PCSK9 Repressor Fusion Proteins With dCas9 for Off-Target Control
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Solution Overview
Problem
Current methods for modulating PCSK9 levels in vivo are ineffective due to off-target effects, genome instability, and lack of safe delivery modalities, necessitating improved gene repressor systems for therapeutic applications.
Innovation Solution
Development of repressor fusion proteins comprising DNA-binding proteins like zinc fingers or catalytically-dead CRISPR proteins linked with repressor domains, along with guide nucleic acids, for targeted transcriptional repression of PCSK9 gene sequences, delivered via vectors and lipid nanoparticles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current methods for modulating PCSK9 levels in vivo are used, then cholesterol levels can be reduced, but off-target effects and genome instability occur
Solution Approach 1:
The patent divides the gene repression function into separate modular components: a DNA-binding module (either zinc finger protein or CRISPR-Cas9 system) and a repressor module (such as KRAB domain or dCas3). This segmentation allows independent optimization of targeting specificity and repression function, reducing off-target effects while maintaining effective PCSK9 suppression.
Solution Approach 2:
The patent introduces an intermediary catalytically inactive Cas9 (dCas9) protein that binds to target DNA sequences without causing double-strand breaks. This intermediary approach enables precise genomic targeting and recruitment of repressor domains to specific loci, achieving gene repression without the harmful genome instability associated with active Cas9.
2Reliability
If current methods for modulating PCSK9 levels in vivo are used, then cholesterol levels can be reduced, but safe delivery modalities are lacking
Solution Approach 1:
The patent employs lipid nanoparticles as intermediary delivery vehicles that encapsulate and protect the gene repression components (dCas9 or zinc finger proteins, guide RNAs, and repressor domains) during in vivo administration. This intermediary delivery system enables safe transport to target tissues, primarily the liver, without causing immune activation or off-target effects.
3Reliability
If repressor fusion proteins with DNA-binding proteins are developed, then specific repression of PCSK9 can be achieved, but device complexity increases
Solution Approach 1:
The patent merges the DNA-binding function and gene repression function into a single fusion protein construct. For example, zinc finger DNA-binding domains are directly fused to repressor domains like KRAB, creating a unified molecule that simultaneously targets and represses the PCSK9 gene. This merging simplifies the overall system architecture compared to using separate proteins that would require complex protein-protein interactions.
Solution Approach 2:
The patent develops universal repressor domains (such as KRAB or dCas3) that can be combined with different DNA-binding modules to target multiple genes. This multi-functional approach allows the same repressor domain to work with various zinc finger proteins or CRISPR systems, reducing the need to develop gene-specific repressors for each target and thereby simplifying the overall system complexity.
Data Source
AI summary
Provided herein are gene repressor systems comprising fusion proteins, such as fusion proteins comprising a DNA binding domain such as a TALE, zinc finger or catalytically-dead CRISPR protein and guide nucleic acid (gRNA), which are useful in the repression of a proprotein convertase subtilisin/kexin Type 9 (PCSK9) gene. Also provided are methods of using such systems to repress transcription of PCSK9.