Engineered Guide RNAs for MAPT RNA Editing
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Solution Overview
Problem
Current RNA editing compositions face challenges in maximizing on-target RNA editing while minimizing off-target editing, particularly for genetic diseases mediated by RNA editing therapies.
Innovation Solution
Engineered guide RNAs that hybridize to the c.1 translation initiation sequence of the MAPT RNA, forming specific structural features like bulges and internal loops, facilitating targeted RNA editing by RNA editing entities such as ADAR1, ADAR2, or ADAR3, with high sequence identity to specific SEQ IDs, to achieve precise editing of adenosines.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional guide RNAs are used for RNA editing, then RNA editing can be achieved, but off-target editing occurs and on-target efficiency is not maximized
Solution Approach 1:
The guide RNA is engineered with specific structural features (bulges, internal loops, hairpins) at localized positions within its sequence. These local structural modifications create distinct thermodynamic and structural properties at specific regions, enabling the guide RNA to form more stable and specific hybridization with the target sequence while reducing affinity for off-target sequences. This local differentiation of structural quality resolves the contradiction between achieving reliable on-target editing and avoiding harmful off-target effects.
Solution Approach 2:
The invention modifies physical and chemical parameters of the guide RNA including its secondary structure configuration, thermodynamic stability profile, and sequence composition. By changing parameters such as creating bulge structures (unpaired nucleotides), internal loops (mismatches), and hairpin formations, the guide RNA achieves optimized binding characteristics that enhance on-target specificity and reduce off-target editing events while maintaining editing efficiency.
2Productivity
If guide RNAs are designed to maximize on-target editing, then editing efficiency improves, but specificity against off-target sequences may be compromised
Solution Approach 1:
The guide RNA sequence is segmented into functionally distinct regions including a seed region for initial target recognition, flanking regions with specific structural elements (bulges at defined positions, internal loops with controlled mismatches), and a 3' end region. This segmentation allows each region to contribute differently to binding kinetics and thermodynamics, enabling high productivity through strong target binding while maintaining precision through differentiated recognition zones that reduce off-target hybridization.
Solution Approach 2:
The guide RNA employs asymmetric structural features where bulges and internal loops are positioned at specific asymmetric locations relative to the target binding region. This asymmetric design creates directional binding characteristics that enhance specificity for the correct target orientation and sequence, preventing mismatched binding that would lead to off-target editing while preserving efficient on-target editing productivity.
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 guide RNAs enhance on-target RNA editing efficiency while reducing off-target edits, effectively addressing the challenges of RNA editing therapies for genetic diseases like Tauopathies by promoting specific protein knockdown and reducing tau protein aggregation.
Implementation Method 1
the engineered guide RNA, upon hybridization to a c.1 translation initiation sequence (TIS) of a target MAPT RNA, forms a guide-target RNA scaffold
Implementation Method 2
formation of the guide-target RNA scaffold substantially forms one or more structural features selected from the group consisting of: a bulge, an internal loop, and a hairpin
Implementation Method 3
the engineered guide RNA facilitates RNA editing of one or more target adenosines in the c.1 TIS of the target MAPT RNA by an RNA editing entity
Data Source
AI summary
Disclosed herein are engineered guide RNAs and compositions comprising the same for treatment of diseases or conditions in a subject. Also disclosed herein are methods of treating diseases or conditions in a subject by administering engineered guide RNAs or pharmaceutical compositions described herein.