Targeted Transgene Integration through Chromatin tethering for Globin Gene Therap

描述（由申请人提供）： 我们的最终目标是实现大（> 25 kb）转基因盒在人造血干细胞（HSC）中的靶向整合。为了达到这个目标，我们构建了辅助病毒依赖的纤维嵌合腺病毒载体（HD-Ad 5/35），其可以转染HSC。HD-Ad 5/35载体在将大的转基因盒递送到HSC的细胞核中非常有效，其中载体基因组保持在附加体阶段，包装成核小体样结构。然而，当HD-Ad 5/35载体含有2-珠蛋白基因座控制区的23 kb片段（HD-Ad 5/35.LCR-1）时，载体基因组以高频率整合到人红系Mo 7 e细胞的染色体DNA中。重要的是，所有整合事件中有20%发生在染色体2-珠蛋白LCR内，特别是进入超敏反应位点2内和下游的区域。我们证明了靶向的HD-Ad 5/35.LCR-1整合涉及“活性”染色质特有的球蛋白LCR特异性蛋白，并且引入的HD-Ad 5/35.LCR-1基因组物理地束缚于涉及染色质蛋白的染色体球蛋白LCR。我们推测，载体和染色体DNA之间的物理接近，连同系留载体DNA附近的DNA断裂，介导我们的载体优先整合到Mo 7 e细胞中的球蛋白LCR中。在这个提议中，我们将尝试在HSC中实现靶向转基因整合。在初步研究中，LCR-染色质拴系和/或载体整合在CD 34+细胞中的效率低于Mo 7 e细胞。我们推测，CD 34+细胞中的2-珠蛋白LCR染色质能够胜任HD-Ad 5/35.LCR-1拴系，并且稳定的CD 34+细胞转导中的限制性步骤是载体整合。我们将在具体目标1中检验这一假设。我们的具体目标2是使用C31-噬菌体整合酶提高系留的HD-Ad 5/35.LCR基因组的整合效率。总的来说，这项工作将使我们更好地了解染色质拴系的机制和结构决定因素及其在促进载体整合中的作用，并为通过逆转录病毒整合酶的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，以实现在人造血干细胞中的大转基因盒的靶向整合。我们的方法结合了将DNA递送到干细胞中的新载体和实现大转基因盒的靶向整合的新想法。具体而言，我们计划利用我们最近的发现，即可以通过转基因盒的染色质拴系来实现优先载体整合到β-珠蛋白LCR中。本研究为整合载体的靶位点特异性工程化提供了理论依据，为珠蛋白基因治疗提供了理论依据。

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.