CRISPR Base Editing PCSK9 Loss-of-Function Variants
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Solution Overview
Problem
Current methods for generating PCSK9 protective variants and loss-of-function mutants in vivo are ineffective due to the need to modify a large number of cells to modulate cholesterol levels, and are associated with off-target effects and genome instability.
Innovation Solution
The use of CRISPR/Cas9-based base-editing technology to precisely target and modify the PCSK9, LDLR, IDOL, or APOC3 genes, resulting in loss-of-function variants that reduce LDL and overall cholesterol levels while minimizing off-target effects and genomic instability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If engineered nucleases are used to create random indels in the PCSK9 genomic locus, then PCSK9 loss-of-function variants can be generated, but off-target effects and genome instability occur
Solution Approach 1:
The patent replaces the mechanical cutting mechanism of engineered nucleases (ZFNs, TALENs, CRISPR-Cas9) with a chemical editing mechanism using base editors. Instead of creating double-strand breaks that require cellular repair machinery (prone to errors and off-target effects), the base editor uses a modified deaminase enzyme to directly convert cytosine to uracil at the target site, which is then processed by cellular repair mechanisms to generate precise C-to-T transitions without requiring DNA cleavage.
Solution Approach 2:
The patent changes the fundamental parameter of genome editing from creating random insertions/deletions (indels) through non-homologous end joining to generating precise point mutations through base substitution. This parameter change from random to precise editing eliminates the need for homology-directed repair and significantly reduces off-target effects while maintaining the ability to generate loss-of-function variants.
2Reliability
If a large number of cells are modified to modulate cholesterol levels, then cholesterol modulation can be achieved, but the complexity and scale of the procedure increases
Solution Approach 1:
The base editing technology enables more efficient gene modification that requires fewer cells to achieve the desired therapeutic effect. The precise C-to-T transitions created by base editors are more likely to produce functional loss-of-function variants compared to random indels, reducing the number of cells that need to be modified and treated to achieve sufficient cholesterol level modulation.
3Object-affected harmful factors
If precise targeting methods are used to modify PCSK9, then loss-of-function variants can be generated with low off-target effects, but the technology complexity increases
Solution Approach 1:
The base editor system replaces complex nuclease-based editing with a simpler deamination chemistry approach. The fusion protein combines a programmable DNA-binding domain (dCas9 or dCpf1) with a cytosine deaminase, creating a more straightforward mechanism that relies on enzymatic conversion rather than DNA cleavage and repair, thereby reducing technology complexity while improving precision.
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 generates loss-of-function variants with cardioprotective functions, effectively lowering LDL and overall cholesterol levels while maintaining genomic stability and reducing the risk of oncogenic modifications.
Implementation Method 1
contacting results in deamination of the target C base by the fusion protein, resulting in a cytosine (C) to thymine (T) change in the PCSK9-encoding polynucleotide
Data Source
AI summary
Provided herein are systems, compositions, and methods of introducing loss-of- function mutations in to protein factors involved in the LDL-R-mediated cholesterol clearance pathway, e.g., PCSK9, APOC3, LDL-R, or IDOL. Loss-of-function mutations may be introduced using a CRISPR/Cas9-based nucleobase editor described in. Further provided herein are compositions and methods of treating conditions related to high circulating cholesterol levels.