Modified Nucleic Acid Construct for Enhanced WT1 Protein Expression
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Solution Overview
Problem
Current immunotherapies face limitations in enhancing the expression level of tumor antigen proteins, particularly the WT1 protein, due to constraints on the amount of RNA that can be introduced into cells, which hampers the efficiency of immune induction.
Innovation Solution
A nucleic acid construct encoding a Wilms tumor gene product or its fragment, optimized with specific mutations and start codon modifications, is introduced into cells to significantly enhance the expression level of the WT1-derived polypeptide independently of expression regulating elements, allowing for improved immune induction with reduced RNA amounts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If the amount of RNA introduced into cells is increased to enhance tumor antigen expression, then the expression level of WT1 protein is improved, but the complexity and quantity of cell preparation increases
Solution Approach 1:
The patent modifies the nucleic acid construct by changing specific parameters: deleting the first ATG start codon, adding a 5' cap structure, and optimizing the 5' UTR region. These parameter changes enable high-level protein expression without requiring large amounts of introduced RNA, thus resolving the contradiction between expression level and preparation complexity
Solution Approach 2:
The patent creates an optimized copy of the WT1 nucleic acid sequence with modified features (deleted ATG, added cap structure). This optimized copy achieves superior expression efficiency compared to the wild-type sequence, allowing high antigen expression with reduced RNA amounts and simplified preparation procedures
2Quantity of substance
If expression vectors are used to introduce tumor antigens into cells, then the antigen expression is improved, but the therapy is classified as gene therapy with limited application range due to regulations
Solution Approach 1:
The patent uses non-integrating nucleic acid constructs (mRNA or plasmid DNA) that provide transient expression rather than permanent genetic modification. This approach achieves sufficient antigen expression for therapy while avoiding the regulatory restrictions associated with permanent gene therapy, thereby expanding application range
3Adaptability or versatility
If RNA is introduced into dendritic cells to avoid gene therapy classification, then the application range is improved, but the introduction efficiency and antigen expression level remain low
Solution Approach 1:
The patent implements multiple parameter changes in the nucleic acid construct: deletion of the first ATG codon to prevent premature translation, addition of a 5' cap structure to enhance translation initiation, and optimization of the 5' UTR region. These changes collectively dramatically improve translation efficiency and antigen expression levels while maintaining the non-integrating nature of the construct, thus resolving the contradiction between application range and expression level
Data Source
AI summary
A cell of the present invention contains a nucleic acid construct encoding a WT1 gene product or a fragment of the WT1 gene product. The nucleic acid construct contains (i) a region encoding a desired fragment of the WT1 gene product and (ii) only AUG as a functional start codon. The present invention can provide a cell into which the nucleic acid construct is introduced so that an expression level of a WT1 gene product or a fragment of the WT1 gene product is remarkably enhanced.