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

表皮角质形成细胞脂质转运体ABCA12的严重缺陷被认为会导致皮肤脂质屏障的缺陷，导致严重的鱼鳞病。在这项研究中，我们打算通过ABCA12毛囊上皮干细胞纠正性基因转移系统来开发一种治疗严重硬皮病的基因治疗方法。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入培养的毛囊干细胞。我们在体外进行了基因导入隆起干细胞的实验。我们解剖了小鼠触须毛囊的突起区域，并建立了原代培养。将ABCA12转基因细胞与培养的毛乳头细胞混合，移植到免疫缺陷小鼠体内。我们成功地从含有转基因报告的细胞中重建了毛囊及其附属物。转基因在毛囊上皮、皮脂腺和表皮中均有表达。此外，我们还利用体外研究中建立的方法，在体内进行了膨大干细胞的基因转染实验，并进行了用于基因治疗的基因构建的转染实验。具体来说，我们利用该载体和从前期实验结果中筛选出的报告基因，克隆了正常的ABCA12基因。在完全知情同意后，我们从携带ABCA12突变的严重鱼鳞病患者身上获得角质形成细胞培养。这些培养的角质形成细胞在裸鼠背部重建出具有典型鱼鳞病表型的表皮。以重建的鱼鳞病皮肤损伤为目标，我们尝试了使用正常ABCA12基因结构的基因治疗实验。该毛囊干细胞靶向ABCA12纠正性基因转移系统提供了可靠的基因治疗。

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.