Hybrid Guide Nucleic Acid for High-Specificity Gene Editing
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Solution Overview
Problem
Current gene editing systems, such as CRISPR, face challenges with off-target editing effects, where the guide RNA binds to non-target sequences, leading to unintended genetic alterations, while maintaining high on-target efficiency is a significant concern for therapeutic applications like treating atherosclerotic cardiovascular disease.
Innovation Solution
A hybrid guide nucleic acid system is developed, comprising a spacer sequence with a combination of deoxyribonucleotides and ribonucleotides, including a 2′ hydroxyl group covalently linked to a methyl group (2′-OMe), within a lipid nanoparticle (LNP) formulation, specifically designed to enhance on-target editing efficiency while reducing off-target effects by optimizing the position and number of deoxyribonucleotides and ribonucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If a standard guide RNA is used in CRISPR gene editing, then on-target editing efficiency is achieved, but off-target editing effects occur leading to unintended genetic alterations
Solution Approach 1:
The guide nucleic acid employs heterogeneous chemical modifications at different positions: 2′-OMe ribonucleotides at positions 1-8 provide enhanced binding affinity and stability at the seed region, while deoxyribonucleotides at positions 9-20 reduce off-target binding. This local differentiation of chemical properties optimizes both on-target efficiency and off-target specificity
Solution Approach 2:
The guide nucleic acid is constructed as a composite molecule combining different nucleotide types (2′-OMe ribonucleotides and deoxyribonucleotides) with distinct chemical properties. This composite structure leverages the strengths of each nucleotide type: 2′-OMe for stability and affinity at the critical binding region, and DNA-like nucleotides for reduced off-target interactions
2Productivity
If the guide RNA sequence is optimized for high on-target binding, then editing efficiency improves, but the risk of off-target binding increases
Solution Approach 1:
The invention changes the chemical parameters of the guide nucleic acid by incorporating 2′-OMe modifications at specific positions (1-8) to enhance thermal stability and binding affinity for on-target sequences, while using unmodified deoxyribonucleotides at positions 9-20 to maintain specificity and reduce off-target binding through reduced chemical reactivity
3Strength
If a fully RNA-based guide is used, then binding affinity is high, but nuclease degradation reduces stability
Solution Approach 1:
The guide nucleic acid applies localized chemical modification strategy: positions 1-8 contain 2′-OMe ribonucleotides that provide both high binding affinity (RNA characteristic) and nuclease resistance (modified sugar), while positions 9-20 use standard deoxyribonucleotides that provide inherent stability and reduced immunogenicity, creating a hybrid structure with optimized local properties
Data Source
AI summary
Disclosed herein are novel gene editing systems capable of being delivered to a subject intravenously through a lipid nano particle pharmaceutical formulation and producing durable in vivo editing of a target gene, such as ANGPTL3, with high on-target gene editing efficiency, reduced or low off-target effect, and no germline editing. The gene editing systems comprise a chemically modified guide nucleic acid sequence with a spacer having a specified arrangement of deoxyribonucleotides and ribonucleotides. The novel gene editing systems comprise mRNA that encodes the gene editor proteins, which may include a modified nickase component. Methods of disease treatments using the gene editing systems are also disclosed.