Kozak Sequence Editing for Protein Level Control in Monogenic Disease
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Solution Overview
Problem
Single-gene disorders caused by functional loss or gain of an allele, such as haploinsufficiency and gene duplication, are challenging to treat due to the impact on protein levels, which existing methods have not effectively addressed.
Innovation Solution
Utilizing CRISPR-Cas genome editing technologies to introduce specific nucleotide conversions in the Kozak sequence to enhance or inhibit translation, thereby modulating protein production, including methods like CRISPR-Cas homology-directed repair, CRISPR-Cas prime editing, and CRISPR-Cas base editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional treatment methods are used for single-gene disorders, then existing therapies can be applied, but they have not effectively addressed the impact on protein levels caused by haploinsufficiency or gene duplication
Solution Approach 1:
The patent applies parameter changes by modifying the Kozak sequence nucleotides (specifically positions -1, -2, and -3 relative to the AUG codon) to alter translational efficiency. This changes the biological parameter of protein production levels, enabling treatment of haploinsufficiency by enhancing translation of the wild-type allele or suppression in case of gene duplication, thereby resolving the contradiction between treatment effectiveness and adaptability to protein level changes
2Quantity of substance
If the Kozak sequence is modified to enhance translation, then protein levels increase for haploinsufficiency diseases, but translation efficiency must be precisely controlled to avoid imbalances
Solution Approach 1:
The patent applies local quality by making specific targeted modifications to the Kozak sequence at precise locations (positions -1, -2, -3 relative to AUG) rather than broad changes. This localized modification allows fine-tuned control of translational efficiency, increasing protein levels for haploinsufficiency while maintaining precision to avoid imbalances, thus resolving the contradiction between quantity increase and manufacturing precision
3Duration of action of stationary object
If genome editing is used to permanently alter genomic DNA, then therapeutic effects are achieved, but the complexity of editing methods must be managed
Solution Approach 1:
The patent applies taking out by extracting and focusing the genome editing approach specifically on the Kozak sequence region rather than performing comprehensive genomic editing. This extraction simplifies the editing process to a targeted region, achieving permanent therapeutic effects while reducing the complexity of the editing method, thus resolving the contradiction between duration of action and device complexity
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
The approach allows for targeted modulation of translational efficiency, permanently altering genomic DNA to rescue HI phenotypes by increasing or decreasing protein levels as needed, without causing imbalances, and is applicable across various cell types and genes.
Implementation Method 1
CRISPR-Cas homology-directed repair
Implementation Method 2
CRISPR-Cas prime editing
Implementation Method 3
CRISPR-Cas base editing
Implementation Method 4
These nucleotide conversions enhance or inhibit the translation of the mRNA produced by the gene
Data Source
AI summary
The present invention relates to the medical field of single-gene disorders caused by functional loss or gain of an allele. The innovative approach developed being based on editing the human genome at the level of the Kozak sequence by means of CRISPR-Cas programmable nucleases. Particularly, the present invention relates to variant Kozak sequences and related in vitro or in vivo methods for obtaining such variant Kozak sequences for therapeutic applications in the treatment of single-gene diseases caused by monoallelic losses or gains. These in vitro and in vivo methods include CRISPR-Cas homology-directed repair, CRISPR-Cas prime editing, CRISPR-Cas base editing or genome editing with other programmable RNA-guided nucleases, and the introduction of specific nucleotide conversions in the Kozak sequence of genes causative of diseases. These nucleotide conversions enhance or inhibit the translation of the mRNA produced by the gene, compensating for the functional loss or gain of one allele in the diseases.