Cell-Stored Barcoded Viral Protein Libraries for High-Throughput Scanning
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Solution Overview
Problem
Existing deep mutational scanning methods for viral entry proteins are limited by low throughput, biosafety challenges, and inefficiencies in barcode association, particularly for viruses requiring high containment facilities, leading to difficulties in studying rapidly evolving viruses and predicting resistance or host adaptation.
Innovation Solution
Development of cell-stored barcoded mutational scanning libraries where virions are produced with a genotype-phenotype link, allowing efficient storage and sequencing without replication, and enabling high-throughput analysis of viral entry protein mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional deep mutational scanning methods are used to study viral entry proteins, then comprehensive mutation analysis can be achieved, but the throughput is low and biosafety challenges arise for high-containment viruses
Solution Approach 1:
The patent divides the viral genome into multiple segments, each containing barcoded mutational libraries. This segmentation allows parallel processing of different viral segments across multiple cell lines, dramatically increasing throughput while maintaining comprehensive mutation analysis coverage. Each segment can be independently barcoded and analyzed, then computationally reassembled to achieve complete viral protein characterization.
Solution Approach 2:
The patent introduces barcodes as intermediary elements that link viral genomic sequences to phenotypic data. These barcodes serve as unique identifiers that enable high-throughput tracking of individual mutational variants through automated sequencing and data analysis pipelines, resolving the throughput limitation while preserving measurement precision.
2Reliability
If replication-competent viral libraries are used for deep mutational scanning, then functional phenotyping can be performed, but biosafety risks increase for dangerous viruses
Solution Approach 1:
The patent extracts the essential functional information from replication-competent viruses by using pseudotyped viral particles that cannot replicate but retain entry function. The barcoded mutational libraries are delivered via non-replicating vectors, allowing functional phenotyping of viral entry proteins without the biosafety risks associated with replication-competent dangerous viruses.
Solution Approach 2:
The patent creates simplified copies of viral entry proteins pseudotyped onto inert viral particles. These copies maintain the functional properties needed for phenotypic analysis (binding, entry) but lack the replication machinery that creates biosafety hazards. The barcodes enable these copies to be tracked and analyzed with the same precision as authentic viruses.
3Productivity
If barcoded mutational libraries are stored in cells, then high-throughput sequencing efficiency improves, but barcode association efficiency becomes a limiting factor
Solution Approach 1:
The patent merges the barcode sequence with the viral genomic sequence in a single integrated construct within the cell. This merging ensures that when DNA is extracted and sequenced, both the barcode and the associated viral variant are captured together in the same sequencing read, eliminating barcode-variant dissociation and enabling direct linkage without additional association steps.
Solution Approach 2:
The patent performs preliminary association of barcodes with viral variants during the library construction and cellular integration phases, before sequencing occurs. By establishing the barcode-variant linkage in advance within the cellular genome, the system eliminates the need for complex post-sequencing association algorithms and prevents information loss during data processing.
Data Source
AI summary
Cell-stored barcoded viral protein deep mutational scanning libraries are described. The libraries can be used to map resistance mutations to therapeutic treatments. The libraries can be used to predict viruses that become resistant to therapeutic compounds and/or may more easily evolve to infect new species. The libraries can also be used to more safely study dangerous viruses that normally require high safety biocontainment facilities. The libraries include features that allow efficient collection and assessment of informative data, obviating many bottlenecks of previous approaches.