Exon 44 Skipping Oligonucleotides for DMD Splicing Specificity
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Solution Overview
Problem
Current treatments for Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD) using antisense-mediated exon skipping face challenges in efficiently skipping exon 44 of the DMD gene without affecting conserved splice sites or inducing immunogenicity, and in achieving optimal RNA binding kinetics and thermodynamic properties.
Innovation Solution
A method involving molecules that bind to specific nucleotide sequences within exon 44, such as SEQ ID NO. 1, 2, 3, or 4, to modulate splicing, using oligonucleotides that are designed to avoid CpG pairs and G-quartet motifs, ensuring efficient skipping of exon 44 while minimizing immunogenicity and optimizing RNA binding properties.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If antisense oligonucleotides target conserved splice sites to induce exon skipping, then exon skipping efficiency is improved, but specificity deteriorates due to co-targeting of additional exons
Solution Approach 1:
The patent applies local quality by transitioning from targeting conserved sequences (uniform quality across all splice sites) to targeting exon-specific non-conserved sequences (differentiated quality for each target). The AONs are designed to bind to unique sequences within exon 44 that do not exist in other exons, thereby achieving high specificity while maintaining skipping efficiency.
Solution Approach 2:
The patent segments the target space by identifying four distinct non-overlapping regions within exon 44 (SEQ ID NO:1-4), each with unique sequences. This segmentation allows selection of region-specific AONs that will not cross-react with other exons, resolving the specificity issue while maintaining efficiency through optimized binding to each segmented region.
2Productivity
If oligonucleotides are designed for high binding affinity to exon 44, then skipping efficiency is improved, but immunogenicity increases due to CpG pairs and G-quartet motifs
Solution Approach 1:
The patent extracts and removes harmful immunogenic elements (CpG pairs and G-quartet motifs) from the oligonucleotide sequence while retaining the essential binding functionality. By carefully selecting sequences from four regions of exon 44 and excluding those containing immunogenic motifs, the patent achieves high skipping efficiency without triggering immune responses.
Solution Approach 2:
The patent changes the chemical and sequence parameters of the oligonucleotides by selecting specific sequences that optimize RNA binding kinetics and thermodynamic properties while eliminating immunogenic features. This parameter optimization allows maintenance of high binding affinity and skipping efficiency without the harmful side effects of CpG pairs and G-quartet structures.
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 method achieves highly efficient skipping of exon 44, restoring the reading frame in DMD patients, producing functional dystrophin protein, and reducing aberrant dystrophin production, thereby alleviating muscle cell characteristics associated with DMD and improving muscle function.
Implementation Method 1
a molecule that binds to a nucleotide sequence comprising SEQ ID NO.1: 5'-GUGGCUAACAGAAGCU; SEQ ID NO.2: 5'-GGGAACAUGCUAAAUAC, SEQ ID NO.3: 5'-AGACACAAAUUCCUGAGA, or SEQ ID NO.4: 5'-CUGUUGAGAAA. This molecule preferably binds or is complementary to any of SEQ ID NO: 1, 2, 3, or 4 when SEQ ID NO:1, 2, 3, or 4 is present within exon 44 of the DMD pre-mRNA.
Data Source
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AI summary
The invention relates to a nucleic acid molecule that binds and/or is complementary to the nucleotide molecule having sequence 5'-GUGGCUAACAGAAGCU (SEQ ID NO 1) and to its use in a method for inducing skipping of exon 44 of the DMD gene in a DMD patient.