Cellular co-factors in stable retroviral transduction

描述(申请人提供)：稳定的逆转录病毒转导的效率是一个关键步骤，它往往决定着基因治疗方法治疗人类疾病的成败。为了成功地将基因转导到宿主细胞，逆转录病毒载体必须整合到宿主细胞基因组中。逆转录病毒DNA整合包括将宿主细胞DNA交错切割，然后通过逆转录病毒酶整合酶将病毒DNA的S末端连接到宿主DNA。因此，整合过程在逆转录病毒DNA两侧产生了短的单链DNA缺口。完整、稳定的病毒DNA转导依赖于整合后修复，整合后修复遵循整合酶介导的3‘端连接反应。我们实验室的长期目标是了解宿主细胞蛋白在病毒生命周期中这一重要但尚未完全确定的修复步骤中的作用。这一建议建立在我们的发现基础上，即整合后修复依赖于非同源末端连接(NHEJ)DNA修复途径的蛋白质(目标1)和细胞组蛋白脱乙酰酶4(HDAC4)蛋白(目标2)。这两项发现都将用于目标3。这项建议中描述的实验将增强我们对蛋白质在整合后修复中的作用的理解，并为利用逆转录病毒载体开发新的方法以提高基因治疗的成功率铺平道路。在目标1中，我们将进一步剖析NHEJ缺陷细胞和正常细胞的整合后修复过程，并确定整合后修复的特定步骤是否需要特定的NHEJ蛋白，这些步骤包括填补DNA缺口，修剪病毒DNA短瓣，将新暴露的病毒DNA 5‘端与宿主DNA连接，以及重新构建适当的染色质结构和组成。拟议的实验结果不仅应该加深我们对NHEJ蛋白如何促进整合后修复的理解，而且还将显著增强我们对NHEJ在重要细胞通路中的功能的理解。在目标2中，我们将继续我们的新发现，即逆转录病毒载体的稳定转导需要细胞内的HDAC4蛋白。此外，我们提出的证据表明，HDAC4积聚在逆转录病毒DNA整合的位置。这些新的发现揭示了HDAC4是逆转录病毒生命周期中的一个新角色。使用为目标1开发的方法，在目标2中，我们将通过增强整合后修复来检验HDAC4对于稳定的转导是必需的假设。分析HDAC4在逆转录病毒转导中的功能将增加我们对HDAC4在DNA修复中的整体作用的理解，并再次为我们提供一条提高逆转录病毒载体效率的新途径。在本提案的最终目标(目标3)中，我们将检验这样一个假设，即稳定的逆转录病毒转导的效率可以通过增加细胞内蛋白质的数量和活性来提高，这是有效的整合后修复所必需的。这一目标中描述的实验应该导致开发新的方法，以提高使用逆转录病毒载体进行基因治疗的效率和成功率。

DESCRIPTION (provided by applicant): The efficiency of stable retroviral transduction is a critical step, which often determines the success or failure of the gene therapy approaches to treatment of human diseases. To successfully transduce a gene into host cells, retroviral vectors must integrate into the host cell genome. Retroviral DNA integration involves a staggered cut of host cell DNA followed by joining of S'-ends of viral DNA to host DNA by the retroviral enzyme integrase. The integration process thus creates short, single-strand DNA gaps that flank retroviral DNA. Complete, stable transduction of the viral DNA then depends on post-integration repair, which follows the integrase-mediated 3'-end joining reaction. The long-term goal of our laboratory is to understand the role of host cell proteins in this essential, but incompletely characterized repair step in the viral life cycle. This proposal builds on our discoveries that post-integration repair depends on the proteins of the non- homologous end joining (NHEJ) DNA repair pathway (Aim 1), and on the cellular histone deacetylase 4 (HDAC4) protein (Aim 2). Both of these discoveries will be put to use in Aim 3. The experiments described in this proposal should enhance our understanding of the roles of the proteins in post-integration repair, and pave the way to development of novel methods to increase success rate of gene therapy using retroviral vectors. In the Aim 1, we will further dissect the process of post-integration repair in NHEJ-deficient and normal cells and determine, if specific NHEJ proteins are required for specific steps of the post-integration repair, which include filling of DNA gaps, trimming of short viral DNA flaps, ligation of the newly exposed 5'- ends of viral DNA to host DNA and reconstitution of appropriate chromatin structure and composition. Results from the proposed experiments should not only deepen our understanding of how NHEJ proteins facilitate post-integration repair, but also significantly enhance our understanding of NHEJ function in important cellular pathways. In the Aim 2, we will pursue our new findings that stable transduction by retroviral vectors requires the cellular HDAC4 protein. In addition, we present evidence indicating that HDAC4 accumulates at sites of retroviral DNA integration. These novel findings reveal HDAC4 as a new player in the retroviral life cycle. Using methodologies developed for the Aim 1, in the Aim 2 we will test the hypothesis that HDAC4 is required for stable transduction through enhancement of post-integration repair. Analysis of the HDAC4 function in retroviral transduction should increase our understanding of the overall role of HDAC4 in DNA repair and again allow us a new avenue to augment the efficiency of retroviral vectors. In the final Aim of this proposal (Aim 3), we will test the hypothesis that the efficiency of stable retroviral transduction can be enhanced by increasing the intracellular amounts and activities of cellular proteins, which are required for efficient post-integration repair. The experiments described in this Aim should lead to development of new methods to increase efficiency and success rate of gene therapy using retroviral vectors.