Targeted Transgene Integration through Chromatin tethering for Globin Gene Therap

描述(由申请人提供)： 我们的最终目标是在人类造血干细胞(HSCs)中实现大(25kb)转基因盒体的定向整合。为了达到这一目标，我们构建了转导HSCs的辅助子依赖、纤维嵌合型腺病毒载体(HD-Ad5/35)。HD-Ad5/35载体可以非常有效地将大的转基因盒运送到HSCs的核中，在那里载体基因组仍处于异构体阶段，包装成核小体样结构。然而，当HD-Ad5/35载体含有23kb的2-珠蛋白基因控制区片段(HD-Ad5/35.LCR-1)时，载体基因组高频整合到人红系Mo7e细胞的染色体DNA中。重要的是，20%的整合事件发生在染色体2-珠蛋白LCR内，特别是进入超敏部位2内和下游的区域。我们证明了靶向HD-Ad5/35整合涉及具有“活性”染色质特征的珠蛋白LCR特异性蛋白质，以及传入的HD-Ad5/35基因组物理上与染色体珠蛋白LCR捆绑在一起，涉及染色质蛋白。我们推测，载体和染色体DNA之间的物理接近，以及拴系载体DNA附近的DNA断裂，介导了我们的载体优先整合到Mo7e细胞中的珠蛋白LCR。在这项提议中，我们将尝试在造血干细胞中实现有针对性的转基因整合。在初步研究中，LCR-染色质连接和/或载体整合在CD34+细胞中的效率低于在Mo7e细胞中的效率。我们推测CD34+细胞中的2-珠蛋白LCR染色质能够与HD-Ad5/35结合，而稳定的CD34+细胞转导的限制性步骤是载体整合。我们将在特定的目标1中验证这一假设。我们的特定目标2是利用a&C31噬菌体整合酶提高拴系的HD-Ad5/35 LCR基因组的整合效率。总体而言，这项工作将使我们更好地理解染色质拴系的机制和结构决定因素，以及它在促进载体整合中的作用，并为通过逆转录病毒整合酶染色质拴系改善现有逆转录病毒载体的靶向整合奠定基础。 公共卫生相关性： 我们提出了一种新的载体系统，以实现人造血干细胞中大的转基因盒的靶向整合。我们的方法结合了一种将DNA输送到干细胞的新载体和一种新的想法，以实现大型转基因盒的靶向整合。具体地说，我们计划利用我们最近的发现，即优先载体整合到β-珠蛋白LCR可以通过转基因盒的染色质捆绑实现。这项研究总体上与整合载体的工程靶点特异性有关，并可能为珠蛋白基因治疗提供依据。

DESCRIPTION (provided by applicant): Our final goal is to achieve targeted integration of large (>25kb) transgene cassettes in human hematopoietic stem cells (HSCs). Towards to goal, we constructed helper-dependent, fiber-chimeric adenovirus vectors (HD-Ad5/35) that transduce HSCs. HD-Ad5/35 vectors are very efficient in delivering large transgene cassettes into the nucleus of HSCs where the vector genomes remain in an episomal stage, packaged into nucleosome-like structures. However, when HD-Ad5/35 vectors contained a 23kb fragment of the 2-globin locus control region (HD-Ad5/35.LCR-1), vector genomes integrated at a high frequency into the chromosomal DNA of human erythroid Mo7e cells. Importantly, 20% of all integration events occurred within the chromosomal 2-globin LCR, particularly into a region within and downstream of hypersensitivity site 2. We demonstrated that targeted HD-Ad5/35.LCR-1 integration involves globin LCR-specific proteins characteristic for "active" chromatin and that incoming HD-Ad5/35.LCR-1 genomes are physically tethered to the chromosomal globin LCR involving chromatin proteins. We speculate that physical proximity between vector and chromosomal DNA, together with DNA breaks within the vicinity of tethered vector DNA, mediates preferential integration of our vector into the globin LCR in Mo7e cells. In this proposal we will attempt to achieve targeted transgene integration in HSCs. In preliminary studies, LCR-chromatin tethering and/or vector integration is less efficient in CD34+ cells than in Mo7e cells. We speculate that the 2-globin LCR chromatin in CD34+ cells is competent for HD-Ad5/35.LCR-1 tethering and the limiting step in stable CD34+ cell transduction is vector integration. We will test this hypothesis in Specific Aim 1. Our Specific Aim 2 is to improve the integration efficiency of tethered HD-Ad5/35.LCR genomes using a &C31-phage integrase. Overall, this work will lead us to a better understanding of mechanisms and structural determinants of chromatin tethering and its role in facilitating vector integration and create the basis for improving targeted integration of existing retrovirus vectors through chromatin tethering of retroviral integrases. PUBLIC HEALTH RELEVANCE: We propose a novel vector system to achieve targeted integration of a large transgene cassette in human hematopoietic stem cells. Our approach combines a new vehicle for DNA delivery into stem cells with a new idea to achieve targeted integration of a large transgene cassette. Specifically, we plan to capitalize on our recent finding that preferential vector integration into the beta-globin LCR can be achieve via chromatin tethering of transgene cassettes. This study is relevant for engineering target site specificity of integrating vectors in general and might provide a basis for globin gene therapy.