High-throughput cell-free production of human genetic variants

Project Summary The rapidly evolving field of human functional genomics has created the promise of personalized medicine. While extensive sequencing has generated valuable genetic association data, our ability to test linked variants in disease has not kept pace. As a result, most Single Nucleotide Polymorphisms (SNPs) are classified as Variants of Unknown Significance (VUS). Following up on function is essential to determine the causal variants and genes for disease biology and drug discovery. There are currently no technologies to produce variants en masse from genomic association data to establish function. We propose using Synvitrobio’s cell-free protein expression and purification platform to rapidly express and purify human SNP variants for downstream functional testing. Cell- free systems take only 8 hours to express, rather than days to weeks in cells, since there is no need for cloning and transformation. They are also at least 10-fold cheaper to run than cells, can be run in high-throughput as reactions (384 well plates), and can be scaled up for protein purification. In short, our approach provides a scalable alternative to cell-based heterologous expression. This Phase I study proposes to produce model human SNP variants using traditional in cellulo methods and compare the specific activity to proteins produced via Synvitrobio’s cell-free platform. We propose two aims that de-risk the strategy of constructing panels of human SNP variants using high-throughput cell-free tools. Aim 1 focuses on a class of protein known to express in E. coli systems, whereas the protein class in Aim 2 is more challenging in typical prokaryotic expression systems. Building a toolbox for variant expression and purification would facilitate understanding the causal variants and genes for disease biology and drug discovery.Project Narrative A high-throughput, cell-free expression tool capable of expressing genetic variants of human coding sequences benefits the public by allowing the exponentially increasing amounts of personal and human genomics data to be interpreted and understood through protein variant production and characterization. This leads to a better understanding of human pathophysiology, the relationship between genotype and phenotype, and ultimately novel and personalized therapeutics.