Mixmer Oligonucleotide Design for Precise ADAR RNA Editing
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Solution Overview
Problem
Current RNA editing techniques for diseases like alpha-1 antitrypsin deficiency (A1AD) are limited in their ability to efficiently correct specific genetic mutations, such as the E342K mutation in the SERPINA1 gene, using endogenous ADAR enzymes.
Innovation Solution
Development of oligonucleotides with a mixmer structure comprising an editing region, a 5′ mixmer region, and a 3′ mixmer region, which are designed to guide ADAR enzymes to specific adenosines in target nucleic acids for precise editing, including the use of conjugates and pharmaceutical compositions for delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If guide oligonucleotides are used to recruit ADAR enzymes to specific target sites, then editing specificity is improved, but editing efficiency is insufficient for severe mutations like E342K in SERPINA1
Solution Approach 1:
The patent employs a composite oligonucleotide structure combining a guide region for ADAR recruitment with a passivation region containing modified nucleosides (2'-O-methyl, 2'-fluoro, or LNA) that are complementary to the target sequence. This composite design simultaneously achieves high editing specificity through the guide region while the passivation region enhances binding affinity and protects the oligonucleotide from degradation, thereby improving overall editing efficiency for severe mutations.
2Object-generated harmful factors
If endogenous ADAR enzymes are used for RNA editing, then ectopic protein expression is avoided, but the editing capability for severe mutations is limited
Solution Approach 1:
The patent uses an engineered guide oligonucleotide as an intermediary that recruits endogenous ADAR enzymes to specific target sites in SERPINA1 mRNA. The oligonucleotide contains a guide region that base-pairs with the target sequence and a passivation region that enhances stability and binding. This intermediary approach enables precise delivery of endogenous ADAR activity to severe mutation sites without requiring exogenous enzyme expression, thus avoiding ectopic protein expression while enhancing editing capability.
3Device complexity
If conventional oligonucleotide structures are used, then simplicity is maintained, but stability and editing performance are insufficient
Solution Approach 1:
The patent applies local quality modifications by incorporating specific nucleoside modifications (2'-O-methyl, 2'-fluoro, or LNA) only in the passivation region of the oligonucleotide, while maintaining standard nucleosides in the guide region. This localized modification approach enhances the stability and binding affinity of the oligonucleotide at critical positions without complicating the overall structure, thereby improving reliability while preserving simplicity.
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 oligonucleotides demonstrate enhanced editing efficiency and specificity in correcting genetic mutations, particularly in SERPINA1 mRNA, thereby potentially treating A1AD effectively.
Implementation Method 1
a guide oligonucleotide forming complementary base pairs with the target nucleic acid, thereby forming a double-stranded molecule to which an ADAR can bind
Implementation Method 2
An ADAR binds to double-stranded RNA and deaminates an adenine nucleobase to form a hypoxanthine nucleobase, thereby converting an adenosine (A) nucleoside to an inosine (1) nucleoside
Data Source
AI summary
The present invention relates to oligonucleotides for editing a target nucleic acid, as well as conjugates, salts and pharmaceutical compositions thereof. The invention also relates to uses of such oligonucleotides, conjugates, salts and pharmaceutical compositions in methods for editing target nucleic acids and in medical uses and methods of treatment of disease.