A Target-Directed Reagent Pipeline via Microfluidic mRNA Display


Year of Award:
2017
Award Type:
R21
Project Number:
CA204708-01A1
RFA Number:
RFA-CA-16-001
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
MALMSTADT, NOAH (contact);
Other PI or Project Leader:
ROBERTS, RICHARD W
Institution:
UNIVERSITY OF SOUTHERN CALIFORNIA
Diagnosing and treating cancer requires having a reliable set of affinity reagents that can specifically and strongly bind to cancer-related protein targets. These reagents are the necessary molecular tools that will enable next-generation technologies for studying, diagnosing, and fighting cancer. Current approaches to producing such reagents, however, are unreliable, expensive, and slow. Existing reagent generation methods (e.g., hybridoma technology, phage display, yeast display) are not readily adapted to leverage high throughput library sequencing. Where reagents do exist, they are often both extremely expensive and poorly characterized. It is these important shortfalls that this research project aims to address. This project combines two powerful existing technologies: 1) mRNA display and 2) Modular Microfluidic and Instrumentation Components (MFIC) to dramatically speed target-directed, renewable reagent development. mRNA display is a molecular selection technology that is uniquely capable of searching libraries of more than a trillion unique compounds to develop ultrahigh affinity reagents against cancer-relevant targets. While this technology has an impressive demonstrated track record of producing such reagents, it has so far been limited to the laboratory scale. This project will adapt it to a true high-throughput format by integrating it into a continous flow microfluidic system based on MFIC technology. By automating mRNA display, this project will make it broadly accessible to the research community while decreasing its cost and increasing its throughput. Once the automated system is developed, we will use it to produce affinity reagents that target two key cancer screening protein markers—PSA and CA125. These markers are broadly used (they were the primary tools used in 154,000 patient Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO)) and renewable, inexpensive reagents that recognize these biomarkers would clearly be of utility. An automated microfluidic mRNA display system will be used to develop novel, specific, and high affinity reagents for these targets. Developing a microfluidic approach for mRNA display selection will involve implementing a magnetophoretic separation system that can both perform affinity selections and purify the products of preparative biochemical reactions. An automated system for preparing mRNA reagents will be combined with an automated target selection system. The DNA-encoded products of this selection will be amplified in a microfluidic PCR system. The entire process workflow will be implemented in a closed loop to enable multiple rounds of selection and amplication, producing an optimal high affinity binding reagent. Each step of the automated mRNA display will be benchmarked for quality assurance by developing affinity reagents for the cancer marker Bcl-xL; standard manual mRNA display has proved resoundingly successful at producing such reagents and there is plentiful existing data against which to benchmark.