Collaborative Research: Manipulating Epigenetic Mechanisms to Enhance Non-Viral Transgene Expression

rege /ElmerDNA或基因传递到哺乳动物细胞在生物技术和医学上有几个应用。然而，外源基因（转基因）的激活，以及随后从它们表达蛋白质的功效，往往受到宿主细胞内部防御机制的阻碍。例如，细胞能够控制转基因蛋白的表达，要么将现有的基因隐藏在细胞核中浓缩的染色体内，以关闭它们，要么放松染色体，以暴露基因并激活它们。细胞内存在的“表观遗传”酶家族通常控制这些基因调控过程；激活基因的酶通常被归类为激活因子，而那些关闭基因的酶被称为抑制因子。在拟议的研究中，研究人员将研究这些表观遗传酶在调节传递转基因表达中的作用。此外，来自合成生物学新兴领域的工具将被用于设计新的DNA序列和激活酶，以增强传递转基因的蛋白质表达。这些研究的结果将对利用DNA或基因传递到哺乳动物细胞的生物技术过程和医学治疗（如基因治疗）产生变革性影响。该项目将通过亚利桑那州立大学量子计划促进的基于pcr的分子生物学项目向K-12学生推广。拟议的外展活动将旨在增加来自亚利桑那州和宾夕法尼亚州代表性不足人群的学生参与STEM活动。就像图书馆里的书页里整齐地排列着单词一样，细胞基因被组蛋白凝聚在一起，形成细胞核中的染色体。表观遗传调控是由多种酶介导的，在细胞内基因表达调控中起着关键作用。由于最近的研究表明表观遗传学机制可以使病毒基因沉默，因此表观遗传学也可能调节以质粒DNA形式传递到哺乳动物细胞的外源基因（即转基因）的表达。该项目的目标是通过(1)使用药物抑制抑制因子并防止转基因失活，(2)在质粒中添加设计好的DNA序列以招募激活酶，以及(3)利用合成生物学原理设计和验证能够选择性结合转基因并增强其表达的新型表观遗传激活酶。这些方法将加深我们对人类细胞中外源转基因（质粒DNA）调节蛋白质表达的表观遗传机制的理解，从而提高医学中基因治疗的有效性，以及生物技术中的细胞工程方法。该奖项由CBET部门的生物技术、生化和生物质工程项目以及分子和细胞生物学部门的系统和合成生物学项目共同资助。

CBET - 1404084/1403214Rege/ElmerDNA or gene delivery to mammalian cells has several applications in biotechnology and medicine. However, the activation of foreign genes (transgenes), and subsequent efficacy of protein expression from them, is often hindered by defense mechanisms inside the host cell. For example, cells are able to control transgene protein expression by either burying existing genes inside condensed chromosomes in the nucleus in order to turn them off, or by relaxing chromosomes in order to expose genes and activate them. A family of 'epigenetic' enzymes present within cells typically controls these processes of gene regulation; enzymes that activate genes are generally classified as activators, while those that turn genes off are called repressors. In the proposed research, the investigators will study the role of these epigenetic enzymes in regulating the expression from delivered transgenes. Furthermore, tools from the emerging field of synthetic biology will be employed to design novel DNA sequences and activator enzymes that enhance protein expression from delivered transgenes. Findings from these studies will have a transformative impact on biotechnology processes and medical treatments (e.g. gene therapy) that utilize DNA or gene delivery to mammalian cells. Outreach from this project will reach K-12 students through a PCR-based molecular biology project facilitated by the Quanta program at ASU. The proposed outreach will be designed to increase participation of students from underrepresented populations in STEM activities in Arizona and Pennsylvania.In the same way that words are neatly organized in the pages of books in a library, cellular genes are condensed by histone proteins to form chromosomes in the nucleus. Epigenetic regulation, mediated by several enzymes, plays a key role in controlling gene expression inside cells. Since recent work has shown that epigenetic mechanisms can silence viral genes, it is likely that epigenetics may also regulate the expression of foreign genes (i.e. transgenes) that are delivered in the form of plasmid DNA to mammalian cells. The goal of this project is to manipulate intracellular epigenetic enzymes in order to enhance transgene expression by (1) using drugs to inhibit repressors and to prevent the deactivation of transgenes, (2) adding designed DNA sequences to the plasmids in order to recruit activator enzymes, and (3) using principles from synthetic biology in order to design and validate novel epigenetic activator enzymes that can selectively bind transgenes and enhance their expression. These approaches will deepen our understanding of epigenetic mechanisms that regulate protein expression from foreign transgenes (plasmid DNA) in human cells, leading to increased efficacy of gene therapy treatments in medicine, and cellular engineering approaches in biotechnology.This award is co-funded by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division and by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.