CRISPRai-regulated stress response modulation for cell factory engineering

Manufacturing therapeutic proteins is challenging. The cells that manufacture proteins often have low productivity and are difficult to culture in large volumes. Cell types with higher productivity can have issues surrounding proper folding and modification of the proteins. High productivity often leads to extreme metabolic stress. This can trigger a cascade of responses that result in cell death. This is a particular problem for the manufacture of antibodies, most of which must be produced in mammalian cells. This experimental project will utilize CRISPR technology to develop appropriate internal sensors and control loops to monitor and respond to stress caused by protein overproduction. The goal will be to develop "smart cells" that can improve their cellular fitness and maintain productivity when stressed. The project will offer training and research opportunities to local high school students and teachers. This will help develop the STEM workforce to support the U.S. biomanufacturing industry. Protein overexpression in mammalian cells leads to incorrectly folded proteins. This activates the unfolded protein response (UPR). The UPR can inhibit protein synthesis and trigger cell death. Current strategies to address this problem amount to trial-and-error attempts to deregulate specific UPR genes. The impact on productivity has been minimal in most cases. To address these issues, the research team developed cell factories that dynamically modulate the UPR in response to stress in the endoplasmic reticulum. This strategy enhances cell viability and productivity. The objective of this research will be to leverage these results and engineer cells with CRISPRai (Clustered Regularly Interspaced Short Palindromic Repeats interference and activation) control systems to modulate the UPR, with the dual objectives of creating platforms for fundamental studies of secretion mechanisms and developing high-producing cell factories with rapid responses to fluctuations in folding requirements. This could provide a platform for studying protein production and secretion. It could also become the basis for processes exhibiting high protein productivity for extended periods. This would be a major step forward for therapeutic protein biomanufacturing. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria. NSF Award ID: 2512961 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Laura Segatori | Institution: William Marsh Rice University, HOUSTON, TX | Award Amount: $521,194 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2512961 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2512961.html