Safe, Silencing-Resistant, Non-Oncogenic Globin Expression Cassettes

安全、抗沉默、非致癌的珠蛋白表达盒

• 批准号: 7248538
• 负责人: ERIC E BOUHASSIRA
• 金额: $ 41.5万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7248538
• 关键词: Address; Adverse effects; Be++ element; Beryllium; Cells; Chickens; Effectiveness; Elements; Enhancers; Erythroid; Erythroid Cells; Event; Gene Activation; Gene Cluster; Globin; Hematopoietic; Hemoglobinopathies; Human; Hybrids; Insertional Activations; Insertional Mutagenesis; Insula of Reil; Knowledge; Measures; Mediating; Methods; Mus; Numbers; Oncogenic; Range; Regulatory Element; Research Personnel; Resistance; Risk; Role; Site; Testing; Therapeutic; Transgenes; Virus; WA01 cell line; WA09 Cell Line; design; embryonic stem cell; gene therapy; human embryonic stem cell; lentiviral-mediated; leukemia/lymphoma; man; non-oncogenic; novel; prevent; promoter; replicator; transgene expression; tumorigenesis

DESCRIPTION (provided by applicant): SAFE, SILENCING-RESISTANT, NON-ONCOGENIC GLOBIN EXPRESSION CASSETTES. A proof of principle that gene therapy for the hemoglobinopathies is feasible has been obtained in mice. The major aim of this project is to move on from this important milestone to address a major remaining issue relating to the potential for oncogenesis by lentiviral-mediated insertional mutagenesis. We propose to design new gene therapy cassettes that will have a minimal impact on the flanking sequences thereby reducing the risk of oncogenic insertional activation events. SPECIFIC AIM 1 is to assess the effectiveness of insulators in blocking activation of genes in the flanking sequences and to test newly discovered insulators. The only known method to protect the flanking sequences near integration sites of enhancers in transgenes is the use of insulators and enhancer-blockers such as DNAsel hyper-sensitive site 4 of the chicken p-globin gene cluster (cHS4). While cHS4 activities are well demonstrated in artificial constructs, whether this element is effective for all promoters is not known. We propose to answer this question and to test other regulatory elements which might have complementary activities. SPECIFIC AIM 2 is to assess the role of the LCR in silencing, its potential to cause insertional mutagenesis in human hematopoietic cells, and to develop novel LCRs with a shorter range of action. We have obtained evidence that paradoxically transgene silencing can be caused by the presence of the LCR. We propose here to determine the generality of this novel mechanism of transgene silencing. SPECIFIC AIM 3 is to determine if replicators incorporated in transgenes can prevent silencing at large number of sites and to measure their effects on the flanking sequences. We have obtained evidence that silencing can be released by inclusion of a replicator in the expression cassette. We propose here to determine if this effect is general. Little is known about the effects of replicators on transgene expression and on the flanking sequences. We propose to fill some of the gaps in our knowledge of these elements. Relevance: Spectacular advances in the field of gene therapy have been made in the last few years but a major possible side-effect remains the risk of treatment-induced leukemias and lymphomas. We propose here to develop therapeutic expression cassettes which will cause less side-effects. hESC line WA01 and WA09 will be used in this project.

描述（由申请方提供）：安全、抗沉默、非致癌性珠蛋白表达盒。 在小鼠中已经获得了血红蛋白病的基因治疗是可行的原理证明。该项目的主要目的是从这个重要的里程碑继续前进，以解决与慢病毒介导的插入诱变致癌潜力相关的主要遗留问题。我们建议设计新的基因治疗盒，其对侧翼序列的影响最小，从而降低致癌插入激活事件的风险。具体目标1是评估绝缘子在阻断侧翼序列基因激活中的有效性，并测试新发现的绝缘子。保护转基因中增强子整合位点附近的侧翼序列的唯一已知方法是使用绝缘子和增强子阻断剂，例如鸡β-珠蛋白基因簇（cHS 4）的DNAsel超敏感位点4。虽然cHS 4活性在人工构建体中得到充分证明，但该元件是否对所有启动子有效尚不清楚。我们建议回答这个问题，并测试其他可能具有互补活性的调节元件。具体目的2是评估LCR在沉默中的作用，其在人造血细胞中引起插入突变的潜力，并开发具有较短作用范围的新型LCR。我们已经获得的证据表明，自相矛盾的转基因沉默可以由LCR的存在引起。我们建议在这里确定这种新的转基因沉默机制的一般性。具体目标3是确定是否复制纳入转基因可以防止沉默在大量的网站，并衡量其对侧翼序列的影响。我们已经获得了沉默可以通过在表达盒中包含复制子来释放的证据。我们在这里建议确定这种效应是否具有普遍性。关于复制子对转基因表达和侧翼序列的影响知之甚少。我们建议填补我们对这些要素的知识中的一些空白。相关性：在过去的几年里，基因治疗领域取得了惊人的进展，但一个主要的可能的副作用仍然是治疗引起的白血病和淋巴瘤的风险。我们在这里建议开发治疗性表达盒，这将导致更少的副作用。hESC细胞系WA 01和WA 09将用于本项目。