Modified Hepatitis PREs for Safe Gene Expression
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Solution Overview
Problem
Current viral vectors for gene expression, particularly those using hepatitis virus-derived post-transcriptional regulatory elements, face challenges in reducing oncogenic and immunogenic potential, as they can express unwanted proteins that promote tumorigenesis and immunogenicity.
Innovation Solution
Development of modified post-transcriptional regulatory elements (PREs) with variant X genes containing stop codons and post-translational degradation sequences, which prevent the expression of oncogenic and immunogenic proteins by introducing modifications such as stop codons and ubiquitination sites not present in wild-type X genes, thereby reducing the risk of oncogenesis and immunogenicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type hepatitis virus PREs are used to enhance gene expression, then expression levels are improved, but oncogenic and immunogenic potential increases due to unwanted protein expression
Solution Approach 1:
The patent extracts and removes the harmful coding regions from the PRE sequence while retaining the beneficial post-transcriptional regulatory functions. Specifically, the wild-type PRE is modified by introducing stop codons and deletions that eliminate the X protein coding capacity, thereby separating the gene expression enhancement function from the oncogenic protein expression function.
Solution Approach 2:
The patent applies local modifications to specific regions of the PRE sequence. Different portions of the PRE are selectively altered - some regions are deleted, stop codons are inserted at specific positions, and ubiquitination sites are introduced - to achieve localized control over protein expression while preserving overall PRE function.
2Reliability
If modifications are introduced to prevent unwanted protein expression, then safety is improved, but gene expression enhancement may be reduced
Solution Approach 1:
The patent changes critical parameters of the PRE sequence including inserting stop codons (UAA, UAG, UGA) at strategic positions, introducing ubiquitination recognition sequences (e.g., Lys48, Lys63), and making point mutations that alter protein stability and expression levels while maintaining RNA regulatory functions.
Solution Approach 2:
The modified PRE combines multiple functional elements into a single composite sequence: post-transcriptional regulatory motifs for gene expression enhancement, stop codons for translation termination, and ubiquitination sites for protein degradation. This composite structure integrates both safety and productivity functions.
Data Source
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AI summary
Provided are polynucleotides containing a modified PRE having a variant X gene that includes one or more stop codons not present in an unmodified, such as wild-type, hepatitis X gene. Also provided are polynucleotides containing a modified PRE having a variant X gene that includes one or more degradation sequences not present in an unmodified, such as wild-type, hepatitis X gene. The modified PRE can be operably linked to a nucleic acid encoding a recombinant protein. Also provided are expression cassettes, viral vectors and cells containing the polynucleotides, and compositions and methods of use thereof.