point mutation in a human cell line using CRISPR/Cas9 gene editing, and corrected this mutation by the additional use of singlestranded oligo DNA nucleotides (ssODNs), simulating a gene correction of a WD point mutation in vitro. By the addition of 0.5 mM of Cu three days after lipofection, a high yield of CRISPR/Cas9-mediated ATP7B repaired cell clones was achieved (60%). Moreover, the repair efficiency was enhanced using ssODNs that incorporated three blocking mutations. The repaired cell clones showed a high resistance to Cu after exposure to increasing Cu concentrations. Our findings indicate that CRISPR/Cas9-mediated correction of ATP7B point mutations is feasible and may have the potential to be transferred to the clinic.

CRISPR/Cas9 systems: Delivery technologies and biomedical applications

“…The field of medical research focused on utilizing the CRISPR/Cas9 system for gene editing therapies is experiencing rapid growth. It is increasingly recognized for its prominent role in addressing diseases with complex etiologies like DMD [147] , liver diseases [148] , and retinal diseases [80] . DMD is a genetic muscle disease caused by mutations in the DMD gene located on the X chromosome [149 , 150] .…”

Wilson disease in children and young adults - State of the art

“…Indications for liver transplantation (LT)[ 67 ] in patients with WD are those with ALF, as defined by the rapid development of severe hepatic insufficiency with coagulopathy and with hepatic encephalopathy, with progression of liver dysfunction to liver failure despite drug therapy, and those with acute on chronic liver failure due to WD. [ 1 17 43 ] LT in patients with neurologic WD is still controversial.…”

“…[ 72 73 74 75 ] A recent study reported the use of CRISPR/Cas9 technology to correct ATP7B point mutation frequently detected in WD patients. [ 67 ] However, clinical studies for WD gene modification are required so that these could become alternative curative strategies in the future.…”

Novel Gene-Correction-Based Therapeutic Modalities for Monogenic Liver Disorders

“…Pöhler and colleagues showed that CRISPR-Cas9 technology is efficient not only in introducing specific ATP7B mutations but also in correcting ATP7B point mutations [ 211 ]. Although the single-stranded oligo DNA nucleotide (ssODN) is limited to non-viral delivery methods, such application can lead to direct and safe modification in the point mutations within the ATP7B gene, providing therapeutic potential [ 212 ].…”