Utilizing TALEN technology to regulate human microRNA's

描述（由申请人提供）：MicroRNAs （miRNAs）由于其对关键蛋白质编码基因的精确调控而迅速成为哺乳动物生理学的重要调节因子。重要的是，特定mirna表达或功能的扰动与多种人类病理状况有关，包括癌症、自身免疫、心血管疾病和神经变性。尽管通过同源重组在小鼠中删除miRNA基因已经提高了对它们在健康和疾病时期如何发挥作用的理解，但仍然需要在人类种系中操纵miRNA基因及其靶结合位点。这将允许在人体中研究它们的生物学，并且可能是一种战略性手段，通过这种手段，mirna可以靶向治疗人类疾病。我们建议开发一种新的方法来激活、抑制或破坏人类miRNA基因，通过工程TALE蛋白，这些蛋白将作为转录因子或核酸酶，特异性靶向miRNA基因或其在关键靶mrna 3' utr中的结合位点。我们将重点关注人类miRNA- 155，一种参与调节免疫反应的“oncomiR”，以及miR- 146a，一种抑制炎症反应的肿瘤抑制miRNA。在适当靶向和破坏这些mirna或其在相关靶mrna的3' utr中的结合位点后，将进行一系列功能分析，以评估人类miR- 155和miR- 146a在调节免疫反应和癌症表型中的作用。除了这两种miRNA，这种方法将与任何人类miRNA兼容，使其成为一种强大的、特异性的和通用的技术，目前尚未用于研究miRNA。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) have quickly emerged as important regulators of mammalian physiology owing to their precise regulation of critical protein coding genes. Importantly, perturbations in expression or function of specific miRNAs has been linked to a plethora of human pathological conditions including cancer, autoimmunity, cardiovascular disease and neurodegeneration. Although miRNA gene deletion in mice via homologous recombination has led to an improved understanding of how they function during times of health and disease, there remains a fundamental need to manipulate miRNA genes and their target binding sites in the human germ line. This would allow for the study of their biology in man, and may be a strategic means by which miRNAs can be targeted therapeutically to combat human disease. We propose to develop a novel approach to activating, repressing or disrupting human miRNA genes in vivo by engineering TALE proteins that will function as transcription factors or nucleases that specifically target miRNA genes or their binding sites in the 3'UTRs of key target mRNAs. We will focus on targeting human miRNA---155, an "oncomiR" involved in regulating immune responses, and miR---146a, a tumor suppressor miRNA that inhibits inflammatory responses. Following proper targeting and disruption of these miRNAs or their binding sites in the 3' UTRs of relevant target mRNAs, a series of functional assays will be carried out to assess the roles of human miR---155 and miR---146a in regulating immune responses and cancer phenotypes. Beyond these two miRNAs, this approach will be compatible with any human miRNA making it a powerful, specific and versatile technology that has not yet been used to study miRNAs.