PCSK9 Gene Editing via Guide RNA and Endonuclease Targeting
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Solution Overview
Problem
Current genome engineering techniques for addressing PCSK9-related disorders lack safety and effectiveness, with random gene insertion leading to severe side effects and limited reproducibility, and existing methods are not sufficient for developing reliable treatments.
Innovation Solution
The use of genome editing methods involving DNA endonucleases to introduce specific single-strand or double-strand breaks in the PCSK9 gene, allowing for permanent insertions, deletions, or mutations to reduce or eliminate PCSK9 gene product expression, using ex vivo and in vivo approaches involving hepatocytes, induced pluripotent stem cells, and mesenchymal stem cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If random gene insertion is used to address PCSK9-related disorders, then gene therapy can be delivered to target cells, but severe side effects occur due to disruption of normal regulation of neighboring genes
Solution Approach 1:
The patent uses guide RNA as an intermediary molecule that directs the nuclease to the specific PCSK9 gene location. The guide RNA binds to the nuclease and the target DNA sequence, mediating precise targeting without random insertion, thereby eliminating side effects while maintaining therapeutic delivery.
Solution Approach 2:
The patent replaces the mechanical/random insertion process with a molecular recognition system based on complementary base pairing between guide RNA and target DNA. This substitution of mechanism enables site-specific gene editing at the PCSK9 locus without the harmful randomness of traditional insertion methods.
2Reliability
If early random insertion technologies are used, then gene therapy can be delivered, but reproducibility is limited as there is no guarantee of insertion at the same place in different cells
Solution Approach 1:
The guide RNA acts as a molecular intermediary that ensures consistent targeting of the PCSK9 gene across different cells. By mediating the interaction between the nuclease and the specific genomic location, it guarantees reproducible insertion at the same place in every cell treated.
Solution Approach 2:
The patent extracts the targeting function from the random insertion process and isolates it into a separate guide RNA component. This extracted targeting mechanism can be precisely controlled and reproduced, independent of the insertion process itself, ensuring consistent genomic location targeting.
3Manufacturing precision
If zinc finger nucleases, TALENs, or homing endonucleases are used to modify specific DNA areas, then precision of alteration is increased, but device complexity and manufacturing difficulty increase
Solution Approach 1:
The patent segments the gene editing system into two independent functional modules: the nuclease (Cas9) and the guide RNA. This segmentation allows the complex targeting function to be separated from the cutting function, simplifying the overall system while maintaining high precision at the PCSK9 gene location.
Solution Approach 2:
The guide RNA provides a universal targeting mechanism that can be applied to any gene sequence by simply changing the RNA sequence, rather than requiring different protein complexes for each target. This universal approach reduces device complexity while maintaining manufacturing precision across different genomic targets.
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 targeted and permanent changes to the PCSK9 gene, potentially providing a safe and effective treatment for PCSK9-related conditions by reducing or eliminating PCSK9 gene product expression, thereby addressing the limitations of existing technologies.
Implementation Method 1
introducing into the cell one or more deoxyribonucleic acid (DNA) endonucleases to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the PCSK9 gene
Data Source
AI summary
The present application provides materials and methods for treating a patient with one or more conditions associated with PCSK9 whether ex vivo or in vivo. In addition, the present application provides materials and methods for editing and/or modulating the expression of PCSK9 gene in a cell by genome editing.