USH2A Exon 13 Skipping to Bypass AAV Gene Size Limits
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Solution Overview
Problem
Current gene therapy approaches for USH2A-associated retinal and cochlear degeneration, particularly for Usher syndrome type II and autosomal recessive retinitis pigmentosa, are challenging due to the large size of the USH2A coding sequence exceeding the packaging capacity of commonly used AAV viral delivery vectors, and there is a need for a more effective method to restore the reading frame and functionality of the usherin protein.
Innovation Solution
A CRISPR/Cas9-based exon-skipping approach is employed to delete exon 13 of the USH2A gene, using dual-guide RNAs to introduce double-strand breaks in introns 12 and 13, facilitating the splicing of exons 12 and 14 in-frame, thereby restoring the reading frame and maintaining the edited gene under its endogenous regulatory elements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If gene therapy is used to treat USH2A-associated disorders, then the usherin protein function can be restored, but the large size of the USH2A coding sequence exceeds the packaging capacity of AAV viral delivery vectors
Solution Approach 1:
The patent extracts and removes exon 13 from the USH2A gene using CRISPR/Cas9-mediated genome editing. This extraction approach eliminates the problematic c.2299delG mutation while retaining the functional portions of the gene, allowing the usherin protein to be restored without requiring delivery of the entire large USH2A coding sequence via AAV vectors.
Solution Approach 2:
The patent segments the USH2A gene by targeting specific intronic regions (intron 12 and intron 13) with guide RNAs to create double-strand breaks. This segmentation allows precise removal of only the affected exon 13 while leaving the rest of the gene intact, solving the packaging capacity limitation by editing the genome in situ rather than delivering the full gene sequence.
2Reliability
If exon 13 is deleted to correct the reading frame, then the usherin protein functionality is restored, but the protein structure loses 4 of 8 Laminin EGF-like domains
Solution Approach 1:
The patent converts the harmful effect of exon 13 deletion (loss of 4 Laminin EGF-like domains) into a benefit by demonstrating that the remaining exons can still produce a functional usherin protein. The CRISPR-mediated deletion corrects the catastrophic frameshift mutation and premature stop codon, restoring the reading frame and enabling production of a truncated but functional protein that rescues the disease phenotype.
3Manufacturing precision
If dual-cut CRISPR approach is used to delete exon 13, then the reading frame is restored, but the complexity of the gene editing process increases
Solution Approach 1:
The patent uses guide RNAs as intermediaries to direct the Cas9 nuclease to specific locations in intron 12 and intron 13. These guide RNAs mediate the interaction between the editing machinery and the target DNA sequences, enabling precise dual-cutting to achieve exon 13 deletion and reading frame restoration while maintaining a relatively simple overall process architecture.
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
This method potentially rescues impaired hair cell structure and auditory function, offering a therapeutic strategy to partially restore sight and hearing in subjects with USH2A-associated disorders by ensuring proper usherin protein expression and function.
Implementation Method 1
A CRISPR/Cas9-based exon-skipping approach is employed to delete exon 13 of the USH2A gene, using dual-guide RNAs to introduce double-strand breaks in introns 12 and 13
Implementation Method 2
facilitating the splicing of exons 12 and 14 in-frame, thereby restoring the reading frame
Data Source
AI summary
Compositions for use in treating subjects with USH2A-associated retinal and/or cochlear degeneration that result from mutations in exon 13 of the USH2A gene by deletion of exon 13 from the USH2A gene or transcripts, and methods of use thereof, as well as genetically modified animals and cells.