Development of a new strategy for gene therapy of autosomal recessive congenital ichthyosis by gene transfer to the bulge stem cells

毛囊、上皮细胞的基因转移不仅是治疗皮肤病的一种有吸引力的方法，而且也是治疗许多全身性疾病的一种有吸引力的方法。在本研究中，我们打算开发一种毛囊上皮干细胞的基因转移系统，以最大限度地发挥有益的治疗效果。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。我们将去年制造的基因结构转染。将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。我们成功地重建了毛囊及其附属物，其中这些细胞含有转基因报告基因。在所有皮肤上皮细胞中均观察到转基因表达， ...更多信息 包括毛囊上皮、皮脂腺和表皮。此外，我们还观察了至少6个月的转基因表达。此外，我们还使用体外研究中建立的方法进行了体内基因转染实验。此外，我们还进行了用于基因治疗的基因构建体的转染实验。本研究利用已构建的转染载体和从前期实验中筛选出的报告基因，克隆了正常的TGM1和ABCA 12的cDNA构建体。我们从携带TGM1突变的板层鱼鳞病患者和在完全知情同意后携带ABCA12突变的丑角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，我们尝试了基因治疗实验，使用正常的TGM1或正常ABCA 12基因构建体在以前的研究。这种毛囊干细胞靶向的基因转移和重建系统提供了可靠的基因功能分析和基因治疗。少

Gene transfer to hair follicle, epithelium is an attractive approach for not only treating skin diseases, but also many systemic disorders. In this study, we intended to develop a gene transfer system for hair follicle epithelial stem cells to maximize the beneficial therapeutic effects. 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. We transfected the gene constructs that we had made last year. The transfected cells were mixed with cultured dermal papilla cells and transplanted on to immunodeficient mice. We succeeded in reconstituting hair follciles and their appendages in which these cells harbored a transgene reporter. The transgene expression was observed in all skin epithelial compartme … More nts including the hair follicle epithelium, sebaceous gland and epidermis. In addition, transgene expression was observed for at least 6 months.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 TGM1 cDNA construct and normal ABCA12 cDNA construct with the transfection vector and the reporter genes selected from the results of previous experiments. We obtained keratinocyte cultures form lamellar ichthyosis patients carrying TGM1 mutations and from harlequin 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 TGM1 or normal ABCA12 gene constructs made in the previous studies. This hair follicle stem cell targeted gene transfer and reconstitution system provides reliable gene-function analysis and gene therapy. Less