Antisense Oligonucleotide RNA Editing With Endogenous ADAR Specificity
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Solution Overview
Problem
Existing RNA editing techniques require genetic modification or delivery of recombinant enzymes to target cells, leading to inefficiencies and challenges in therapeutic applications, particularly in multicellular organisms like humans, and suffer from promiscuous editing that targets multiple adenosines instead of specific ones.
Innovation Solution
A single-stranded antisense oligonucleotide (AON) that forms a double-stranded complex with target RNA, utilizing endogenous ADAR enzymes for specific adenosine deamination, with optional mismatches, wobbles, and bulges, and sugar modifications to enhance specificity and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If genetically engineered fusion proteins or recombinant enzymes are delivered to target cells, then RNA editing can be achieved, but the complexity of the system increases and therapeutic application becomes difficult
Solution Approach 1:
The patent extracts and utilizes only the essential endogenous ADAR enzyme activity needed for RNA editing, removing the need for complex genetically engineered fusion proteins. By designing AONs that can recruit and activate endogenous ADAR enzymes directly at the target site, the system eliminates the need for delivering entire recombinant enzyme complexes, thereby reducing system complexity while maintaining editing capability
Solution Approach 2:
The invention enables the cell's own endogenous ADAR enzymes to perform the editing function without requiring external delivery of recombinant enzymes. The AONs serve as guides that direct these self-existing cellular enzymes to the correct target sequences, allowing the system to utilize the cell's natural machinery rather than imposing complex external enzyme delivery systems
2Device complexity
If endogenous ADAR enzymes are utilized with simple AONs, then system complexity is reduced, but specificity may be compromised leading to promiscuous editing
Solution Approach 1:
The patent applies local quality by introducing specific chemical modifications at precise locations within the AON sequence. The 2′-O-methyl modifications are placed at specific positions to enhance binding stability and specificity at the target site, while 5′-phosphorothioate modifications provide localized protection against nucleases. This localized modification strategy enhances editing specificity without requiring complex system changes
Solution Approach 2:
The invention utilizes parameter changes by systematically varying the pattern and position of chemical modifications along the AON sequence. By adjusting the degree and location of 2′-O-methyl and phosphorothioate modifications, the patent optimizes both the specificity of target recognition and the stability of the AON, thereby achieving high editing specificity with a relatively simple system
3Measurement precision
If long guide RNAs (>50 nucleotides) are used for specific recognition, then editing specificity improves, but manufacturing difficulty and cell entry problems increase
Solution Approach 1:
The patent employs shorter AONs (15-50 nucleotides) with strategically placed chemical modifications that provide enhanced stability and specificity comparable to longer unmodified sequences. The 2′-O-methyl and phosphorothioate modifications effectively compensate for the reduced length, allowing the use of shorter, easier-to-manufacture oligonucleotides that maintain high target recognition specificity while improving manufacturability and cell delivery
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 AON achieves targeted RNA editing in human cells without genetic modification, reducing off-target effects and enhancing editing efficiency, applicable for treating various genetic disorders.
Implementation Method 1
A single-stranded antisense oligonucleotide (AON) that forms a double-stranded complex with target RNA
Implementation Method 2
utilizing endogenous ADAR enzymes for specific adenosine deamination
Data Source
AI summary
The invention relates to antisense oligonucleotides that are capable of bringing about specific editing of a target nucleotide (adenosine) in a target RNA in a eukaryotic cell, wherein said oligonucleotide does not, in itself, form an intramolecular hair-pin or stem-loop structure, and wherein said oligonucleotide comprises a cytidine (a non-complementary nucleotide) or a uridine in a position opposite to the target adenosine to be edited in the target RNA region.