Library Screening for Novel Enhancer Blockers Derived from Human T Lymphocytes

描述（由申请人提供）：最近免疫缺陷疾病的临床基因治疗试验已经利用整合逆转录病毒载体来提供各种治疗基因到造血干细胞中的稳定传递。虽然这些试验已经证明了在严重联合免疫缺陷和Wiscott-Aldrich综合征中的有效性，但由于载体诱导的细胞原癌基因（最明显的是LMO 2）活化，已经有6例T细胞恶性肿瘤病例。在大多数情况下，如果不是所有这些情况下，载体编码的增强子，位于病毒长末端重复序列，与相邻的细胞原癌基因启动子的相互作用，导致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子和其他病毒转录元件、使用自失活慢病毒载体和使用含有增强子阻断序列的染色质绝缘子元件来消除或降低这种严重副作用的发生率。本申请集中于鉴定新的、更有效的增强子阻断元件，其将在人T细胞中提供最大活性，并因此允许用于T细胞免疫缺陷病症的更安全的基因治疗载体。具体目标1的重点是开发用于筛选潜在绝缘子元件库的灵敏且相关的细胞测定;在具体目标2中，新开发的绝缘子筛选系统将用于鉴定在人T淋巴细胞中具有高水平活性的新型增强子阻断元件。该领域目前的一个重要障碍是相对缺乏用于此目的的有效绝缘子元件;该领域的大部分使用来自鸡的珠蛋白基因座（cHS 4）的元件，该元件相对具有组织特异性，并且在T细胞中仅具有部分活性。我们将在圣裘德开发的用于Jurkat T细胞同源基因靶向的新型系统的基础上，筛选来自T细胞的CTCF结合序列库的增强子阻断活性。我们的目标是确定在T细胞中高度活跃的新的阻断元件。 公共卫生相关性：成功鉴定新型绝缘体可以增加缺乏合适治疗选择的免疫缺陷患者的基因治疗方法的安全性。

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. PUBLIC HEALTH RELEVANCE: Successful identification of novel insulators could increase the safety of gene therapy approaches for immunodeficiency patients that lack suitable treatment options.