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

安全、抗沉默、非致癌珠蛋白表达盒 在小鼠身上已经获得了基因治疗血红蛋白疾病是可行的原则的证据。这个 这个项目的主要目标是从这个重要的里程碑前进，以解决一个主要的遗留问题 与慢病毒介导的插入突变致癌的可能性有关。我们建议 设计新的基因治疗盒，从而对侧翼序列产生最小的影响 降低致癌插入激活事件的风险。具体目标1是评估有效性 绝缘体在阻止侧翼序列中基因激活方面的作用，并测试新发现的 绝缘体。唯一已知的保护增强子整合位点附近侧翼序列的方法 转基因是使用绝缘体和增强子阻断剂，如Dnasel的超敏感部位4 鸡p-珠蛋白基因簇(CHS4)。虽然cHS4活性在人工构建物中得到了很好的展示， 这一要素是否对所有推动者都有效尚不清楚。我们建议回答这个问题，并 测试可能具有补充活动的其他监管要素。具体目标2是评估 LCR在沉默中的作用，它在人类造血细胞中引起插入突变的可能性， 并开发具有更短作用范围的新型LCR。我们已经获得了自相矛盾的证据 转基因沉默可由LCR的存在引起。我们在此建议确定 这一转基因沉默的新机制的一般性。具体的目标3是确定复制者是否 整合到转基因中可以防止大量位点的沉默，并测量它们对 侧翼序列。我们已经获得了证据，可以通过包括一个 表达盒中的复制物。我们建议在这里确定这种影响是否具有普遍性。鲜为人知 关于复制子对转基因表达和侧翼序列的影响。我们建议填补 我们对这些元素认识上的一些空白。相关性：在以下领域的惊人进展 在过去的几年里，基因治疗已经取得了进展，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。我们建议在这里开发治疗性表达 盒式磁带的副作用会更小。HESC线路WA01和WA09将用于本项目。

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 of 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.