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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070851-03
Application #
7051442
Study Section
Special Emphasis Panel (ZGM1-MBP-9 (ME))
Program Officer
Jones, Warren
Project Start
2004-02-01
Project End
2008-01-31
Budget Start
2006-02-01
Budget End
2008-01-31
Support Year
3
Fiscal Year
2006
Total Cost
$180,725
Indirect Cost
Name
University of MD Biotechnology Institute
Department
Type
Organized Research Units
DUNS #
603819210
City
Baltimore
State
MD
Country
United States
Zip Code
21202
Hebert, Colin G; Valdes, James J; Bentley, William E (2009) In vitro and in vivo RNA interference mediated suppression of Tn-caspase-1 for improved recombinant protein production in High Five cell culture with the baculovirus expression vector system. Biotechnol Bioeng 104:390-9
Hebert, Colin G; Valdes, James J; Bentley, William E (2009) Investigating apoptosis: characterization and analysis of Trichoplusia ni-caspase-1 through overexpression and RNAi mediated silencing. Insect Biochem Mol Biol 39:113-24
Hebert, Colin G; Valdes, James J; Bentley, William E (2008) Beyond silencing--engineering applications of RNA interference and antisense technology for altering cellular phenotype. Curr Opin Biotechnol 19:500-5
Kim, Eun Jeong; Kramer, Shannon F; Hebert, Colin G et al. (2007) Metabolic engineering of the baculovirus-expression system via inverse ""shotgun"" genomic analysis and RNA interference (dsRNA) increases product yield and cell longevity. Biotechnol Bioeng 98:645-54
March, John C; Bentley, William E (2007) RNAi-based tuning of cell cycling in Drosophila S2 cells--effects on recombinant protein yield. Appl Microbiol Biotechnol 73:1128-35
March, John C; Bentley, William E (2006) Engineering eukaryotic signal transduction with RNAi: enhancing Drosophila S2 cell growth and recombinant protein synthesis via silencing of TSC1. Biotechnol Bioeng 95:645-52
Yung, Chong Wing; Barbari, Timothy A; Bentley, William E (2006) Counteracting apoptosis and necrosis with hypoxia responsive expression of Bcl-2Delta. Metab Eng 8:483-90
March, John C; Bentley, William E (2004) Insulin stimulates double-stranded RNA uptake in Drosophila S2 cells. Biotechniques 37:898, 900