Administrative Supplement (NOT-CA-20-042) SNAP-X: Development of a Mutagenesis Strategy and High Density Protein Array to Comprehensively Display Protein Variants PIs: Christopher L. Warren and Mary S. Ozers This application is being submitted as an Administrative Supplement in response to the Notice of Special Interest (NOSI) identified as NOT-CA-20-042. Study of highly infectious diseases, especially those caused by viruses, poses unique biosafety challenges to the scientific research community. Rather than lose the insights this research can provide, common workarounds exist to reduce the risk of studying infectious virus by examining the proteins encoded by the virus in the absence of a fully infectious particle. This approach has been employed for many other viruses and has been proven effective in obtaining valuable data. However, viruses like SARS-CoV-2 mutate leading to many different strains and sequence variations, and it can be difficult to predict which mutations alter virus transmission, infection symptoms, or vaccine / treatment efficacy. Given that cancer patients constitute an at-risk population for Covid-19, this is even more complicated in the context of how cancer cells affect virus infection or how the virus affects cancer progression. Methods to examine virus protein mutations in a high throughput, systematic, and comprehensive (i.e. every mutation) manner are completely lacking. To help address this challenge, we propose to harness our innovative high throughput mutagenesis strategy to comprehensively generate plasmids of every possible point mutation of the SARS-CoV-2 S ?spike? protein as the key viral recognition protein of the human entry receptor, towards accelerating functional studies and vaccine development. The resulting plasmid set is especially useful for functional assays to identify critical S protein variants, which is a vital area of current Covid-19 research. The S protein mutant plasmid set will be validated by expression of the variant plasmids in human cells and determination of which variants bind to different commercially available SARS-CoV-2 S protein antibodies. These results will be confirmed by immunofluorescence staining. While we envision these variants being employed by the cancer research community in their model systems, they can be used equally effectively by the SARS-CoV-2 research community in general. The proposed method significantly improves on random mutagenesis by error-prone PCR by avoiding its substantial mutational bias and ensuring exactly one mutation per plasmid for streamlined analysis. This strategy will be confirmed by next generation sequencing (NGS), comparing against error-prone PCR. Following successful validation assays for the S protein variant pool, we will generate mutant plasmid sets for three additional key proteins encoded by the SARS-CoV-2 genome. This Administrative Supplement is within the scope of our parent IMAT award, which is a technology development R21 mechanism, to generate all mutants of three key oncoproteins and link the expressed protein variants back to specific feature locations on a microarray. Upon successful completion of this Administrative Supplement, we intend to prepare a SBIR Phase I to sort each individual SARS-CoV-2 mutant plasmid into distinct wells of multiwell plates for additional availability to the Covid-19 research community.
Administrative Supplement (NOT-CA-20-042) SNAP-X: Development of a Mutagenesis Strategy and High Density Protein Array to Comprehensively Display Protein Variants PIs: Christopher L. Warren and Mary S. Ozers The spike protein of the recently emerging SARS-CoV-2 virus is responsible for making critical contacts with the human host cell receptor, and the virus adapts its infectivity through mutations of this protein. Our novel cell-free mutagenesis method, being developed in the parent award, will facilitate creation of all possible single amino acid substitutions for the large spike protein, as well as for key viral structural proteins. These comprehensive collections of variant plasmids for important viral proteins will be made widely available to the cancer research community, in addition to the SARS-CoV-2 research community, towards mutational screens and functional assays that can identify variants that result in significant changes in Covid-19 pathogenicity and impact drug / vaccine development.
