Targeted Integration Vector for Constitutively Expressed Genes
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Solution Overview
Problem
Current methods for targeted integration of exogenous nucleic acids into the genome face challenges, particularly low frequency of homologous recombination and limited safe harbor sites in the human genome, which hinders predictable and safe expression of transgenes without adverse effects on host cells.
Innovation Solution
A vector design that includes a 5' nucleic acid homologous to a genomic sequence 5' of a constitutively expressed gene's stop codon, an exogenous nucleic acid, a 3' nucleic acid homologous to the genomic sequence 3' of the stop codon, and a translation interruption-reinitiation signal, allowing for targeted integration and expression of exogenous nucleic acids by replacing the genomic stop codon, thereby enabling constitutive expression without disrupting endogenous gene function.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If homologous recombination is used for targeted integration, then integration can occur at desired locations, but the frequency of targeted integration is extremely low
Solution Approach 1:
The patent introduces a double-stranded break at the target genomic location before introducing the exogenous polynucleotide. This preliminary action creates a pre-condition that dramatically stimulates homologous recombination, transforming it from a rare event into a highly efficient process. The double-stranded break is created using nucleases such as CRISPR-Cas9, TALENs, or zinc-finger nucleases targeting a specific site within or near the constitutively expressed gene, ensuring the genomic location is primed and ready for precise integration of the therapeutic transgene.
2Productivity
If exogenous polynucleotide is introduced without targeted integration, then insertion can occur at various genomic locations, but the frequency of random insertion exceeds targeted integration by several orders of magnitude
Solution Approach 1:
The patent employs nucleases with highly specific binding domains that recognize and bind to unique DNA sequences at the desired integration site. This creates a localized effect where the double-stranded break occurs only at the precise target location within the constitutively expressed gene, such as the GAPDH gene. The specificity of the nuclease binding ensures that integration is confined to this local genomic region, preventing random insertions throughout the genome while maintaining high integration frequency at the target site.
3Reliability
If transgenes are integrated into safe harbor sites, then predictable expression can be achieved, but the number of available safe harbor sites is limited
Solution Approach 1:
The patent identifies and utilizes constitutively expressed genes such as GAPDH, ACTB, and HSP90 as universal integration targets that function as safe harbor sites across multiple cell types and differentiation stages. By targeting these universally expressed genes, the approach creates a multi-functional integration strategy that works across hematopoietic cells, endothelial cells, and other cell types, effectively expanding the number of available safe integration sites from the traditional three to numerous constitutively expressed genes that can accommodate transgene integration with predictable expression.
4Productivity
If transgenes are integrated into constitutively expressed genes, then constitutive expression can be achieved, but there is concern about adverse effects on host cell function
Solution Approach 1:
The patent performs preliminary validation by integrating transgenes into the constitutively expressed genes of cells that have already differentiated into various cell types, including hematopoietic and endothelial cells. This preliminary action in differentiated cells ensures that the integration and expression do not cause adverse effects on cell function, as these cells have already committed to their specific fates. The approach demonstrates that integration into constitutively expressed genes like GAPDH does not disrupt essential cell functions or cause malignant transformation, thereby validating the safety of this strategy before broader application.
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 enables stable and predictable expression of exogenous nucleic acids in cells, maintaining endogenous gene function and avoiding malignant transformation, with demonstrated expression in various cell types and differentiation stages, including hematopoietic and endothelial cells.
Implementation Method 1
Attempts have been made to alter genomic sequences in cultured cells by taking advantage of the natural phenomenon of homologous recombination. If a polynucleotide has sufficient homology to the genomic region comprising the sequence to be altered, it is possible for part or all of the sequence of the polynucleotide to replace the genomic sequence by homologous recombination.
Implementation Method 2
a translation interruption-reinitiation signal operably linked to the 5' nucleic acid and the exogenous nucleic acid, wherein the translation interruption-reinitiation signal is capable of replacing the genomic stop codon of the constitutively expressed gene
Data Source
AI summary
The invention relates to a vector comprising: a 5′ nucleic acid that is homologous to a genomic sequence 5′ of a stop codon of a constitutively expressed gene; an exogenous nucleic acid; a 3′ nucleic acid that is homologous to a genomic sequence 3′ of the stop codon of the constitutively expressed gene; a translation interruption-reinitiation signal operably linked to the 5′ nucleic acid and the exogenous nucleic acid, wherein the translation interruption-reinitiation signal is capable of replacing the stop codon of the constitutively expressed gene.