hCMV-MIE Promoter C to G Mutations Prevent Silencing

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Solution Overview

Problem

The gradual loss of productivity in long-term cell cultures for recombinant protein expression is a common issue due to promoter silencing caused by epigenetic modifications, particularly methylation of CpG sites in the hCMV-MIE promoter, leading to unstable protein production.

Innovation Solution

Introducing specific C to G point mutations at positions -41 and/or -179 relative to the transcription start site in the hCMV-MIE promoter reduces silencing and enhances production stability, maintaining high yields over extended cultivation times without affecting promoter strength.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If the hCMV-MIE promoter is used for recombinant protein expression, then high initial productivity is achieved, but promoter silencing occurs over time due to methylation at CpG sites

Engineering Contradiction:

Improveinitial protein expression levelVSAvoidproduction stability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent applies parameter changes by introducing point mutations at specific CpG sites (positions -41 and -179) in the hCMV-MIE promoter sequence. These mutations alter the chemical structure of the DNA at methylation-prone sites, preventing methyltransferase enzymes from adding methyl groups. This parameter change in the promoter sequence maintains high productivity while eliminating the silencing effect, resolving the contradiction between initial expression level and long-term stability

Inventive Principle:

Principle #35Parameter changes

2Reliability

If the promoter sequence is mutated to prevent silencing, then production stability is improved, but promoter strength may be affected

Engineering Contradiction:

Improveproduction stabilityVSAvoidpromoter strength

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent applies local quality by making targeted point mutations only at specific CpG sites (positions -41 and -179) within the promoter sequence, rather than altering the entire promoter. This localized modification prevents methylation at these critical sites while preserving the overall promoter architecture and function. The selective mutation approach maintains promoter strength while achieving production stability

Inventive Principle:

Principle #3Local quality

3Reliability

If C to G point mutations are introduced at positions -41 and/or -179, then silencing is reduced and production stability is improved, but the promoter sequence is altered

Engineering Contradiction:

Improveproduction stabilityVSAvoidpromoter sequence integrity

Core Design Contradiction:

ReliabilityVSStability of the object's composition

Solution Approach 1:

The patent converts the harmful effect of CpG sites (which are naturally prone to methylation and silencing) into a benefit by introducing C to G mutations at these same positions. The mutated sites no longer contain the problematic CpG dinucleotide sequence, thereby eliminating the silencing mechanism while maintaining the promoter's position and overall structure. This transforms the original vulnerability into a protective feature

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

Data Source

PatentEP3146057B1Method for production of polypeptides

Publication Date: 2020.07.29 F HOFFMANN LA ROCHE & CO AG

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AI summary

The current invention reports a promoter that has the nucleic acid sequence of SEQ ID NO: 02 or SEQ ID NO: 03 which is a human CMV major immediate-early (hCMV-MIE) promoter/enhancer with C to G point mutation at position -41 and/or -179 relative to the transcription start site. This new promoter is especially useful for the production of polypeptides at large scale as it shows reduced promoter silencing and improved polypeptide production.