Translating TCRa locus control region activity to T cell gene therapy vectors

描述(由申请人提供)：T细胞是一种非常重要的细胞类型，可以作为基因治疗的靶点。12 T细胞受体(TCR)复合体被大多数循环中的T细胞用来识别抗原和启动免疫反应。T细胞可以被改造成表达特定的、克隆的TCR基因，编码受体，使它们能够启动所需的免疫反应。原则上，这种方法将产生针对各种病原体和肿瘤的直接免疫疗法。基因疗法还可以治疗不可治愈的遗传和获得性T细胞疾病，其中几种疾病对美国少数族裔社区的影响不成比例，包括由艾滋病毒和HTLV-1引起的疾病。胚胎和成体干细胞以及慢病毒载体是将治疗性基因产品引入T细胞的很有前途的载体。但这种方法需要在体内T细胞发育过程中调控转录的DNA序列方面取得重大进展。目前用于转导干细胞的病毒载体不能产生T系特异性或发育控制的转录。此外，它们经常在染色体整合时被沉默。T细胞受体(TCR)-1位点控制区(LCR)DNA的特性可以直接应用于克服这些显着的限制。然而，携带治疗性基因的干细胞转导病毒载体对额外的外源基因调控DNA的空间非常有限。因此，将TCR1 LCR活性应用于基因治疗载体的开发将需要定位和鉴定支持其强大功能的DNA亚序列。本实验室一直在研究TCR1 LCR的体内活性。该项目的目标是创造一种TCR1 LCR的“微型化”版本，以最少的DNA支持最大的LCR活性。实现这一目标的一个障碍是缺乏两个关键LCR元件(称为HS1‘和HS4)的基本分子知识。我们假设HS1‘和HS4区包含多个对TCR1 LCR活性至关重要的局部功能子序列元件。利用我们实验室建立的一系列分子、生化、细胞和体内功能分析，我们建议在这里定位HS1‘和HS4区域的这些功能亚元件。根据这些信息，我们进一步假设构建一个最小尺寸但完全活跃的TCR1迷你LCR是可以实现的。在这个项目中，我们将基于从HS1‘和HS4分析中获得的知识，以及另外两个关键LCR亚区的分子组成的先验知识，构建和测试TCR1迷你LCR版本。这项工作对于将TCR1 LCR DNA的功能分子成分应用于设计安全、有效的载体，从而在T细胞中产生细胞类型受限的治疗性基因表达是至关重要的。 与公共卫生相关：干细胞和慢病毒载体是将治疗性基因产品引入T细胞以纠正遗传缺陷或使其能够启动针对各种病原体和癌症的特定免疫反应的有前途的载体。此外，艾滋病毒和HTLV-1会导致毁灭性的、目前无法治愈的T细胞疾病(艾滋病和成人T细胞白血病)，对少数群体和未得到充分服务的人群成员造成不成比例的影响。该项目将揭示T细胞受体-1基因位点控制区强大的基因调控活性的DNA元件，可用于将安全、有效的治疗性基因表达专门针对T细胞来治疗这些疾病。

DESCRIPTION (provided by applicant): T cells are a highly significant cell type to target for gene therapy. The 12 T cell receptor (TCR) complex is used by most circulating T cells to recognize antigen and initiate immune responses. T cells can be engineered to express specific, cloned TCR genes encoding receptors enabling them to initiate a desired immune response. In principle, this approach would yield direct immunotherapy against a wide variety of pathogen and tumors. Gene therapy can also address incurable genetic and acquired diseases of T cells, several of which disproportionately impact minority communities in the U.S., including those caused by HIV and HTLV-1. Embryonic and adult stem cells and lentiviral vectors are promising vehicles for introducing therapeutic gene products into T cells. But this approach will require major advances in knowledge of DNA sequences regulating transcription during T cell development in vivo. Viral vectors currently used to transduce stem cells cannot generate T lineage-specific or developmentally controlled transcription. Furthermore, they are frequently silenced upon chromosomal incorporation. The properties of the T cell receptor (TCR)-1 Locus Control Region (LCR) DNA can be directly applied towards overcoming these significant limitations. However, stem cell-transducing viral vectors carrying therapeutic genes have very limited space for additional exogenous gene regulatory DNA. Thus, the application of TCR1 LCR activity to gene therapy vector development will require localization and characterization of the DNA sub-sequences that support its powerful function. This laboratory has been studying the in vivo activity of the TCR1 LCR. The goal of this project is to enable the creation of a "miniaturized" version of the TCR1 LCR that supports maximal LCR activity using the minimum amount of DNA. A hurdle to achieving this goal is the lack of essential molecular knowledge of two key LCR elements (named HS1' and HS4). We hypothesize that the HS1' and HS4 region contain multiple, localized functional sub-sequence elements critical to TCR1 LCR activity. Using an array of molecular, biochemical, cellular and in vivo functional assays well established in our laboratory, we propose here to map these functional sub-elements of the HS1' and HS4 regions. With this information, we further hypothesize that construction of a minimally sized, but fully active, TCR1 mini-LCR is achievable. In this project, we will construct and test TCR1 mini-LCR versions based on the knowledge obtained here from the analyses of HS1' and HS4, and prior knowledge of the molecular components of two other critical LCR sub-regions. The proposed work is essential to applying the functional molecular components of TCR1 LCR DNA towards designing safe, effective vectors producing cell type-restricted therapeutic gene expression in T cells. PUBLIC HEALTH RELEVANCE: Stem cells and lentiviral vectors are promising vehicles for introducing therapeutic gene products into T cells to correct genetic deficiencies, or enable them to initiate specific immune responses against a wide variety of pathogens and cancers. In addition, HIV and HTLV-1 cause devastating and currently incurable diseases of T cells (AIDS and Adult T cell leukemia) that disproportionately affect members of minority and underserved populations. This project will reveal DNA elements of the powerful gene regulatory activity of the T cell receptor-1 gene locus control region that can be used to direct safe, effective therapeutic gene expression specifically to T cells to treat these conditions.