PILOT--SILENCING OF TRANSGENES BY HISTONE DEACETYLASE

描述（改编自应用程序）： 囊性纤维化基因治疗的方法包括使用来源于细胞的载体。 来自腺相关病毒和鼠或其它逆转录病毒。这些载体 由于它们具有长期表达的潜力， 和/或整合到宿主染色体中。小鼠表达持续时间 逆转录病毒载体受到宿主失活的限制， 这些病毒结构。整合病毒DNA的沉默发生在 在上皮细胞和非上皮细胞中持续数周至数月。我们已经确定 腺相关病毒灭活的潜在机制的一部分， 载体包括载体DNA的选择性脱乙酰化。并非所有整合AAV 带菌者都沉默了。在非选择性细胞中，约80%的整合 HeLa细胞中的病毒基因组，以及K562（红白血病细胞）中的50%， 在这个过程中失去活性。用曲古抑菌素A（3：M）治疗， 组蛋白脱乙酰酶抑制剂，以一种方式重新激活转基因表达 这似乎不是启动子或细胞类型特异性的。此次试点 项目，我们将研究报告基因转移的持续时间和效率 通过AAV和MuLv对气道上皮细胞系和原代气道上皮 从囊性纤维化患者身上提取的细胞。我们将评估 整合事件，并将该数据与 发生的基因沉默。与重组AAV载体的整合不是 在人类气道上皮细胞中完全了解，但许多研究都是在 优化整合和/或附加型持久性的进展， vector.通过研究AAV或MuLv基因转移后的非选择性细胞，我们将 检查整合的AAV结构的频率和性质，并帮助 描述整合基因组在原发性肝癌中沉默的机制。 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是否 在体内参与哺乳动物气道中AAV的转基因失活。的 沉默的病毒转导基因的重新激活暗示着基因 疗法有效的基因转移后，药物治疗，以减轻 抑制可以为各种遗传病提供有效联合治疗， 和传染病。该试点和可行性项目的目的是 为了测试能够增加组蛋白的化合物的安全性和有效性， 高度乙酰化对基因转移至气道的可能影响 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.