Library Screening for Novel Enhancer Blockers Derived from Human T Lymphocytes

描述(申请人提供)：最近免疫缺陷疾病的临床基因治疗试验利用整合的逆转录病毒载体提供稳定的各种治疗基因进入造血干细胞。虽然这些试验已经证明对严重的联合免疫缺陷和Wiscott-Aldrich综合征有效，但已经有6例T细胞恶性肿瘤是由载体诱导的细胞原癌基因激活引起的，最明显的是LMO2。在大多数情况下，如果不是全部，位于病毒长末端重复序列的载体编码的增强子与相邻的细胞原癌基因启动子的相互作用导致了无意中的基因激活事件和转化。基因治疗领域的一个主要目标是通过消除增强剂和其他病毒转录元件，使用自失活慢病毒载体，以及使用含有增强子阻断序列的染色质绝缘体元件来消除或减少这种严重副作用的发生。这项应用侧重于识别新的、更有效的增强子阻断元件，这些元件将在人类T细胞中提供最大活性，从而允许更安全的T细胞免疫缺陷疾病的基因治疗载体。具体目标1的重点是建立一种灵敏和相关的细胞方法来筛选潜在的绝缘子元件文库；在特定目标2中，新开发的绝缘子筛选系统将用于在人T淋巴细胞中鉴定新的具有高活性的增强子阻断元件。该领域的一个重要电流障碍是相对缺乏有效的绝缘体元素来实现这一目的；该领域的大部分领域使用来自鸡珠蛋白基因(CHS4)的元素，该元素相对组织特有，仅在T细胞中部分活跃。我们将建立一个在圣犹大开发的用于Jurkat T细胞同源基因打靶的新系统，以筛选来自T细胞的CTCF结合序列文库，以增强启动子阻断活性。我们的目标是确定在T细胞中高度活跃的新的阻断元件。

DESCRIPTION (provided by applicant): Recent clinical gene therapy trials for immunodeficiency disorders have utilized integrating retroviral vectors to provide stable transmission of various therapeutic genes into hematopoietic stem cells. While these trials have demonstrated proof of efficacy in severe combined immunodeficiencies and Wiscott-Aldrich syndrome, there have been six cases of Tcell malignancies due to vector-induced activation of cellular proto-oncogenes, most notably LMO2. In most if not all of these cases, the interaction of vector-encoded enhancers, located in the viral long terminal repeats, with adjacent cellular proto-oncogene promoters resulted in inadvertent gene activation events and transformation. A major goal in the field of gene therapy is to eliminate or reduce the incidence of this serious side effect through the elimination of enhancers and other viral transcriptional elements, the use of self-inactivating lentiviral vectors, and the use of chromatin insulator elements that contain enhancer blocking sequences. This application focuses on identifying new, more effective enhancer blocking elements that would provide maximum activity in human T cells and therefore allow safer gene therapy vectors for T cell immunodeficiency disorders. The focus of specific aim 1 is to develop a sensitive and relevant cellular assay for screening libraries of potential insulator elements; in specific aim 2, the newly developed insulator screening system will be used to identify novel enhancer blocking elements with high level activity in human T lymphocytes. An important current obstacle for the field is the relative lack of effective insulator elements for this purpose; much of the field uses an element from the chicken ¿-globin locus (cHS4) that is relatively tissue specific and only partially active in T cells. We will build upon a novel system developed here at St. Jude for homologous gene targeting in Jurkat T cells to screen a library of CTCF binding sequences derived from T cells for enhancer blocking activity. Our goal is to identify new blocking elements that are highly active in T cells.