Barcoded Modular Polypeptide Libraries for High-Throughput Phenotype Screening
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Solution Overview
Problem
Current methods for constructing and screening synthetic modular proteins are low-throughput and cumbersome, limiting the development of new proteins due to the need for individual constructs and separate screening, especially when transitioning from in vitro to in vivo assays.
Innovation Solution
A method involving the introduction of a barcoded library of nucleic acids into host cells to generate a heterogeneous population, allowing for high-throughput screening of synthetic modular polypeptides, including chimeric antigen receptors and modular receptors, by identifying phenotypes through sequencing barcodes and using detectable reporters.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If individual constructs are screened one at a time or in small parallel groups, then identification of well-performing constructs is facilitated, but throughput and efficiency are severely limited
Solution Approach 1:
The patent combines multiple individual construct screenings into a single pooled library screening system. Multiple synthetic modular polypeptide constructs are co-transfected into host cells along with barcoded nucleic acids, allowing simultaneous screening of many constructs in one experiment rather than individually or in small groups
Solution Approach 2:
The patent introduces barcoded nucleic acids as intermediaries that link each synthetic modular polypeptide construct to its identifying signature. These barcodes serve as mediators that enable tracking and identification of individual constructs within the pooled library, allowing high-throughput screening without losing the ability to identify specific performant constructs
2Measurement precision
If large numbers of new proteins are screened individually, then accurate phenotype identification is achieved, but the process becomes prohibitively cumbersome and expensive
Solution Approach 1:
The patent merges multiple individual screening processes into a single pooled experiment. Hundreds or thousands of synthetic modular polypeptide constructs are screened simultaneously in one transfection and screening event, dramatically reducing the time required compared to sequential individual screening while maintaining phenotype identification accuracy through barcode tracking
3Adaptability or versatility
If synthetic modular polypeptides are developed for in vivo assays, then therapeutic application potential is achieved, but the cumbersome individual screening process becomes even more prohibitive
Solution Approach 1:
The patent combines multiple construct developments into pooled libraries that can be screened in parallel, accelerating the development rate of synthetic modular polypeptides for therapeutic applications. Multiple candidates for in vivo assays are evaluated simultaneously rather than sequentially
Solution Approach 2:
The patent performs preliminary high-throughput screening of large libraries of synthetic modular polypeptides to identify top candidates before advancing to complex in vivo assays. This preliminary filtering step reduces the number of constructs that need to undergo time-consuming and expensive in vivo testing, thereby accelerating overall development
Data Source
AI summary
The present disclosure provides synthetic modular polypeptide libraries and nucleic acids encoding such synthetic modular polypeptide libraries. Also provided are methods of making synthetic modular polypeptide libraries and nucleic acids encoding synthetic modular polypeptide libraries. Methods of screening a synthetic modular polypeptide library to identify a selected phenotype associated with a member of a synthetic modular polypeptide library are also provided where such methods find use in both in vitro and in vivo assays.