Library Screening for Novel Enhancer Blockers Derived from Human T Lymphocytes

描述（由申请人提供）：最近针对免疫缺陷疾病的临床基因治疗试验利用整合逆转录病毒载体将各种治疗基因稳定地传递到造血干细胞中。虽然这些试验已经证明了对严重联合免疫缺陷和Wiscott-Aldrich综合征的有效性，但由于载体诱导的细胞原癌基因激活，其中最明显的是LMO2，已经有6例t细胞恶性肿瘤。在大多数情况下（如果不是全部的话），位于病毒长末端重复序列的载体编码增强子与邻近的细胞原癌基因启动子的相互作用导致无意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子和其他病毒转录元件，使用自我灭活的慢病毒载体，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率。该应用程序的重点是识别新的、更有效的增强子阻断元件，这些元件将在人类T细胞中提供最大的活性，从而为T细胞免疫缺陷疾病提供更安全的基因治疗载体。具体目标1的重点是开发一种敏感和相关的细胞检测方法，用于筛选潜在绝缘体元素文库；在特定目标2中，新开发的绝缘子筛选系统将用于识别人类T淋巴细胞中具有高水平活性的新型增强子阻断元件。目前该领域的一个重要障碍是相对缺乏用于此目的的有效绝缘体元件；该领域的大部分研究都使用鸡血红蛋白位点（cHS4）中的一种元素，这种元素相对具有组织特异性，仅在T细胞中部分活跃。我们将在St. Jude开发的一个新系统的基础上，在Jurkat T细胞中进行同源基因靶向，筛选来自T细胞的CTCF结合序列库，以获得增强子阻断活性。我们的目标是识别在T细胞中高度活跃的新阻断元件。

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.