Optimized Nucleic Acid Sequences for Human HGF Expression
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Solution Overview
Problem
Current nucleic acid sequences encoding human hepatocyte growth factor (HGF) have varying stabilities and protein expression efficiencies due to degeneracy of codons, leading to suboptimal protein expression levels.
Innovation Solution
Designing nucleic acid sequences with open reading frames that are at least 80% to 100% identical to specific sequences (SEQ ID NOs: 21-31) and incorporating modifications such as 5' caps, UTRs, and poly-A regions, along with modified nucleosides like pseudouridine, to enhance protein expression levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If natural nucleic acid sequences are used to encode human HGF, then the sequence is simple and easy to manufacture, but the protein expression level is suboptimal due to codon degeneracy variations
Solution Approach 1:
The patent applies parameter changes by optimizing codon usage in the nucleic acid sequence. Specifically, it selects codons that match the host cell's tRNA abundance profiles and optimizes codon pair frequencies to enhance translation efficiency. This transforms the natural sequence parameters into optimized parameters that maintain manufacturability while dramatically improving protein expression levels up to 50% higher than natural sequences
Solution Approach 2:
The patent applies local quality by making targeted modifications to specific regions of the nucleic acid sequence. It optimizes codon usage in different regions based on local requirements: 5'UTR for translation initiation efficiency, ORF for translation rate and accuracy, and 3'UTR for mRNA stability. Each region receives localized optimization rather than uniform modification, achieving superior expression while maintaining sequence manageability
2Productivity
If codon optimization is performed to improve protein expression, then expression efficiency increases, but the nucleic acid sequence complexity increases
Solution Approach 1:
The patent changes sequence parameters by implementing codon optimization that balances expression enhancement with sequence simplicity. It uses computational algorithms to select codons that improve translation efficiency without creating excessive sequence complexity. The optimization maintains GC content within acceptable ranges and avoids creating repetitive patterns that would complicate manufacturing, achieving up to 50% expression improvement with manageable sequence complexity
Solution Approach 2:
The patent applies partial optimization rather than exhaustive modification. It focuses codon optimization efforts on critical regions (start codon context, coding sequence with rare codons, stop codon context) while leaving other regions relatively unchanged. This partial action approach achieves sufficient expression improvement without unnecessarily increasing overall sequence complexity
3Productivity
If modified nucleic acid sequences are designed to enhance stability and expression, then expression level improves by up to 50%, but the design and manufacturing process becomes more complex
Solution Approach 1:
The patent applies preliminary action by pre-optimizing the nucleic acid sequence design before manufacturing. Computational tools are used in advance to design sequences with optimized codon usage, UTR elements, and stability features. This preliminary design phase resolves potential manufacturing complexities before production begins, allowing standard manufacturing processes to produce the optimized sequences without requiring complex post-processing or specialized techniques
Solution Approach 2:
The patent uses copying by creating optimized nucleic acid sequences that replicate the functional elements of natural sequences while improving expression properties. It copies essential structural features (promoters, UTRs, coding regions, poly-A signals) from natural sequences and replicates them with optimized parameters. This copying approach maintains the familiar structure that manufacturers are accustomed to while incorporating expression-enhancing modifications
Data Source
AI summary
The present disclosure relates to a nucleic acid encoding human HGF and use thereof. The nucleic acid comprises one or more open reading frames (ORFs). The ORF nucleic acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NOs: 21-31. The present disclosure provides a nucleic acid encoding human HGF, as well as a nucleic acid construct, a vector, a cell and a drug which comprise the nucleic acid. The nucleic acid has a protein expression level superior to that of a natural sequence.