Nanoparticle-Mediated CRISPR-Cas9 Delivery for Gene Therapy
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Solution Overview
Problem
Current gene therapy methods for hereditary diseases, particularly those using adeno-associated virus (AAV) vectors, face limitations such as limited packaging capacity and the generation of neutralizing antibodies, which hinder the efficient delivery of CRISPR-Cas9 machinery into target cells, especially in post-mitotic cells and those with non-dividing cells.
Innovation Solution
A gene delivery system utilizing nanoparticles, specifically supramolecular nanoparticles (SMNP), to encapsulate CRISPR-Cas9 components and employ the homology-independent targeted integration (HITI) strategy for effective genome editing, enabling precise gene knock-in in both dividing and non-dividing cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If AAV vectors are used to deliver CRISPR-Cas9 machinery, then gene delivery is achieved, but the packaging capacity is limited and neutralizing antibodies are generated
Solution Approach 1:
The CRISPR-Cas9 system is divided into multiple separate AAV vectors (AAV-Cas9, AAV-sgRNA, AAV-donor DNA), allowing each component to be packaged independently within the AAV capacity limit. This segmentation enables delivery of the complete CRISPR-Cas9 machinery despite the limited packaging capacity of individual AAV vectors.
Solution Approach 2:
Split AAV vectors serve as intermediaries to deliver CRISPR-Cas9 components that cannot be packaged in a single AAV vector. The multiple vectors work together as a system, with each vector carrying a specific component (Cas9, sgRNA, donor DNA) that combines to form the functional CRISPR-Cas9 editing system in target cells.
2Manufacturing precision
If HDR strategy is used for CRISPR-Cas9 editing, then precise gene knock-in is achieved, but it is not accessible to post-mitotic cells
Solution Approach 1:
The patent employs a dynamic approach by providing multiple repair strategies (HDR and NHEJ) that can be selected based on cell type and experimental needs. The system can adapt between precise HDR-mediated knock-in in dividing cells and NHEJ-mediated editing in post-mitotic cells, making the CRISPR-Cas9 system versatile across different cell types.
Solution Approach 2:
The patent changes the DNA repair pathway parameter by offering both HDR and NHEJ strategies. By adjusting the repair mechanism parameter (HDR for precision in dividing cells, NHEJ for accessibility in post-mitotic cells), the system optimizes gene editing outcomes across different cell types and experimental requirements.
3Quantity of substance
If split AAV vectors are used to deliver CRISPR-Cas9 components, then packaging capacity limitation is overcome, but transduction efficiency decreases
Solution Approach 1:
Multiple split AAV vectors are merged/combined to deliver the complete CRISPR-Cas9 machinery. The vectors are co-delivered to target cells where their components (Cas9, sgRNA, donor DNA) come together to form the functional editing system, achieving both increased capacity and maintained transduction efficiency through coordinated action of multiple vectors.
Data Source
AI summary
The present invention is directed to an integrated conceptual strategy for a gene delivery system, using the combination of nanoparticles, CRISPR-Cas9, and the HITI strategy to deliver CRISPR-Cas9 and achieve effective genome editing; wherein the advanced nanoparticles to overcome the limited packaging size of AAV-based vehicles. Also provided is a promising therapeutic solution for the treatment of hereditary diseases via gene therapy