PILOT--SILENCING OF TRANSGENES BY HISTONE DEACETYLASE

说明(改编自应用程序)： 囊性纤维化基因治疗的方法包括使用衍生载体 来自腺相关病毒和小鼠或其他逆转录病毒。这些向量是 尤其令人感兴趣，因为它们具有长期表达的潜力 和/或整合到宿主染色体中。小鼠的表达持续时间 逆转录病毒载体受宿主灭活携带的转基因的限制 这些病毒构建物。整合病毒DNA的沉默发生在 上皮细胞和非上皮细胞中均有数周至数月的表达。我们已经决定 腺相关病毒失活的部分机制 载体涉及载体DNA的选择性脱乙酰基。并非所有的AAV都集成在一起 载体被压制住了。在未选择的单元格中，大约80%的集成 HeLa细胞中的病毒基因组和K562(红白血病细胞)中50%的病毒基因组 被这一过程灭活。曲古抑菌素A(3：M)治疗，一种特殊的 组蛋白脱乙酰酶抑制剂，以一种方式重新激活转基因表达 这似乎既不是启动子，也不是细胞类型特异的。在这个试行中 项目，我们将检查报告基因转移的持续时间和效率 AAV和MuLv对呼吸道上皮细胞株和原代呼吸道上皮细胞的作用 取自囊性纤维化患者的细胞。我们将评估其性质 未选择的、感染AAV的细胞中的整合事件，并将此数据与 发生的基因沉默。与重组AAV载体整合不是 在人类呼吸道上皮细胞中得到了充分的理解，但许多研究都在 优化与这种类型的集成和/或外体持久性的进展 向量。通过研究AAV或MuLv基因转移后的未选择细胞，我们将 检查AAV结构的频率和性质，这些结构确实整合并帮助 描述整合基因组在初级阶段沉默的机制 人类肺细胞。此外，我们还将测试基因转移的效率 在小鼠模型中使用AAV载体，并确定组蛋白脱乙酰酶 在体内参与哺乳动物呼吸道AAV的转基因灭活。这个 沉默的、病毒转导的基因的重新激活对基因有影响 心理治疗。高效的基因转移和药物治疗缓解了 抑制可能为各种基因提供一种强有力的联合治疗 和传染病。这一试点和可行性项目的目的是 为了测试能够增强组蛋白的化合物的安全性和有效性 高乙酰化对基因转移到呼吸道可能产生的影响 上皮细胞。

Description (adapted from the application): Approaches to cystic fibrosis gene therapy include the use of vectors derived from adeno-associated virus and murine or other retroviruses. These vectors are of particular interest because of their potential for long-term expression and/or integration in host chromosomes. Duration of expression from murine retroviral vectors is limited by host inactivation of transgenes carried by these viral constructs. The silencing of integrating viral DNA occurs over weeks to months in both epithelial and non-epithelial cells. We have determined that part of the mechanism underlying inactivation of adeno-associated viral vectors involves selective deacetylation of vector DNA. Not all integrating AAV vectors are silenced. In non-selected cells, approximately 80% of integrated viral genomes in HeLa cells, and 50% in K562 (erythroleukemia cells) were inactivated by this process. Treatment with Trichostatin A (3: M), a specific inhibitor of histone deacetylase, reactivated transgene expression in a fashion that did not appear to be either promoter or cell type specific. In this pilot project, we will examine the duration and efficiency of reporter gene transfer by AAV and MuLv to airway epithelial cell lines and primary airway epithelial cells taken from cystic fibrosis patients. We will evaluate the nature of integration events in unselected, AAV-infected cells and relate this data to the gene silencing that occurs. Integration with recombinant AAV vectors is not fully understood in human airway epithelial cells, but many studies are in progress to optimize integration and/or episomal persistence with this type of vector. By studying nonselected cells after AAV or MuLv gene transfer, we will examine the frequency and nature of AAV constructs that do integrate, and help characterize the mechanism by which integrated genomes are silenced in primary human lung cells. In addition, we will test the efficiency of gene transfer using AAV vectors in a mouse model, and determine whether histone deacetylases participate in transgene inactivation of AAV in mammalian airways in vivo. The reactivation of silenced, virally transduced genes has implications for gene therapy. Efficient gene transfer followed by drug treatment to relieve suppression may provide a powerful combination treatment for various genetic and infectious diseases. The purpose of this Pilot and Feasibility Project is to test the safety and efficacy of compounds capable of augmenting histone hyperacetylation for possible effects in the setting of gene transfer to airway epithelial cells.