Nucleic Acid Assemblies for CRISPR Delivery
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for delivering CRISPR-Cas RNPs to cells face challenges such as cytotoxicity, off-target editing, and lack of control over stoichiometry and intracellular trafficking, limiting their effectiveness for gene editing and biotechnological applications.
Innovation Solution
Nucleic acid assemblies that enclose and protect cargo, such as CRISPR-Cas effector proteins and guide molecules, with designed physiochemical properties for targeted delivery, enhanced stability, and reduced immunogenicity, allowing for controlled stoichiometry and intracellular trafficking.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current delivery methods (viral vectors, liposomal delivery) are used to deliver CRISPR-Cas RNPs, then delivery efficiency is improved, but cytotoxicity increases and off-target editing occurs
Solution Approach 1:
The invention segments the delivery system into distinct functional modules: a core RNP complex (Cas protein + guide RNA) and a separate delivery vehicle (polymer nanoparticle or liposome). This segmentation allows the toxic delivery components to be separated from the therapeutic RNP, reducing cytotoxicity while maintaining delivery efficiency. The RNP can be delivered in a controlled manner without exposure to excessive toxic reagents.
Solution Approach 2:
The invention introduces an intermediary delivery vehicle (polymer nanoparticle or liposome) that mediates between the RNP cargo and the cell membrane. This intermediary protects the RNP from degradation, facilitates cellular uptake through endocytosis, and enables controlled release inside the cell, thereby improving delivery efficiency while reducing direct cytotoxic effects on cells.
2Productivity
If viral vectors are used for RNP delivery, then transduction efficiency is improved, but off-target editing and genome instability increase due to Cas protein overexpression
Solution Approach 1:
The invention performs preliminary assembly of the RNP complex extracellularly before delivery into the cell. The Cas protein and guide RNA are pre-assembled into a stable complex with defined stoichiometry outside the cell, ensuring that only the correct RNP ratio is delivered. This prevents Cas protein overexpression and misassembly that would occur with viral vector delivery of separate components, thereby maintaining editing fidelity.
Solution Approach 2:
The invention changes the delivery parameters by using non-viral vectors (polymer nanoparticles or liposomes) with controlled physical and chemical properties. By adjusting particle size, surface charge, and composition, the delivery system achieves high transduction efficiency while controlling Cas protein expression levels, preventing off-target editing and genome instability associated with viral overexpression.
3Reliability
If intact RNP delivery is implemented, then gene editing fidelity is improved, but control over stoichiometry of multiple sgRNAs and intracellular trafficking is lost
Solution Approach 1:
The delivery vehicle (polymer nanoparticle or liposome) is designed with multi-functionality: it can accommodate multiple different RNP complexes with different guide RNAs, control their stoichiometry through controlled incorporation during assembly, and provide targeted delivery to specific cell types or organelles. This universal platform maintains RNP fidelity while restoring control over stoichiometry and trafficking that was lost in simple delivery methods.
Solution Approach 2:
The invention incorporates feedback mechanisms through the design of the delivery vehicle that responds to cellular conditions. The polymer nanoparticles or liposomes are engineered to release their RNP cargo in response to specific intracellular triggers (such as endosomal acidification or enzymatic conditions), ensuring controlled release at the right time and place. This feedback control maintains editing fidelity while enabling precise manipulation of delivery timing and location.
4Productivity
If conventional transfection techniques are used for RNP delivery, then nuclear delivery is achieved, but cytotoxicity occurs due to plasma membrane disruption
Solution Approach 1:
The invention replaces mechanical disruption methods (conventional transfection that physically disrupts the plasma membrane) with a biochemical delivery mechanism. The polymer nanoparticle or liposome vehicle facilitates cellular uptake through receptor-mediated endocytosis, a gentle biochemical process that does not disrupt the membrane. The RNP is then released into the cytoplasm and trafficked to the nucleus through natural cellular pathways, achieving nuclear delivery without cytotoxic membrane disruption.
Data Source
AI summary
Disclosed are compositions and methods involving nucleic acid assemblies that enclose and/or protect cargo. Disclosed are compositions that include a nucleic acid assembly comprising one or more nucleic acid molecules and cargo comprising two or more cargo molecules. The nucleic acid assembly can have physiochemical properties that: (i) enhance targeting of the composition to one or more types of cells, tissues, organs, or microenvironments relative to other types of cells, tissues, organs, or microenvironments in vivo; (ii) enhance stability and/or half-life of the composition in vivo; and/or (iii) reduce immunogenicity of the composition. The nucleic acid assembly and/or cargo can have features that enhance intracellular trafficking of nucleic acid assembly and/or its cargo. The cargo can be enclosed and/or protected by the nucleic acid assembly. Some or all of the cargo molecules in the composition can be present in a defined stoichiometric ratio.