■威尔逊病(WD)是一种由ATP7B功能丧失突变引起的铜代谢常染色体隐性遗传疾病,它编码一种铜转运蛋白。它的特点是组织中过量的铜沉积,主要在肝脏和大脑。我们试图研究基因校正的患者特异性诱导多能干细胞(iPSC)衍生的肝细胞(iHeps)是否可以作为WD细胞移植治疗的自体细胞来源。
■我们首先使用CRISPR/Cas9和iHeps(来自WD患者)中的单链寡脱氧核苷酸(ssODN)比较了ATP7B在基因校正前后的体外表型和细胞功能。ATP7778L突变(ATP7BR8L/R778L)。接下来,我们在免疫缺陷型WD小鼠模型(Atp7b-/-/Rag2-/-/Il2rg-/-;ARG)中评估了WD基因校正iHeps细胞移植的体内治疗潜力.
■我们使用CRISPR/Cas9和ssODN成功地创建了携带野生型ATP7B基因(ATP7BWT/-)的1个等位基因的杂合基因校正的iPSC。与ATP7BR778L/R778LiHP相比,基因校正的ATP7BWT/-iHeps在体外恢复ATP7B亚细胞定位,其响应铜过载的亚细胞运输及其铜出口功能。此外,通过脾内注射将ATP7BWT/-iHeps体内细胞移植到ARG小鼠中,可显着减轻WD的肝脏表现。肝功能改善和肝纤维化减少由于减少肝铜积累和因此铜诱导的肝细胞毒性。
■我们的发现表明,基因校正的患者特异性iPSC来源的iHeps可以挽救WD的体外和体内疾病表型。这些原理证明数据表明,源自基因校正的WDiPSC的iHeps具有作为体内治疗WD以及其他遗传性肝病的自体离体细胞来源的潜在用途。
■基因校正恢复了源自威尔逊氏病患者的诱导多能干细胞的肝细胞中的ATP7B功能。这些基因校正的肝细胞是Wilson病患者自体细胞治疗的潜在细胞来源。
UNASSIGNED: Wilson\'s disease (WD) is an autosomal recessive disorder of copper metabolism caused by loss-of-function mutations in ATP7B, which encodes a copper-transporting protein. It is characterized by excessive copper deposition in tissues, predominantly in the liver and brain. We sought to investigate whether gene-corrected patient-specific induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps) could serve as an autologous cell source for cellular transplantation therapy in WD.
UNASSIGNED: We first compared the in vitro phenotype and cellular function of ATP7B before and after gene correction using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) in iHeps (derived from patients with WD) which were homozygous for the ATP7B R778L mutation (ATP7BR778L/R778L). Next, we evaluated the in vivo therapeutic potential of cellular transplantation of WD gene-corrected iHeps in an immunodeficient WD mouse model (Atp7b -/- / Rag2 -/- / Il2rg -/- ; ARG).
UNASSIGNED: We successfully created iPSCs with heterozygous gene correction carrying 1 allele of the wild-type ATP7B gene (ATP7BWT/-) using CRISPR/Cas9 and ssODNs. Compared with ATP7BR778L/R778L iHeps, gene-corrected ATP7BWT/- iHeps restored i n vitro ATP7B subcellular localization, its subcellular trafficking in response to copper overload and its copper exportation function. Moreover, in vivo cellular transplantation of ATP7BWT/- iHeps into ARG mice via intra-splenic injection significantly attenuated the hepatic manifestations of WD. Liver function improved and liver fibrosis decreased due to reductions in hepatic copper accumulation and consequently copper-induced hepatocyte toxicity.
UNASSIGNED: Our findings demonstrate that gene-corrected patient-specific iPSC-derived iHeps can rescue the in vitro and in vivo disease phenotypes of WD. These proof-of-principle data suggest that iHeps derived from gene-corrected WD iPSCs have potential use as an autologous ex vivo cell source for in vivo therapy of WD as well as other inherited liver disorders.
UNASSIGNED: Gene correction restored ATP7B function in hepatocytes derived from induced pluripotent stem cells that originated from a patient with Wilson\'s disease. These gene-corrected hepatocytes are potential cell sources for autologous cell therapy in patients with Wilson\'s disease.