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

描述（由申请人提供）：安全，抗沉默，非致癌的珠蛋白表达盒。在小鼠实验中获得了基因治疗血红蛋白病是可行的原理证明。该项目的主要目的是从这一重要里程碑出发，解决与慢病毒介导的插入突变致癌潜力有关的主要遗留问题。我们建议设计新的基因治疗盒，将对侧翼序列产生最小的影响，从而降低致癌插入激活事件的风险。特异性目的1是评估绝缘子阻断侧翼序列中基因激活的有效性，并测试新发现的绝缘子。唯一已知的保护转基因中增强子整合位点附近侧翼序列的方法是使用绝缘子和增强子阻断剂，如鸡p-珠蛋白基因簇（cHS4）的DNAsel超敏感位点4。虽然cHS4的活性在人工构建物中得到了很好的证明，但该元件是否对所有启动子都有效尚不清楚。我们建议回答这个问题，并测试其他可能具有补充作用的监管要素。具体目标2是评估LCR在沉默中的作用，其在人类造血细胞中引起插入突变的潜力，并开发具有更短作用范围的新型LCR。我们已经获得的证据表明，矛盾的是，转基因沉默可以由LCR的存在引起。我们在此建议确定这种转基因沉默新机制的普遍性。特异性目的3是确定在转基因中加入的复制子是否可以防止大量位点的沉默，并测量它们对侧翼序列的影响。我们已经获得证据表明沉默可以通过在表达盒中加入复制子来释放。我们在此提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知识的一些空白。相关性：在过去几年中，基因治疗领域取得了惊人的进展，但一个可能的主要副作用仍然是治疗引起的白血病和淋巴瘤的风险。我们在此建议开发治疗性表达磁带，以减少副作用。本项目将使用hESC线WA01和WA09。

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.