Guide RNA-Cas RNP Conjugates for Targeted Viral Genome Editing
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Solution Overview
Problem
Current CRISPR-Cas9 therapies for viral infections and gene editing face challenges such as off-target effects, host immunity, and inefficient delivery, particularly in addressing latent and chronic viral infections like HIV, HBV, and HSV, which existing treatments fail to clear viral genetic materials.
Innovation Solution
Development of chemically modified guide RNA-Cas protein (RNP) complexes conjugated with PEG, polymers, ligands, lipids, antibodies, or peptides to enhance specificity, reduce off-target effects, and improve delivery, using viral vectors for targeted gene editing and regulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR-Cas9 is used for viral infection treatment, then viral genetic materials can be targeted, but off-target effects occur and host immunity is triggered
Solution Approach 1:
The guide RNA is chemically modified with substitutions at specific positions (e.g., 2'-F-ribonucleotides, LNA, UNA, PACE, thioPACE, phosphoromonothioates) to change its physical and chemical properties. These modifications increase thermal stability, enhance target recognition specificity, and reduce off-target effects while minimizing host immune recognition.
Solution Approach 2:
The guide RNA is conjugated with multiple types of molecules including PEG polymers, non-PEG polymers, ligands for cellular receptors, lipids, oligonucleotides, antibodies, polysaccharides, and peptides. This creates a composite conjugate structure that combines the target-specific binding capability of guide RNA with the beneficial properties of each conjugate component, such as improved stability, reduced immunogenicity, and enhanced cellular uptake.
2Productivity
If conventional CRISPR-Cas9 delivery methods are used, then gene editing can be performed, but delivery efficiency is poor especially for latent and chronic viral infections
Solution Approach 1:
Various conjugate molecules serve as intermediaries to facilitate delivery. PEG polymers improve circulation time and reduce clearance by the reticuloendothelial system. Cell-penetrating peptides and lipids enhance cellular uptake. Receptor ligands mediate targeted delivery to specific cell types infected with latent or chronic viruses, ensuring the CRISPR-Cas9 system reaches its target.
Solution Approach 2:
The conjugate system is designed to address multiple delivery challenges simultaneously. The same guide RNA-Cas9 conjugate platform can target different viral infections (HIV, HBV, HSV) and different infection states (acute, latent, chronic) by adjusting the conjugate components and guide RNA sequences, providing universal applicability across various viral therapies.
3Reliability
If guide RNA is chemically modified to increase stability and reduce toxicities, then specificity is enhanced, but manufacturing complexity increases
Solution Approach 1:
The guide RNA is synthesized in segmented portions with specific chemical modifications introduced at defined positions during the synthesis process. The conjugation of different molecules (PEG, lipids, peptides) is performed at specific sites on the guide RNA structure. This segmentation approach allows for systematic introduction of modifications and simplifies the manufacturing process by breaking down the complex modification task into manageable steps.
Data Source
AI summary
Provided herein are compositions of conjugates of a guide RNA(s)-CRISPR Cas protein (RNP) complex. The conjugate comprises a guide RNA(s)-CRISPR Cas protein (RNP) complex and one or more molecules selected from PEG, non-PEG polymers, ligands for cellular receptors, lipids, oligonucleotides, polysaccharides and peptides and chemically linked to the Cas protein and/or guide RNA(s). The conjugates are delivered to targeted cells as RNP complexes, or formed in targeted cells from guide RNA conjugates and a mRNA or a viral vector encoding a Cas protein, or formed in targeted cells from a crRNA conjugates and a viral vector encoding both a Cas protein and a tracrRNA. Also provided are preparation methods and uses of these conjugates.