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

描述(申请人提供)：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。该项目的主要目标是从这一重要的里程碑向前迈进，以解决与慢病毒介导的插入突变导致肿瘤的可能性相关的一个主要遗留问题。我们建议设计新的基因治疗盒，它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。具体目的1是评估绝缘子在阻断侧翼序列中基因激活方面的有效性，并测试新发现的绝缘子。虽然cHS4活性在人工构建物中得到了很好的证明，但这个元件是否对所有启动子都有效尚不清楚。我们建议回答这一问题，并测试其他可能具有补充作用的监管要素。特异性目的2是评估LCR在沉默中的作用及其对人造血细胞的插入突变的可能性，并开发作用范围更短的新型LCR。我们已经获得的证据表明，自相矛盾的是，转基因沉默可以由LCR的存在引起。我们建议在这里确定这一新的转基因沉默机制的普遍性。特异目的3是确定转基因中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。我们已经获得了证据表明，通过在表达盒中加入复制子可以解除沉默。我们建议在这里确定这种影响是否具有普遍性。关于复制子对转基因表达和侧翼序列的影响，人们知之甚少。我们建议填补我们对这些要素知识的一些空白。相关性：基因治疗领域在过去几年中取得了惊人的进步，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。我们建议开发一种副作用较小的治疗性表达盒。HESC线路WA01和WA09将用于本项目。

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.