CAR Variant Library Synthesis via Microarray Oligonucleotide Assembly
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Solution Overview
Problem
Current synthetic biology methods for generating custom DNA sequences are hindered by high costs and low throughput, limiting the ability to efficiently explore sequence space and optimize chimeric antigen receptor (CAR) therapies for cancer treatment due to bottlenecks in DNA synthesis and oligonucleotide production.
Innovation Solution
Development of nucleic acid libraries comprising thousands to millions of nucleic acids or oligonucleotides, each encoding variants of CAR domains, synthesized using advanced methods that include PCR mutagenesis and surface-based oligonucleotide synthesis to achieve high diversity and accuracy, enabling efficient screening for improved affinity, specificity, and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional oligonucleotide synthesis and gene synthesis methods are used, then DNA sequences can be generated, but the throughput is low and the cost is high
Solution Approach 1:
The patent divides the gene synthesis process into modular components: oligonucleotide synthesis on microarrays, PCR amplification, and assembly into full-length genes. This segmentation enables parallel processing of multiple oligonucleotides simultaneously, dramatically increasing throughput while reducing per-unit cost through economies of scale
Solution Approach 2:
The patent uses PCR amplification to generate multiple copies of DNA sequences from synthesized oligonucleotides. This copying process enables the production of large quantities of DNA at low marginal cost, resolving the contradiction between high throughput and low cost by decoupling the expensive synthesis step from the inexpensive amplification step
2Reliability
If molecular cloning is used for gene variant synthesis, then robust gene production is achieved, but the method is not scalable
Solution Approach 1:
The patent replaces the manual, labor-intensive molecular cloning process with automated microarray-based oligonucleotide synthesis and high-throughput PCR assembly. This substitution maintains the reliability of robust gene production while enabling scalability to thousands of gene variants simultaneously
Solution Approach 2:
The patent creates a universal platform that can synthesize any gene variant by combining standardized oligonucleotide synthesis modules with flexible PCR assembly protocols. This universal approach allows the same system to produce diverse gene variants at scale, resolving the scalability limitation of traditional cloning methods
3Productivity
If microarray-based oligonucleotide synthesis is used, then throughput increases to 96 genes, but the cost efficiency and turnaround time remain insufficient for large-scale sequence space exploration
Solution Approach 1:
The patent performs preliminary oligonucleotide synthesis on microarrays, creating pools of synthesized oligonucleotides that can be stored and reused. This preliminary action eliminates the need to re-synthesize oligonucleotides for each experiment, dramatically reducing turnaround time while maintaining high throughput capability
Solution Approach 2:
The patent merges multiple oligonucleotide synthesis reactions into pooled reactions on microarrays, then combines these pools through PCR assembly into full-length genes. This merging approach processes thousands of sequences in parallel, achieving the throughput necessary for large-scale sequence space exploration within practical timeframes
Data Source
AI summary
Disclosed herein are methods for the generation of highly accurate nucleic acid libraries encoding for predetermined variants of a nucleic acid sequence. The nucleic acid sequence may encode for all or part of a reference domain of a CAR. The degree of variation may be complete, resulting in a saturated variant library, or less than complete, resulting in a non-saturating library of variants. The variant nucleic acid libraries described herein may be designed for further processing by transcription or translation. The variant nucleic acid libraries described herein may be designed to generate variant RNA, DNA and/or protein populations. Further provided herein are method for identifying variant species with increased or decreased activities, with applications in regulating biological functions and the design of therapeutics for treatment or reduction of a disease, such as cancer.