RNA Interference as a Metabolic Engineering Tool

描述(申请人提供)：我们已经开始探索可控制的体内策略，试图操纵宿主功能，具体目的是改变生物合成格局，有利于重组蛋白质合成。在本提案中，RNA干扰(RNAi)或翻译后基因沉默被探索为一种代谢工程控制器，为此目的下调靶向宿主基因的表达。到目前为止，还没有任何表达系统的相关报道。 我们期望这种方法将补充代谢工程中已经证实的互补、突变、缺失和等位基因替换的策略。RNAi片段的长度、靶序列、时间和给药方法都是我们调查的对象。此外，我们还将讨论蛋白质表达的两个关键方面：(1)产品产量和(2)产品质量。在这两种情况下，初步证据表明，RNA特异的基因沉默方法将奏效。在第一种情况下，我们将以真核细胞周期为目标。在第二种情况下，我们将针对昆虫系统中产生的重组蛋白的N-糖基化。为拟议的RNAi工作选择昆虫系统是基于以下几个因素：(1)存在大量与昆虫遗传学有关的知识，包括细胞周期电路和调控，(2)许多开创性的RNAi工作已在昆虫(如果蝇)中完成，(3)我们的实验室已经开发出利用昆虫细胞和昆虫幼虫的跟踪记录，以及(4)DNA微阵列将使人们能够更全面地了解基于RNAi的代谢工程的机制和有效性。 拟议活动的智力价值-RNAi作为敏感和特定的控制器的发展将为细胞、组织和动物的代谢工程开辟新的途径。也就是说，特定基因的下调，包括那些在复杂途径中的基因，补充了现有的基于增加基因的方法。重要的是，当RNAi处理得当时，已知会引起最小的副作用或多效性。

DESCRIPTION (provided by applicant): We have begun to explore controllable in vivo strategies that attempt to manipulate host function with the specific intention of altering the biosynthetic landscape in favor of recombinant protein synthesis. In this proposal, RNA interference (RNAi) or post-translational gene silencing, is explored as a metabolic engineering controller that downregulates targeted host gene expression for this purpose. To date, there have been no related reports in any expression system. We expect that this approach will complement the already proven strategies in metabolic engineering of complementation, mutation, deletion, and allele substitution. The RNAi segment length, target sequences, timing, and method of administration are all subjects of our investigation. Also, we will address two key aspects of protein expression: (1) product yield and (2) product quality. In both cases, preliminary evidence suggests that an RNA-specific gene silencing approach will work. In the first case, we will target the eukaryotic cell cycle. In the second case we will target N-glycosylation of recombinant proteins produced in insect systems. The selection of insect systems for the proposed RNAi work is predicated on several factors: (1) there exists a significant body of knowledge pertaining to insect genetics, including cell cycle circuitry and regulation, (2) much of the pioneering RNAi work has been accomplished in insects (e.g., Drosophila), (3) our laboratory has developed a track record for exploiting insect cells and insect larvae, and (4) DNA microarrays are available that will enable more comprehensive understanding of the mechanisms and efficacy of the RNAi-based metabolic engineering. Intellectual Merit of the Proposed Activity - The development of RNAi as a sensitive and specific controller will open new avenues for metabolic engineering of cells, tissues, and animals. Namely, the downregulation of specific genes, including those in complex pathways, complements existing approaches based on augmenting genotype. Importantly, RNAi is known to elicit minimal side effects or pleiotropy when it is processed properly.