Development of a new therapy of severe ichthyosis caused by ABCA12 mutations

已知表皮角质形成细胞脂质转运蛋白ABCA 12的严重缺陷会导致皮肤脂质屏障缺陷，从而导致严重的鱼鳞病。本研究旨在通过毛囊上皮干细胞ABCA 12基因转移系统，建立一种治疗重度鱼鳞癣的基因治疗方法。为了在毛囊干细胞中持续稳定地表达转基因，我们将编码报告基因的逆转录病毒载体转移到培养的毛囊干细胞中。我们在体外对隆突干细胞进行了基因转染实验。我们解剖了小鼠触须毛囊的隆突区，并建立了原代培养。将ABCA 12转染的细胞与培养的毛乳头细胞混合并移植到免疫缺陷小鼠上。我们成功地从携带转基因报告基因的细胞中重建毛囊及其附属物。在包括毛囊上皮、皮脂腺和表皮的所有皮肤上皮区室中观察到转基因表达。此外，我们使用体外研究中建立的方法进行了体内基因转染到隆突干细胞中的实验。此外，我们还进行了基因治疗用基因构建体的转染实验。具体地，我们用转染载体和从先前实验结果中选择的报告基因克隆了正常ABCA 12 cDNA构建体。我们在完全知情同意的情况下，从携带ABCA12突变的严重鱼鳞病患者中获得角质形成细胞培养物。从这些培养的角质形成细胞在裸鼠背部重建显示特征性鱼鳞病表型的表皮。针对重建的鱼鳞病皮肤病变，我们尝试使用正常ABCA 12基因构建体的基因治疗实验。该毛囊干细胞靶向ABCA12基因治疗系统为毛囊干细胞治疗提供了可靠的基因治疗手段。

Serious defects in the epidermal keratinocyte lipid transporter ABCA12 are known to result in a deficient skin lipid barrier, leading to severe ichthyosis. In this study, we intended to develop a gene therapy method of severe icththyosis by an ABCA12 corrective gene transfer system for hair follicle epithelial stem cells. For persistent and stable transgene expression in hair follicle stem cells, we transferred retroviral vectors encoding reporter genes into cultured hair follicle stem cells. We performed gene transfection experiments into the bulge stem cells in vitro. We dissected bulge areas from mice vibrissa hair follicles and established primary cultures. The ABCA12 transfected cells were mixed with cultured dermal papilla cells and transplanted on to immunodeficient mice. We succeeded in reconstituting hair follciles and their appendages from the cells harboring a transgene reporter. The transgene expression was observed in all skin epithelial compartments including the hair follicle epithelium, sebaceous gland and epidermis. In addition, we performed gene transfection experiments into the bulge stem cells in vivo using the methods that had been established from in vitro studies.Furthermore, we performed transfection experiments of gene constructs for gene therapy. In detail, we cloned normal ABCA12 cDNA construct with the transfection vector and the reporter genes selected from the results of previous experiments. We obtained keratinocyte cultures form severe ichthyosis patients harboring ABCA12 mutations after fully informed consents. Epidermis showing characteristic ichthyosis phenotype was reconstituted from these cultured keratinocytes form the patients on the back of nude mice. Targetting the reconstructed ichthyosis skin lesions, we tried gene therapy experiments using normal ABCA12 gene constructs. This hair follicle stem cell targeted ABCA12 corrective gene transfer system provided reliable gene therapy.