APPROACHES TO CORRECT DISEASES WITH GENETICALLY MODIFIED CELLS OF THE SKIN

该计划项目的目标是将潜力定义为 以及使用遗传修饰细胞的局限性， 皮肤治疗疾病。从我们过去五年的工作来看， 很明显，通过遗传学途径丧失转基因表达 在体内的修饰细胞，无论是因为细胞死亡的死亡 基因沉默是基因治疗在临床应用中的一个主要限制因素 疗法例如，通过人成纤维细胞表达转基因 用重组逆转录病毒转导的重组逆转录病毒在体外是稳定的，但 是当这些细胞被置于体内时表达的快速丧失。这 表达缺失是一个有组织的过程，但以前没有 被充分地描述、调查或研究。因为失去 通过遗传修饰的细胞的稳定转基因表达是一种体内转基因技术， 事件，则必须在 vivo.我们提出，决定稳定性的两个主要因素 转基因在体内的表达是细胞衰老和细胞凋亡。 微环境我们认为，这些方面既影响分数 移植后存活的转基因细胞， 存活细胞中转基因表达的水平。评价 细胞微环境对两种细胞存活的相对作用 转基因细胞及其转基因表达，我们将 比较开放和开放环境中转基因细胞的比率， 封装器件。这两个设备将使我们能够独立地 修饰细胞-宿主和细胞-细胞外相互作用。我们还将 确定是否增加细胞的寿命用于 转基因治疗将增加转基因持续时间， 伴随正常细胞衰老的表达具有以下作用： 沉默转基因表达。因此，抑制细胞衰老 将增加转基因表达的持续时间。这将是 使用表达E6/E7基因的角质形成细胞检测。这些方法 将允许我们定义影响细胞的关键变量的作用 存活和持续的转基因表达，并将用于 旨在避免转基因在体内丢失的指导未来策略 来自皮肤和其他器官的基因修饰细胞的表达， 就像皮肤一样。

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.