PCSK9 Gene Repression Using dCas-KRAB for Familial Hypercholesterolemia
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Solution Overview
Problem
Modifying aberrant expression of a disease-causing allele, such as PCSK9, is insufficient to treat conditions like Familial Hypercholesterolemia, necessitating a more effective approach to reduce PCSK9 expression in cells.
Innovation Solution
A heterologous polypeptide comprising a Cas protein with reduced DNA cleavage activity (dCas) coupled to a transcriptional repressor, such as KRAB, is used to bind to the PCSK9 gene, reducing its expression through histone modification, delivered via viral or non-viral vectors like AAV or LNPs, guided by specific gRNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a heterologous polypeptide with dCas and transcriptional repressor is used to reduce PCSK9 expression, then PCSK9 expression levels are significantly decreased, but the device complexity increases due to the multi-component system
Solution Approach 1:
The patent combines dCas (a deactivated Cas protein that retains DNA binding ability but lacks cleavage activity) with a transcriptional repressor domain into a single heterologous polypeptide fusion protein. This merging allows the system to simultaneously bind to the target PCSK9 gene and recruit chromatin-modifying enzymes, achieving reliable gene expression suppression without requiring multiple separate components.
Solution Approach 2:
The dCas protein serves as an intermediary that bridges the guide RNA (which provides target specificity) and the transcriptional repressor (which executes the gene suppression function). The dCas binds to the guide RNA and uses it to locate the target gene, then recruits the transcriptional repressor to modify chromatin structure and suppress transcription, thereby mediating the entire gene silencing process.
2Productivity
If viral vectors like AAV are used to deliver the heterologous polypeptide, then delivery efficiency is improved, but safety concerns arise due to potential immunogenicity and insertional mutagenesis
Solution Approach 1:
The patent employs lipid nanoparticles (LNPs) as an intermediary delivery vehicle to transport the heterologous polypeptide or encoding nucleic acid into target cells. LNPs are non-viral carriers that protect the cargo from degradation, facilitate cellular uptake, and avoid the immunogenicity and insertional mutagenesis risks associated with viral vectors, while still achieving efficient delivery to the desired tissues.
3Ease of operation
If the dCas protein size is reduced to less than or equal to 800 amino acids, then ease of delivery is improved, but the binding affinity and stability may be compromised
Solution Approach 1:
The patent extracts and uses only the essential functional domains of the Cas protein, creating a truncated or miniaturized dCas variant that retains DNA binding capability but removes unnecessary regions. This extraction of core functionality reduces the protein size to ≤800 amino acids, facilitating delivery, while preserving the critical binding and repressor recruitment functions needed for reliable gene suppression.
Solution Approach 2:
The patent modifies the physical parameters of the dCas protein by reducing its size through truncation or using engineered variants. This parameter change (size reduction) improves deliverability across biological barriers, while the preserved functional domains maintain sufficient binding affinity and stability for effective target gene recognition and repression.
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 significantly decreases PCSK9 expression, enhancing LDL uptake and reducing serum cholesterol levels, potentially normalizing them to healthy levels.
Implementation Method 1
the actuator moiety binds to an endogenous target gene encoding proprotein convertase subtilisin/kexin type 9 (PCSK9) or a regulatory region thereof in a cell
Implementation Method 2
the transcriptional repressor comprises a histone modifier. In some cases, the histone modifier comprises a histone methylation modifier
Data Source
AI summary
Described herein are compositions, systems, and methods for modulating gene expression. Also described herein are systems and methods for treating a disease or a condition by modulating gene expression. In some embodiments, the compositions, systems, and methods provided herein are used to treat familial hypercholesterolemia. In some embodiments, the compositions, systems, and methods provided herein involve suppression of endogenous PCSK9.