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

描述（由申请人提供）：T细胞是基因治疗靶向的高度重要的细胞类型。12 T细胞受体（TCR）复合物被大多数循环T细胞用于识别抗原并启动免疫应答。T细胞可以被工程化以表达编码受体的特异性克隆TCR基因，使它们能够启动所需的免疫应答。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。基因疗法还可以解决无法治愈的遗传性和获得性T细胞疾病，其中一些疾病对美国的少数民族社区造成了不成比例的影响，包括HIV和HTLV-1引起的疾病。胚胎和成体干细胞和慢病毒载体是将治疗性基因产物引入T细胞的有前途的载体。但是这种方法需要在体内T细胞发育过程中调节转录的DNA序列的知识方面取得重大进展。目前用于扩增干细胞的病毒载体不能产生T谱系特异性或发育控制的转录。此外，它们在染色体掺入时经常沉默。T细胞受体（TCR）-1基因座控制区（LCR）DNA的特性可以直接应用于克服这些重大限制。然而，携带治疗基因的干细胞转导病毒载体对于额外的外源基因调控DNA具有非常有限的空间。因此，将TCR 1 LCR活性应用于基因治疗载体开发将需要定位和表征支持其强大功能的DNA亚序列。该实验室一直在研究TCR 1 LCR的体内活性。该项目的目标是创建一个“小型化”版本的TCR 1 LCR，使用最少量的DNA支持最大的LCR活性。实现这一目标的障碍是缺乏两个关键LCR元件（命名为HS 1 ′和HS 4）的基本分子知识。我们假设HS 1 ′和HS 4区含有对TCR 1 LCR活性至关重要的多个局部功能亚序列元件。利用我们实验室建立的一系列分子、生物化学、细胞和体内功能测定，我们在这里提出绘制HS 1 ′和HS 4区的这些功能子元件。有了这些信息，我们进一步假设构建最小尺寸但完全活性的TCR 1 mini-LCR是可以实现的。在这个项目中，我们将构建和测试TCR 1 mini-LCR版本的基础上，从HS 1 '和HS 4的分析，和其他两个关键的LCR亚区的分子组成的先验知识在这里获得的知识。所提出的工作对于将TCR 1 LCR DNA的功能性分子组分应用于设计安全、有效的载体在T细胞中产生细胞类型限制的治疗性基因表达是必不可少的。

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.