Targeted Transgene Integration through Chromatin tethering for Globin Gene Therap

通过染色质束缚进行靶向转基因整合用于球蛋白基因治疗

• 批准号: 7570551
• 负责人: ANDRE Michael LIEBER
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-27 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7570551
• 关键词: Active Sites; Adenovirus Vector; Bacteriophages; CD34 gene; Cell Nucleus; Cell Proliferation; Cells; Characteristics; Chromatin; DNA; DNA Repair; DNA delivery; Engineering; Erythroid; Event; Fiber; Frequencies; Genes; Genome; Globin; Goals; Hematopoietic stem cells; Human; Hypersensitivity; Integrase; Lead; Locus Control Region; Mediating; Nonhomologous DNA End Joining; Nucleosomes; Proteins; Retroviral Vector; Role; Site; Specificity; Staging; Stem cells; Structure; System; Testing; Transgenes; Work; base; beta Globin; cellular transduction; chromatin protein; gene therapy; human DNA; improved; novel; public health relevance; vector; vector genome

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.

描述（由申请人提供）： 我们的最终目标是实现大型（>25kb）转基因盒在人类造血干细胞（HSC）中的靶向整合。为了实现这一目标，我们构建了转导 HSC 的辅助依赖性纤维嵌合腺病毒载体 (HD-Ad5/35)。 HD-Ad5/35 载体能够非常有效地将大型转基因盒递送至 HSC 细胞核，其中载体基因组保持游离状态，包装成核小体样结构。然而，当 HD-Ad5/35 载体含有 2-球蛋白基因座控制区 (HD-Ad5/35.LCR-1) 的 23kb 片段时，载体基因组以高频率整合到人红系 Mo7e 细胞的染色体 DNA 中。重要的是，所有整合事件的 20% 发生在染色体 2-珠蛋白 LCR 内，特别是进入超敏位点 2 内和下游的区域。我们证明，靶向 HD-Ad5/35.LCR-1 整合涉及珠蛋白 LCR 特异性蛋白质特征对于“活性”染色质，传入的 HD-Ad5/35.LCR-1 基因组在物理上与涉及染色质的染色体珠蛋白 LCR 相连蛋白质。我们推测载体和染色体 DNA 之间的物理接近性，以及系留载体 DNA 附近的 DNA 断裂，介导我们的载体优先整合到 Mo7e 细胞中的珠蛋白 LCR 中。在本提案中，我们将尝试在 HSC 中实现靶向转基因整合。在初步研究中，LCR-染色质束缚和/或载体整合在 CD34+ 细胞中的效率低于 Mo7e 细胞。我们推测 CD34+ 细胞中的 2-珠蛋白 LCR 染色质能够进行 HD-Ad5/35.LCR-1 束缚，稳定 CD34+ 细胞转导的限制步骤是载体整合。我们将在具体目标 1 中测试这一假设。我们的具体目标 2 是使用 &C31 噬菌体整合酶提高束缚 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了解染色质束缚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束缚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： 我们提出了一种新型载体系统，以实现大型转基因盒在人类造血干细胞中的靶向整合。我们的方法将 DNA 递送到干细胞的新载体与实现大型转基因盒的靶向整合的新想法相结合。具体来说，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束缚来实现载体优先整合到β-珠蛋白LCR中。这项研究与整合载体的靶位点特异性工程相关，并可能为珠蛋白基因治疗提供基础。