USH2A Exon 13 Skipping snRNA for Precise Pre-mRNA Splicing
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Solution Overview
Problem
Conventional gene therapy delivery methods struggle to effectively target and modify the large coding sequence of the USH2A gene, particularly Exon 13, due to packaging limitations, and existing approaches for Exon skipping are inefficient or risky, leading to potential frameshift mutations.
Innovation Solution
Development of snRNA that targets USH2A pre-mRNA, specifically designed with a recognition domain complementary to Exon 13, to induce splicing skipping using U7-snRNP, which is modified to enhance binding and safety, reducing the risk of double skipping and ensuring efficient Exon 13 exclusion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional gene therapy delivery methods are used to target USH2A gene, then the large coding sequence can be delivered, but packaging limitations prevent effective targeting and modification
Solution Approach 1:
The patent extracts only the critical Exon 13 region (642 bp) from the entire USH2A coding sequence for targeted modification, rather than attempting to deliver or modify the complete large gene. This extraction approach overcomes packaging limitations by focusing therapeutic action on the specific mutated exon causing disease, while leaving the rest of the gene intact.
Solution Approach 2:
The patent segments the USH2A gene into individual exons for targeted treatment, specifically isolating Exon 13 as the therapeutic target. This segmentation allows precise modification of only the problematic exon containing pathogenic mutations, avoiding the need to handle the entire large coding sequence and its packaging constraints.
2Measurement precision
If existing Exon skipping approaches are used, then Exon 13 can be targeted, but efficiency is low and frameshift mutations may occur
Solution Approach 1:
The patent introduces snRNA as an intermediary molecule that mediates Exon 13 skipping by binding to the pre-mRNA and recruiting splicing factors. This intermediary approach provides more precise control over the splicing process compared to direct antisense oligonucleotide methods, reducing off-target effects and frameshift mutations while improving skipping efficiency.
Solution Approach 2:
The patent modifies the U7 snRNA structure by changing its 5' region sequence to be complementary to USH2A Exon 13, and optimizing the Sm binding site sequence. These parameter changes in the snRNA molecular structure enable specific recognition of the target exon and enhance binding affinity, thereby improving skipping precision and reliability.
3Productivity
If U7 snRNA is modified to enhance binding, then therapeutic efficacy improves, but potential off-target effects may increase
Solution Approach 1:
The patent applies local quality by making only the 5' region of U7 snRNA complementary to the specific USH2A Exon 13 sequence, while keeping other regions of the snRNA structure intact for proper function. This localized modification ensures high specificity for the target exon, enhancing therapeutic efficacy while minimizing off-target effects through precise spatial restriction of the binding domain.
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 snRNA effectively induces splicing skipping of Exon 13, producing a functional Usherin protein without frameshift mutations, addressing Usher syndrome and related retinitis pigmentosa by enhancing therapeutic efficacy and safety.
Implementation Method 1
The snRNA comprises a recognition domain that is reversely complementary to the USH2A pre-mRNA sequence. The snRNA binds to the USH2A pre-mRNA to induce splicing skipping of Exon 13.
Data Source
AI summary
Provided is snRNA which targets USH2A pre-mRNA. The recognition domain of the snRNA is the reverse complement of a USH2A pre-mRNA sequence. The snRNA binds to the USH2A pre-mRNA to splice and jump exon 13. The snRNA promotes a higher exon 13 skipping efficiency than AON.