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

毛囊、上皮基因转移不仅是治疗皮肤病，而且是治疗许多全身性疾病的一种有吸引力的方法。在这项研究中，我们打算开发一个毛囊上皮干细胞的基因转移系统，以最大限度地提高有益的治疗效果。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。我们在体外对鼓包干细胞进行了基因转染实验。我们解剖了小鼠毛囊的凸起区域并建立了原代培养。我们转染了去年制作的基因结构。将转染的细胞与培养的真皮乳头细胞混合，移植到免疫缺陷小鼠身上。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。该基因在毛囊上皮、皮脂腺和表皮等所有皮肤上皮区室均有表达。此外，转基因表达观察至少6个月。此外，我们使用体外研究中建立的方法在体内对凸起干细胞进行了基因转染实验。此外，我们还进行了基因转染实验，用于基因治疗。我们利用转染载体和前期实验结果中选择的报告基因克隆正常TGM1 cDNA构建体和正常ABCA12 cDNA构建体。在完全知情同意后，我们从携带TGM1突变的板层状鱼鳞病患者和携带ABCA12突变的丑角状鱼鳞病患者中获得了角质细胞培养物。从裸鼠背部培养的患者角质形成细胞中重建了具有鱼鳞病表型的表皮。针对重建鱼鳞病皮损，我们尝试使用以往研究中构建的正常TGM1或正常ABCA12基因进行基因治疗实验。该毛囊干细胞靶向基因转移和重组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