Mutant IRES Sequences for Precise Gene Expression Control

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Solution Overview

Problem

Current methods for controlling the simultaneous expression of multiple genes in mammalian cells lack precision and stability, with existing strategies facing issues such as variable transfection efficiency, transcriptional interference, and the formation of undesirable fusion proteins, and they fail to provide individual control over gene expression ratios across a wide range.

Innovation Solution

The use of mutant IRES sequences, specifically modifying the 10th, 11th, and 12th AUG codons of the Encephalomyocarditis virus IRES, allows for systematic control of gene expression by varying the type and number of mutations, enabling independent regulation of each gene linked to its own IRES sequence, thereby achieving precise and stable differential expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If co-transfection of multiple vectors is used to control multiple gene expression, then different genes can be introduced into cells, but the relative amount of different genes incorporated into cells varies from cell-to-cell due to variations in transfection efficiency

Engineering Contradiction:

Improveability to control multiple gene expressionVSAvoidprecision of gene incorporation ratio

Core Design Contradiction:

Adaptability or versatilityVSManufacturing precision

Solution Approach 1:

The patent divides a single polycistronic mRNA into multiple independent cistrons, each with its own IRES element. This segmentation allows each gene to be independently controlled at the translational level, ensuring precise and consistent expression ratios across cells while maintaining the advantage of single-vector delivery.

Inventive Principle:

Principle #1Segmentation

2Manufacturing precision

If a single vector with multiple promoters is used to ensure introduction of different genes into each cell at identical amounts, then gene introduction is controlled, but the expression ratio between the products of the different genes still varies between cells in a stably transfected cell pool as the arrangement of multiple promoters in close proximity causes transcriptional interference

Engineering Contradiction:

Improveprecision of gene introductionVSAvoidstability of expression ratio

Core Design Contradiction:

Manufacturing precisionVSReliability

Solution Approach 1:

The patent introduces IRES elements as intermediary sequences between each gene's promoter and coding region. These IRES elements act as translational mediators that independently initiate protein synthesis for each cistron, eliminating the transcriptional interference problem while maintaining precise gene introduction through single-vector delivery.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Manufacturing precision

If splicing signals with varied splicing efficiencies are inserted to control relative gene expression, then stricter control of relative gene expression is achieved in both transient and stable transfections, but cryptic splicing sites in protein coding sequences need to be eliminated making the method difficult to use

Engineering Contradiction:

Improveprecision of relative gene expression controlVSAvoidease of construct design

Core Design Contradiction:

Manufacturing precisionVSEase of manufacture

Solution Approach 1:

The patent replaces the splicing mechanism with a translational mechanism. Instead of using splicing signals that require complex design to eliminate cryptic sites, the patent uses IRES elements that directly control translation initiation. This substitution simplifies construct design while maintaining precise expression control.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

4Manufacturing precision

If 2A elements are used for co-expression of multiple genes from one mRNA, then equal amounts of co-expressed proteins are achieved, but this method does not allow modulation of the expression ratio between the proteins of interest and incomplete cleavage of 2A peptides often results in the attachment of unwanted residues to the proteins of interest and formation of fusion proteins

Engineering Contradiction:

Improveprecision of protein expression ratioVSAvoidformation of fusion proteins

Core Design Contradiction:

Manufacturing precisionVSObject-generated harmful factors

Solution Approach 1:

The patent changes the control parameter from post-translational (2A cleavage) to translational (IRES-mediated initiation). By using IRES elements, the system achieves precise modulation of expression ratios through translational control without the harmful side effects of 2A cleavage, including fusion protein formation and unwanted residue attachment.

Inventive Principle:

Principle #35Parameter changes

Data Source

PatentUS9790488B2Mutated internal ribosomal entry site (IRES) for controlled gene expression

Publication Date: 2017.10.17 AGENCY FOR SCI TECH & RES

AI summary

The present invention relates to a nucleic acid molecule comprising one or multiple mutant IRES elements. Further, the present invention relates to methods of enhancing gene expression and to methods of differentially controlling expression of one or multiple gene(s) of interest. In addition, the present invention relates to a kit for studying interactions or any application requiring co-expression of multiple genes.