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

将 TCRa 基因座控制区活性转化为 T 细胞基因治疗载体

• 批准号: 8418752
• 负责人: BENJAMIN D., ORTIZ
• 金额: $ 29.53万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-09 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8418752
• 关键词: Acquired Immunodeficiency Syndrome; Address; Adult; Affect; Antigens; Applications Grants; Binding Sites; Biochemical; Biological Assay; CD4 Positive T Lymphocytes; Characteristics; Clinical; Collaborations; Communities; Complex; DNA; DNA Sequence; Deoxyribonucleases; Development; Disease; Elements; Embryo; Engineering; Environment; Funding; Gene Expression; Gene Transduction Agent; Genes; Genetic; Genetic Engineering; Genetic Transcription; Genome; Goals; HIV; Health; Human T-lymphotropic virus 1; Immune response; Immunotherapeutic agent; Immunotherapy; Institutes; Knowledge; Laboratories; Lentivirus Vector; Locus Control Region; Malignant Neoplasms; Maps; Mediating; Minority; Minority Groups; Molecular; Names; Production; Productivity; Property; Proteins; Regulator Genes; Reporter; Research Personnel; Site; Solid; Specificity; Stem cells; Students; System; T-Cell Development; T-Cell Leukemia; T-Cell Receptor; T-Cell Receptor Genes; T-Lymphocyte; Testing; Training; Transgenes; Translating; Translational Research; Underserved Population; United States; Viral Vector; Work; adult stem cell; base; cell type; chromatin immunoprecipitation; college; design; ethnic minority population; gene therapy; in vivo; member; miniaturize; molecular array; mutant; new technology; pathogen; prevent; receptor; therapeutic gene; tumor; vector

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.

描述（由申请人提供）：T细胞是基因治疗中非常重要的靶细胞类型。大多数循环T细胞使用12t细胞受体（TCR）复合物来识别抗原并启动免疫反应。T细胞可以被改造成表达特异性的、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多种病原体和肿瘤的直接免疫疗法。基因疗法还可以治疗无法治愈的遗传和获得性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，包括由HIV和HTLV-1引起的疾病。胚胎干细胞和成体干细胞以及慢病毒载体是将治疗性基因产物引入T细胞的有希望的载体。但这种方法需要在体内T细胞发育过程中调节转录的DNA序列知识方面取得重大进展。目前用于转导干细胞的病毒载体不能产生T谱系特异性或发育控制转录。此外，它们经常在染色体合并后沉默。T细胞受体(TCR)-1基因座控制区（LCR） DNA的特性可以直接用于克服这些显著的限制。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的空间非常有限。因此，将TCR1 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。本实验室一直在研究TCR1 LCR的体内活性。该项目的目标是创建一个“小型化”版本的TCR1 LCR，使用最少的DNA量来支持最大的LCR活性。实现这一目标的障碍是缺乏两个关键LCR元素（称为HS1'和HS4）的基本分子知识。我们假设HS1'和HS4区域包含多个局部功能亚序列元件，对TCR1 LCR活性至关重要。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，我们建议在这里绘制HS1'和HS4区域的这些功能亚元件。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的分析中获得的知识，以及对其他两个关键LCR子区域的分子成分的先验知识，构建和测试TCR1 mini-LCR版本。这项工作对于应用TCR1 LCR DNA的功能分子成分来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至关重要。