APPROACHES TO CORRECT DISEASES WITH GENETICALLY MODIFIED CELLS OF HUMAN SKIN

对于许多遗传性疾病，异常基因和正常基因都有 在体外进行了定义、克隆和表达。这些分子方面的进展 遗传学已经使纠正遗传性疾病成为可能，通过将 非缺陷基因通过体细胞(体细胞)导入受害宿主 基因疗法)。这种方法似乎特别适合于 获得性畸形和异常的遗传性皮肤病 形态发生，如Darier病、色素性干皮病、表皮松解症 大疱性皮肤病，以及那些以缺乏 血浆蛋白，例如血友病、糖尿病等。一个中心问题， 现在处于生物医学界的前沿，专注于如何最好地 将这些无缺陷的基因引入到患病的个体中。这是我们的 通过对皮肤细胞进行基因改造进行矫正的论断 风险相对较低，应用广泛，具有相当大的 临床应用。我们相信，这种方法的可行性、局限性和 适用的方法可以通过移植在临床前得到回答 转基因人类皮肤的等效物或其成分 无性系啮齿动物，并通过实验分析选定的参数 皮肤作为时间的函数。 具体目标： 1.基因移植临床前技术的发展 将皮肤改良成人类。 A.替换已有的人皮肤移植到无性系上的表皮 具有由转基因产生的表皮的啮齿动物 角质形成细胞。 B.产生携带正常或正常的转基因皮肤等价物 可移植的转基因成纤维细胞或角质形成细胞 到无性系啮齿动物身上。 C.生成浸透转基因的真皮基质 可以放置在啮齿动物皮下的成纤维细胞，可能 在人类的皮内。 2.确定包含这些的皮肤的长期功能 转基因成分；具体地说是一剂的能力 反应，转基因的产生和稳定性，以及 转基因对皮肤固有结构和功能的影响。 3.确定隐性X连锁鱼鳞病是否可以矫正 转导类固醇基因的角质形成细胞或成纤维细胞 硫酸盐酶，并移植到无性系啮齿动物身上。这些实验是 旨在定义这项技术的潜力和局限性。

For many genetic disorders, the abnormal, as well as the normal gene have been defined, cloned, and expressed in vitro. These advances in molecular genetics have made it possible to correct genetic disease by bringing the non-defective gene into the afflicted host via somatic cells (somatic cell gene therapy). This approach appears to be particularly suitable to heritable skin diseases of acquired malformation and abnormal morphogenesis, e.g., Darier's disease, xeroderma pigmentosa, epidermolysis bullosa, and those disorders which are characterized by a deficiency of a plasma protein, e.g., hemophilia, diabetes mellitus, etc. A central issue, now at the forefront of the biomedical community, focuses on how best to introduce these non-defective genes into afflicted individuals. It is our thesis that correction by genetically modifying cells of the skin has relatively low risk, has broad application and thus has considerable clinical utility. We are persuaded that the feasibility, limitations and methods for applicability can be answered preclinically by transplanting genetically modified human skin equivalents or components thereof to athymic rodents and by experimentally analyzing selected parameters of such skin as a function of time. Specific aims: 1. Development of the preclinical technology for transplanting genetically modified skin to humans. a. Replacing the existing epidermis of human skin grafted onto athymic rodents with an epidermis generated from genetically modified keratinocytes. b. Generating a genetically modified skin-equivalent carrying normal or genetically modified fibroblasts or keratinocytes which can be transplanted onto athymic rodents. c. Generating a dermal matrix impregnated with genetically modified fibroblasts that can be placed subcutaneously in rodents, and possibly intradermally in humans. 2. To determine the long-term functionality of skin containing these genetically modified components; specifically the capacity for a dose response, the production by and stability of the transfected gene, and the effect of transfection on structure and function inherent to skin. 3. To determine whether recessive X-linked ichthyosis, can be corrected with keratinocytes or fibroblasts transfected with the gene for steroid sulfatase, and transplanted to athymic rodents. These experiments are designed to define the potential and the limitations of this technology.