APPROACHES TO CORRECT DISEASES WITH GENETICALLY MODIFIED CELLS OF THE SKIN

该方案项目的目标是将潜力定义为 以及使用转基因细胞的局限性 用皮肤来治病。从我们过去五年的工作来看， 越来越清楚的是，转基因表达的丧失是由于遗传原因 体内修饰细胞，无论是因为细胞死亡还是死亡 “沉默”是基因临床应用的一大局限。 心理治疗。例如，通过人成纤维细胞表达转基因 转导重组逆转录病毒在体外是稳定的，但有 当这些细胞被放置在体内时，表达会迅速丧失。这 表达的丧失是一个有组织的过程，但以前从未出现过 充分描述、调查或调查。因为损失了 转基因细胞的稳定转基因表达是体内的一种 事件，则控制表达式丢失的参数必须在 活着。我们认为有两个主要因素决定了 转基因在体内的表达是细胞衰老和细胞 微环境。我们认为这些方面对分数都有影响 在移植后存活的转基因细胞和 转基因在存活细胞中的表达水平。评估 细胞微环境在两者存活中的相对作用 转基因细胞及其转基因的表达，我们将 比较开放和开放环境中转基因细胞的比率 封装的设备。这两个设备将使我们能够独立地 修改细胞-宿主和细胞-细胞外相互作用。我们还将 确定延长电池的使用寿命是否 转基因基因治疗将延长转基因持续时间 伴随着正常细胞衰老的表达具有以下效果 沉默转基因表达。因此，抑制细胞衰老 会增加转基因表达的持续时间。这将是 用表达E6/E7基因的角质形成细胞进行检测。这些方法 将使我们能够定义影响细胞的关键变量的角色 体内存活和持续的转基因表达，将有助于 旨在避免体内转基因丢失的直接未来策略 来自皮肤和其他器官的转基因细胞表达不 像皮肤一样平易近人。

The objective of the program project has been to define the potential as well as the limitations of the use of genetically modified cells of the skin to treat disease. From our work over the last five years, it has become clear that loss of expression of transgenes by genetically modified cells in vivo, either because of cell death of death "silencing", is a major limitation in the clinical application of gene therapy. For example, expression of transgenes by human fibroblasts transduced with recombinant retroviruses is stable in vitro, but there is rapid loss of expression when these cells are placed in vivo. This loss of expression is an organized process, but has not previously been adequately described or investigated or investigated. Because loss of stable transgene expression by genetically modified cells is an in vivo event, the parameters that govern loss of expression must be defined in vivo. We propose that two major factors that dictate the stability of transgene expression in vivo are cell senescence and cell microenvironment. We propose that these aspects affect both the fraction of genetically modified cells that survive following transplantation and the level of transgene expression in the surviving cells. To evaluate the relative roles of cell microenvironment on both survival of genetically modified cells and expression of their transgenes, we will compare the rate of genetically modified cells in both open and encapsulated devices. These two devices will allow us to independently modify cell-host and cell-extracellular interactions. We will also determine whether increasing the lifespan of cells used for transkaryotic gene therapy will increase the duration of transgene expression that accompanies normal cell senescence has the effect of silencing transgene expression. Therefore, suppressing cell senescence will increase the duration of transgene expression. This will be examined using keratinocytes expressing E6/E7 genes. These approaches will allow us to define the roles of key variables affecting cell survival and continued transgene expression in vivo and will serve to direct future strategies designed to avoid in vivo loss of transgene expression by genetically modified cells from skin and other organs not as accessible as skin.