Episomal Expression Vector with Inverted Repeat Elements for Gene Amplification

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Solution Overview

Problem

Conventional gene expression methods in mammalian cells, particularly in human cells, face challenges such as time-consuming step-by-step selection processes and inability to perform post- and peri-translational modifications, leading to inefficient production of biopharmaceutical proteins and suppression of gene amplification due to mismatch repair systems.

Innovation Solution

An expression vector organized as an inverted dimer (ID) with a nuclear anchoring element and inverted repeat elements is used to enhance gene expression in mammalian cells, allowing for rapid and effective protein production even in mismatch repair-proficient cells by transfecting the vector into cells and culturing them under selective pressure.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional step-by-step selection method is used to enhance gene expression in mammalian cells, then gene expression can be enhanced, but the process becomes tedious and time-consuming taking months to complete

Engineering Contradiction:

Improvegene expression enhancementVSAvoidtime for selection process

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent introduces an expression vector containing an inverted repeat element that is pre-designed to trigger gene amplification and expression enhancement directly upon transfection, eliminating the need for prolonged step-by-step selection. The inverted repeat element is prepared in advance within the vector structure, allowing immediate activation of the gene amplification mechanism when introduced into mammalian cells, thus reducing the time required from months to a much shorter period.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent changes the molecular structure parameter of the expression vector by incorporating an inverted repeat element with specific nucleic acid sequences. This structural modification enables the vector to activate gene amplification through a different mechanism than conventional selection methods, transforming the time parameter from months to a significantly shorter duration while maintaining reliable gene expression enhancement.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If conventional gene amplification method is used in human cells, then gene expression can be enhanced, but the mismatch repair system strongly suppresses gene amplification

Engineering Contradiction:

Improvegene expression enhancementVSAvoidsuppression by mismatch repair system

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent exploits the inverted repeat element to create a beneficial effect that overcomes the harmful suppression by the mismatch repair system. The inverted repeat structure, when present in the expression vector, triggers a mechanism that promotes gene amplification and expression enhancement, effectively converting the potential harm of MMR suppression into a benefit by using the vector's structural features to activate alternative pathways for gene expression enhancement that are not inhibited by MMR.

Inventive Principle:

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

Solution Approach 2:

The inverted repeat element acts as an intermediary mechanism between the expression vector and the cell's gene expression machinery. It mediates the interaction by providing a specific structural feature that initiates gene amplification and expression enhancement, serving as a bridge that overcomes the inhibitory effect of the mismatch repair system and enables reliable gene expression enhancement in human cells.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Ease of manufacture

If prokaryotic expression system is used, then protein production is simplified, but post- and peri-translational modifications cannot be performed

Engineering Contradiction:

Improveprotein production simplicityVSAvoidpost-translational modification capability

Core Design Contradiction:

Ease of manufactureVSAdaptability or versatility

Solution Approach 1:

The patent creates a universal expression system by combining features that enable both simplified protein production and post-translational modification capabilities. The expression vector is designed to function in mammalian cells, which provides the necessary enzymatic machinery for post- and peri-translational modifications while maintaining the efficiency and simplicity of modern expression systems. This multi-functional approach allows the same system to handle both basic protein production and complex modified proteins.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Data Source

PatentUS8530233B2Expression vector with enhanced gene expression capacity and method for using the same

Publication Date: 2013.09.10 NAT CHENG KUNG UNIV

AI summary

The present invention provides a novel expression vector which comprises a gene of interest, a nuclear anchoring element, and at least one inverted repeat element, preferably two inverted repeat elements. The expression vector is an episomal vector capable of transfecting a mammalian cell. The present invention further provides a method for enhancing gene expression by transfecting the expression vector to a mammalian cell, preferably a human cell.