Bifunctional Oligonucleotides for Alternative Splice Site Modulation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods are inadequate for effectively targeting and modulating alternative splicing pathways to address disease-associated splicing patterns, particularly in tissue-specific or development stage-specific contexts, which are crucial for treating conditions like Huntington's disease.
Innovation Solution
Development of bifunctional oligonucleotides that can bind to alternative splice sites and recruit spliceosome components to modulate splicing, thereby regulating the production of transcription products and proteins, including those associated with diseases like Huntington's disease.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional single-function oligonucleotides are used to target splice sites, then binding to alternative splice sites is achieved, but recruitment of spliceosome components and modulation of splicing is insufficient
Solution Approach 1:
The patent combines two distinct functions into a single oligonucleotide molecule: (1) binding to alternative splice sites through a splice site targeting sequence, and (2) recruiting spliceosome components through a separate spliceosome targeting sequence. This merging of functions resolves the technical contradiction by achieving reliable splicing modulation without requiring multiple separate agents, thereby improving effectiveness while maintaining manageable complexity through integrated design.
Solution Approach 2:
The bifunctional oligonucleotide is designed to perform multiple functions simultaneously: it acts as both a binding agent for alternative splice sites and a recruiting agent for spliceosome components. This multi-functionality allows a single molecule to modulate splicing pathways effectively, addressing the contradiction between achieving reliable splicing modulation and the complexity of implementing multiple separate mechanisms.
2Reliability
If bifunctional oligonucleotides are designed with both splice site targeting and spliceosome recruitment sequences, then splicing modulation is enhanced, but oligonucleotide design complexity increases
Solution Approach 1:
The oligonucleotide is segmented into distinct functional regions: a splice site targeting sequence (5-50 nucleotides) that binds to alternative splice sites and a spliceosome targeting sequence (5-50 nucleotides) that recruits spliceosome components. This segmentation allows each region to be optimized independently for its specific function while maintaining overall manufacturability through standardized synthesis protocols for modular sequences.
Solution Approach 2:
The patent specifies parameter ranges for the oligonucleotide sequences, including length (5-50 nucleotides for each functional region) and positioning relative to the alternative splice site. These parameter changes provide design flexibility that simplifies manufacturing by allowing optimization within defined ranges rather than requiring precise fixed values, thereby enhancing splicing modulation effectiveness while maintaining ease of manufacture.
3Reliability
If oligonucleotides bind to alternative splice sites in mutant genes with repeated trinucleotides, then disease-associated splicing patterns are targeted, but specificity and off-target effects become challenging to control
Solution Approach 1:
The splice site targeting sequence is designed with local quality by being positioned specifically 5' or 3' to the alternative splice site and containing specific sequence motifs (e.g., 5-25 nucleotides long with particular nucleotide compositions). This localized design ensures high specificity for disease-associated splice sites in mutant genes with repeated trinucleotides, thereby improving disease-specific targeting while controlling off-target effects through precise local sequence selection.
Solution Approach 2:
The bifunctional oligonucleotide acts as an intermediary between the alternative splice site and the spliceosome. By binding to the alternative splice site and simultaneously recruiting spliceosome components, it mediates the splicing process with high specificity. This intermediary role allows precise control over splicing outcomes at disease-associated sites while minimizing off-target effects through the specific sequence design and positioning of the oligonucleotide.
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 bifunctional oligonucleotides effectively regulate splicing at alternative splice sites, providing therapeutic potential for conditions such as Huntington's disease by altering the production of target proteins and RNA isoforms, thus offering a novel approach to treat neurological disorders.
Implementation Method 1
binding to a target sequence (e.g., an RNA, e.g., a pre-mRNA or mRNA) comprising an exonic element, such as an alternative splice site
Implementation Method 2
recruiting a spliceosome component... capable of modulating splicing of a target sequence, e.g., at an alternative splice site
Data Source
AI summary
The present disclosure features bifunctional oligonucleotides and related compositions that, inter alia, modulate nucleic CA acid splicing, e.g., splicing of a pre-mRNA, as well as methods of use thereof.