| Wilson’s disease(WD)is an autosomal recessive inherited copper storage disorder caused by mutations in the ATP7B gene.ATP7B encodes a transmembrane copper-transporting ATPase,leading to impaired copper homeostasis and copper overload in the liver,brain,and other organs.In China,the Arg778Leu mutation in exon 8 and the Pro992Leu mutation in exon 13 are found more frequently in patients with WD.However,the pathogenesis of these mutations remains unclear.It is necessary to generate animal models for these mutations.CRISPR/Cas9 has recently been developed as an efficient genetic engineering tool.It has been widely used in the generation of gene-modified animal models and has shown great potential for the generation of specific gene-modified animal models.Current treatments are based on lifelong copper-chelating drugs and zinc salts,which may cause side effects and do not restore normal copper metabolism.In this study,we generated ATP7B site-directed point mutation mice to simulate a major mutation type in Chinese(p.Arg778Leu and p.Pro992Leu)with WD by using the CRISPR/Cas9 system combined with single-stranded DNA oligonucleotides(ssODNs).Successfully,the R778L and P992L mutation mice of ATP7B were generated.In addition,the clinical phenotypes of the two mutant mice were compared with those of patients with WD.The results showed that serum holo-ceruloplasmin levels were reduced in WD mice.Furthermore,R778L and P992L mutations had significantly increased serum non-ceruloplasmin binding copper,liver,and brain copper content,accompanied by liver injury and behavioral abnormalities.The protein levels of inflammasome adaptor molecule apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain(ASC),cleaved Caspase-1,and interleukin-1β were upregulated in the brain regions of WD mice.The NLRP3 inflammasome is activated in WD mouse brains.Furthermore,activation of the NLRP3 inflammasome was noted in primary microglia treated with CuCl2,accompanied by increased levels of cleaved Caspase-1,ASC,and interleukin-1β.Blocking NLRP3 inflammasome activation with siNlrp3 or MCC950 reduced interleukin-1β and interleukin-18 production,thereby effectively mitigating cognitive decline,locomotor behavior impairment,and neurodegeneration in WD mice.In addition,we assessed the efficacy of gene therapy in WD mice.We transduced the livers of a WD mouse model with an adeno-associated vector serotype 9(AAV9)encoding human ATP7B cDNA.After vector administration,we performed periodic evaluation of variables associated with copper metabolism and disease progression.We observed a therapeutic effect of AAV9-ATP7B manifested as a reduction of serum transaminase levels and urinary copper excretion,normalization of serum holo-ceruloplasmin,and restoration of physiological biliary copper excretion in response to copper overload.The livers of treated animals showed normalization of copper content and absence of histological alterations.Furthermore,tail vein injection of AAV9-ATP7B in WD mice inhibited NLRP3 inflammasome activation and effectively mitigated motor deficits,neurodegeneration,and accumulation of copper in the brain.In summary,this study successfully developed a precision point mutation mouse disease model using CRISPR/Cas9 technology following gene knockout and gene knock-in in mice.The present study developed a potential mouse model for human WD disease in Chinese with potential applications in pathological analysis,clinical treatment,and gene therapy research.Furthermore,gene therapy using AAV-ATP7B provides long-term correction of copper metabolism in a clinically relevant WD mouse model.These results pave the way for the implementation of gene therapy in patients with WD. |
