Exon-Skipping via Dual AON Targeting of ESE Sites
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Solution Overview
Problem
Current exon skipping methods using single anti-sense oligonucleotides (AONs) are inefficient for skipping certain exons, particularly dystrophin exon 45, as they often result in low or zero skipping efficiency due to the presence of multiple independent exonic splicing enhancer (ESE) sites, which can still allow exon inclusion.
Innovation Solution
Employing a combination of two or more exon-internal AONs that target multiple independent ESE sites on the same exon, preventing SR protein binding and enhancing exon skipping efficiency by using a method that includes hybridizing AONs with non-overlapping sequences to block all ESE sites, thereby increasing the exclusion of targeted exons from mature mRNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If a single anti-sense oligonucleotide (AON) is used to target an exon, then the method is simple and easy to implement, but the exon skipping efficiency is low or zero due to multiple independent ESE sites
Solution Approach 1:
The patent divides the targeting strategy into multiple independent AONs, each targeting a specific ESE site within the exon. Instead of relying on a single AON to block all ESE sites, the method segments the task across multiple oligonucleotides that collectively cover all independent ESE sites, thereby achieving high skipping efficiency while maintaining experimental simplicity
Solution Approach 2:
The patent changes the parameter of oligonucleotide concentration by using equimolar combinations of multiple AONs. This parameter change ensures that all ESE sites are simultaneously blocked with adequate oligonucleotide coverage, overcoming the limitation of single AON approaches without significantly complicating the protocol
2Reliability
If multiple AONs are used to target multiple ESE sites, then the exon skipping efficiency is significantly increased, but the complexity of the method increases
Solution Approach 1:
The patent establishes a universal approach for exon skipping that can be applied to any exon with multiple ESE sites. The method uses a standardized protocol of combining multiple AONs in equimolar ratios, making the approach universally applicable across different exons and genes without requiring custom optimization for each case
Solution Approach 2:
The patent merges multiple individual AON functions into a single combined treatment protocol. By combining several AONs that target different ESE sites and applying them together in equimolar concentrations, the method achieves synergistic exon skipping effects while simplifying the overall experimental workflow
3Quantity of substance
If a single AON targets a single ESE site, then the oligonucleotide concentration can be kept low, but the exon exclusion level is insufficient due to remaining functional ESE sites
Solution Approach 1:
The patent segments the ESE blocking task across multiple AONs, allowing each oligonucleotide to be used at lower concentrations while collectively achieving complete ESE site coverage. This segmentation enables effective exon skipping without requiring high concentrations of any single AON, reducing potential off-target effects
Solution Approach 2:
The patent changes the concentration parameter by using equimolar combinations of multiple AONs at moderate concentrations. This parameter change ensures adequate coverage of all ESE sites while avoiding the need for high concentrations that might cause non-specific effects or increase cost
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
Significantly increases the efficiency and robustness of exon skipping by ensuring higher exclusion levels and longer duration of exon exclusion, improving upon single AON methods by targeting multiple ESE sites, which are often independent and require simultaneous blocking to achieve effective skipping.
Implementation Method 1
The small molecules are thought to act by hybridising to specific locations on the pre-mRNA. The hybridisation is thought to interfere with either the enzymatic process of splicing or the recognition of exons.
Data Source
Figure 1A
Figure 1A
Figure 1B~1E
AI summary
In the present invention means and method are provided for optimising exon- skipping using exon-internal AON. We show that skipping efficiencies are improved by targeting putative splicing regulatory sequences (ESEs) within an exon. Such double targeting may be particularly useful for exons with which efficient skipping was difficult to obtain prior to the invention.