Lipid Nanoparticle RNP Encapsulation for Flow-Channel Genome Editing
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Solution Overview
Problem
Existing methods for producing lipid nanoparticles that encapsulate RNPs for genome editing are ineffective due to the inactivation of proteins like RNP by physical parameters during preparation, and there are no reports on using alcohol dilution methods for such formulations.
Innovation Solution
A lipid nanoparticle composition containing pH-sensitive cationic lipid, neutral phospholipid, and polyalkylene glycol-modified lipid, with specific ratios, is used to encapsulate RNPs, and is prepared via an alcohol dilution method using a flow channel structure with a particular design to efficiently load RNP complexes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If viral vectors or non-viral vectors are used to express Cas9 protein in target cells, then delivery is relatively easy, but Cas9 protein expresses for a relatively long-term period causing off-target mutations
Solution Approach 1:
The invention extracts only the necessary functional component (RNP complex) from the full Cas9 expression system, delivering pre-formed RNP directly to cells rather than delivering Cas9 gene that would be expressed long-term. This removes the harmful long-term expression while maintaining delivery feasibility through lipid nanoparticle encapsulation
Solution Approach 2:
The RNP complex is prepared in advance outside the cell, with Cas9 protein and guide RNA pre-assembled into the active complex. This preliminary formation of the functional complex allows direct delivery of ready-to-action editing machinery, eliminating the need for long-term cellular expression and reducing off-target effects
2Object-affected harmful factors
If RNP is directly introduced into target cell, then off-target mutation is suppressed to the minimum, but RNP rapidly degrades and disappears after mutation is introduced
Solution Approach 1:
The invention provides beforehand cushioning by encapsulating the RNP complex within lipid nanoparticle carriers that protect it from degradation. The lipid nanoparticle shell acts as a protective barrier during delivery, and the pH-sensitive composition enables controlled release at the target site, extending RNP persistence without increasing off-target effects
3Productivity
If high concentration of Cas9 is required to induce gene knockdown in cultured cells, then genome editing efficiency is insufficient, but protein inactivation occurs during preparation
Solution Approach 1:
The invention changes the physical-chemical parameters of the delivery system by using pH-sensitive cationic lipids with specific pKa values (6.0-9.0). This parameter optimization allows the lipid nanoparticle to remain stable at preparation pH while automatically releasing RNP at the lower pH of endosomes, achieving both high editing efficiency and protein stability without requiring excessive Cas9 concentration
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 lipid nanoparticles enable efficient genome editing by suppressing off-target mutations and maintaining high editing efficiency, as the RNP is stably encapsulated and delivered to target cells.
Implementation Method 1
a pH-sensitive cationic lipid of general formula (I)... the ratio of the pH-sensitive cationic lipid relative to the total amount of lipids constituting the lipid nanoparticle is 30% by mol to 50% by mol
Implementation Method 2
a flow channel structure having a simple two-dimensional structure in which baffles (baffle plates) having a fixed width relative to a flow channel width are disposed alternately from two sides on a micro-sized flow channel through which a feedstock solution is passed makes it possible to form a nano-sized lipid particle formation system
Data Source
AI summary
The present invention addresses the problem of providing a lipid nanoparticle in which a nucleic acid, etc., required in genome editing is encapsulated and which can be produced by an alcohol dilution method using flow channels and contributes to high genome editing efficiency. The present invention pertains to a lipid nanoparticle which comprises a lipid component, a DNA nuclease, a guide RNA and a single-stranded oligonucleotide, wherein: the lipid component comprises a pH-sensitive cationic lipid, a neutral phospholipid and a polyalkylene glycol-modified lipid; the ratio of the pH-sensitive cationic lipid relative to the total lipids constituting the lipid nanoparticle is 30-50 mol %; the ratio of the neutral phospholipid relative to the total lipids constituting the lipid nanoparticle is 20-50 mol %; and the ratio of the polyalkylene glycol-modified lipid relative to the total lipids constituting the lipid nanoparticle is 1-4 mol %.