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.
