Cationic Lipid Nanoparticles for Large CRISPR Cargo Delivery
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Solution Overview
Problem
Delivering functional genome editing agents, such as CRISPR/Cas9, to cells is challenging due to their large size and inefficient encapsulation and delivery using both viral and non-viral systems, with existing lipid nanoparticles failing to efficiently deliver large nucleic acid sequences like mRNAs or plasmids.
Innovation Solution
Development of lipid particles comprising a cationic lipid encapsulating nucleic acid sequences encoding proteins of at least 500 amino acids, using specific cationic lipids represented by Formula I, which co-encapsulate Cas9 mRNA and sgRNA, forming CRISPR LNPs that efficiently deliver and edit genes in cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If viral vectors like AAV are used to deliver CRISPR/Cas9, then delivery efficiency is improved, but carrying capacity is limited and immune responses occur
Solution Approach 1:
The patent segments the delivery system into lipid nanoparticles that can carry multiple nucleic acid components (Cas9 mRNA and gRNA) separately or together, overcoming the single-packaging limitation of viral vectors. The lipid-based system divides the therapeutic payload into manageable encapsulated units that can be delivered without the immune constraints of viral delivery.
Solution Approach 2:
The patent introduces lipid nanoparticles as an intermediary delivery vehicle between the CRISPR/Cas9 components and target cells. These LNPs serve as a non-viral mediator that protects the nucleic acids from degradation, facilitates cellular uptake, and avoids the immune system restrictions that limit viral vector carrying capacity.
2Productivity
If lipid nanoparticles optimized for siRNA are used, then siRNA delivery is efficient, but large nucleic acid sequences like Cas9 mRNA are not efficiently delivered
Solution Approach 1:
The patent modifies key parameters of the lipid nanoparticle formulation, including lipid composition (ionizable lipids with specific pKa values), particle size, and encapsulation methods, to accommodate larger nucleic acid sequences like Cas9 mRNA while maintaining delivery efficiency. The ionizable lipid properties are specifically tuned to facilitate endosomal escape for large payloads.
Solution Approach 2:
The patent develops a universal lipid nanoparticle platform that can deliver multiple types of nucleic acids (siRNA, shRNA, mRNA, plasmid DNA) of varying sizes through optimized formulations. This multi-functional system adapts to different therapeutic nucleic acids while maintaining efficient cellular delivery, overcoming the siRNA-specific optimization limitation.
3Adaptability or versatility
If large genome editing agents like CRISPR/Cas9 are delivered, then gene editing capability is achieved, but cellular internalization becomes difficult
Solution Approach 1:
The patent replaces mechanical or physical delivery methods (electroporation, microinjection) with a chemical-biological lipid nanoparticle system that facilitates cellular internalization through endocytosis. The LNPs chemically interact with cell membranes and endosomal compartments to deliver large CRISPR/Cas9 agents without requiring complex mechanical intervention.
Solution Approach 2:
The patent optimizes physical-chemical parameters of the lipid nanoparticles, including surface charge, hydrophobicity, and phase transition temperature, to enhance cellular internalization of large genome editing agents. The ionizable lipid properties are specifically engineered to promote membrane fusion and endosomal escape, making cellular uptake of large Cas9 complexes efficient.
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 CRISPR LNPs achieve up to 98% gene editing efficiency in vitro across multiple cell types, reduce tumor growth, and improve survival in cancer mouse models, demonstrating effective therapeutic genome editing.
Implementation Method 1
a cationic lipid encapsulating a nucleic acid sequence
Data Source
AI summary
Lipid particles for nucleic acid delivery and clinical applications of same are provided. Accordingly there is provided a lipid particle comprising a cationic lipid encapsulating a nucleic acid sequence, wherein said nucleic acid sequence encodes a protein having a length of at least 500 amino acids, the cationic lipid being represented by Formula I, as defined in the specification.