Multi-site SSI Mammalian Cells for Difficult Protein Expression
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Solution Overview
Problem
Current site-specific integration (SSI) methods for recombinant protein expression in mammalian cells are limited by the use of single genome insertion sites, which restricts the number of integrated gene copies and requires repetitive rounds of recombinase-mediated cassette-exchange (RMCE) for multi-protein production, leading to inefficiencies and potential toxicity issues with difficult-to-express proteins.
Innovation Solution
The development of tandem SSI landing pads, where multiple distinct recombination target sites are chromosomally-integrated at specific loci, allowing for independent addressing and increased gene copy integration, enabling more efficient production of recombinant proteins by facilitating the integration of multiple genes of interest at different loci.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If random integration method is used to integrate recombinant protein expression cassettes into host cell genome, then integration can occur at existing double strand breaks, but the number of gene copies integrated and expression characteristics are highly variable leading to position variegation effect
Solution Approach 1:
The genome is segmented into specific integration loci (landing pads) with defined recombination target sites, dividing the integration process into controlled segments rather than random integration throughout the genome
Solution Approach 2:
Site-specific recombinase systems (FLP-Frt or Cre-loxP) act as intermediaries to mediate precise integration at predetermined landing pads, eliminating the randomness of integration at double strand breaks
2Manufacturing precision
If site-specific integration with single landing pad is used, then recombination occurs at predetermined sites, but the number of integrated gene copies is limited requiring repetitive rounds of RMCE for multi-protein production
Solution Approach 1:
Multiple independent landing pads are created at different genomic loci, segmenting the integration capacity across multiple sites rather than relying on repetitive use of a single landing pad
Solution Approach 2:
Multiple gene copies and different proteins are integrated simultaneously across multiple landing pads, combining the production capacity in parallel rather than sequentially
3Quantity of substance
If multiple vectors are transfected to increase integrated gene copies, then more genes can be integrated, but integration efficiency decreases and expression of some difficult-to-express proteins results in toxicity
Solution Approach 1:
The integration capacity is segmented across multiple independent landing pads, allowing simultaneous integration of multiple vectors at different loci rather than requiring multiple transfections at a single site
Solution Approach 2:
The integration strategy moves from a single-dimension (single landing pad) to multi-dimension (multiple landing pads at different genomic loci), enabling parallel integration of multiple gene copies
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enhances the production capacity for recombinant proteins, particularly difficult-to-express proteins, by allowing for higher titers and reducing the complexity of multi-protein production, thereby improving the homogeneity and efficiency of recombinant protein expression.
Implementation Method 1
the recombination enzyme or recombinase is responsible for recombination events between donor and target DNA containing compatible recombination sites (Frt or loxP respectively)
Data Source
AI summary
The present disclosure relates to a site-specific integration (SSI) mammalian cell that comprises at least two distinct recombination target sites (RTS) wherein two RTS are chromosomally-integrated within the NL1 locus or the NL2 locus. The disclosure also relates to a SSI mammalian cell comprising at least four distinct RTS wherein two RTS are chromosomally-integrated within the NL1 or the NL2 locus and two RTS are chromosomally-integrated within a separate locus. The disclosure also relates to methods for using the SSI mammalian cell to produce recombinant protein expression cell lines that can express difficult to express proteins.