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人造血干细胞（HSC）中大型（> 25kb）盒的目标整合。为了实现目标，我们构建了转导HSC的助手依赖性，纤维智病毒载体（HD-AD5/35）。 HD-AD5/35载体在将大型转基因盒输送到HSC的核中非常有效，在该核中，载体基因组保持在偶发阶段，包装到核体样结构中。但是，当HD-AD5/35载体包含2-珠蛋白基因座控制区域（HD-AD5/35.LCR-1）的23KB片段时，载体基因组以高频整合到人类红细胞MO7E细胞的染色体DNA中。重要的是，所有整合事件中有20％发生发生在染色体2-蛋白LCR中，特别是在高敏位点的区域内和下游区域2。我们证明，靶向的HD-AD5/35.LCR-1整合涉及Globin LCR LCR特异性蛋白的特征，用于“活性HD-AD-AD-AD-AD-AD-AD-AD-5/35.LCR-1”。涉及染色质蛋白的球蛋白LCR。我们推测，载体和染色体DNA之间的物理接近，以及在束缚载体DNA附近的DNA断裂，介导了我们的载体在MO7E细胞中的Globin LCR中的优先整合。在此提案中，我们将尝试实现HSC中有针对性的转基因整合。在初步研究中，LCR-染色质的束缚和/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染色质具有HD-AD5/35.LCR-1的束缚，并且稳定的CD34+细胞转导中的限制步骤是矢量积分。我们将在特定目标1中检验这一假设。我们的特定目的2是使用A＆C31-phage积分酶提高束缚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理解染色质束缚的机制和结构决定因素及其在促进矢量整合中的作用，并通过逆转录病毒整合酶的染色质链球酶为改善现有逆转录病毒载体的有针对性整合创造了基础。 公共卫生相关性： 我们提出了一个新型的矢量系统，以实现人类造血干细胞中大型转基因盒的有针对性整合。我们的方法结合了一种新车辆，将DNA递送到干细胞中的新车和一个新想法，以实现大型转基因盒的目标整合。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绑扎来实现优先的向量整合到β-珠蛋白LCR中。这项研究与一般整合向量的工程目标位点特异性有关，并可能为球蛋白基因疗法提供基础。