Circularized Guide RNA for Enhanced Editing Efficiency
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Solution Overview
Problem
Current RNA-editing technologies face inefficiencies in editing RNA sequences, particularly in terms of synthesis, stability, targeting, and localization of guide RNAs, which hampers their therapeutic potential for diseases.
Innovation Solution
Engineered polynucleotides, such as precursor engineered linear polynucleotides, are designed with targeting domains complementary to specific RNAs and spacer domains that facilitate circularization upon insertion into mammalian cells, enhancing RNA-editing efficiency by stabilizing the guide RNA and recruiting RNA editing enzymes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If linear guide RNA is used for RNA editing, then the structure is simple and easy to synthesize, but the stability and editing efficiency are insufficient
Solution Approach 1:
The patent applies circularization of the guide RNA structure, transforming the linear form into a closed circular configuration. This curvature eliminates free ends that are susceptible to degradation, thereby enhancing stability while maintaining synthesis feasibility through self-ligation mechanisms or ribozyme-mediated circularization
Solution Approach 2:
The patent creates composite structures by combining the guide RNA with spacer domains and potentially other functional elements to form a circularized composite molecule. This composite approach integrates multiple functions (guiding, stabilizing, and potentially recruiting editing enzymes) into a single enhanced structure
2Duration of action of stationary object
If guide RNA is extended to improve stability, then the half-life increases, but the synthesis cost and complexity increase
Solution Approach 1:
Circularization provides a compact closed structure that protects the entire molecule from exonuclease degradation without requiring extensive length extensions. The circular topology inherently increases resistance to degradation while maintaining a relatively compact size
Solution Approach 2:
The guide RNA is divided into functional domains (targeting domain, spacer domains, and circularization elements). This segmentation allows each component to perform its specific function while the overall circular structure provides stability, balancing complexity and performance
3Reliability
If circularized guide RNA is used, then the stability and editing efficiency are improved, but the design and synthesis process becomes more complex
Solution Approach 1:
The linear precursor guide RNA is designed with pre-positioned circularization elements (such as complementary overhangs or ribozyme sites) that enable automatic circularization upon entry into the cell. This preliminary arrangement of circularization capabilities in the linear form simplifies the overall design process while achieving the stability benefits of circular structures
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 engineered polynucleotides significantly increase RNA-editing efficiency, stability, and targeting specificity, leading to effective editing of target RNAs and potential therapeutic benefits for diseases like Rett syndrome, Huntington's disease, and others.
Implementation Method 1
hybridization of the targeting domain with the target RNA facilitates an edit of a base of a nucleotide of the target RNA by an RNA editing enzyme
Data Source
AI summary
Disclosed herein are engineered guide RNAs, engineered polynucleotides, precursor engineered polynucleotide, vectors omprising engineered polynucleotide, nucleic acids of engineered polynucleotide, pharmaceutical compositions thereof, methods of making the engineered polynucleotides and methods of treating or preventing a disease or condition by administering above described thereof.