Allele-Specific Splice Switching Oligonucleotides for Pseudoexon Incorporation
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Solution Overview
Problem
Current technologies face challenges in efficiently incorporating pseudoexons into mature mRNA transcripts, which can be crucial for modulating gene expression and treating diseases.
Innovation Solution
The development of allele-specific splice switching oligonucleotides (SSOs) that target specific sequence parameters in genes with pseudoexons, allowing for the incorporation of pseudoexons into mature mRNA transcripts in a controlled manner.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If splice switching oligonucleotides are used to incorporate pseudoexons into mature mRNA, then gene expression can be modulated and disease-causing proteins inactivated, but the complexity of identifying suitable target sequences and designing effective SSOs increases
Solution Approach 1:
The patent establishes specific parameter ranges for pseudoexon characteristics (length 30-300 nt, MaxEnt scores within defined ranges, pyrimidine content 20-80%, guanine content 10-50%) and SSO binding regions (+9 to +39 downstream of 5' splice site) to predict SSO effectiveness. These quantitative parameters provide objective criteria for identifying target sequences and designing SSOs, reducing the complexity of the design process while maintaining reliability of gene expression modulation.
Solution Approach 2:
The patent performs preliminary computational analysis of gene sequences to identify pseudoexons meeting specific criteria before SSO design. By pre-screening for pseudoexons with appropriate MaxEnt scores, lengths, and nucleotide compositions, and identifying suitable binding regions downstream of 5' splice sites, the method prepares target candidates in advance, streamlining the subsequent SSO design and validation process.
2Object-affected harmful factors
If allele-specific SSOs are designed to target specific alleles, then selective inactivation of disease-causing proteins can be achieved, but the precision required for allele-specific binding increases the difficulty of SSO design
Solution Approach 1:
The patent exploits local sequence variations (allele-specific nucleotides) within the pseudoexon or flanking regions to design SSOs with allele-specific binding. By focusing on localized differences rather than requiring genome-wide specificity, the method achieves selective targeting of disease alleles while minimizing off-target effects. The SSOs are designed to bind to specific nucleotide sequences that differ between alleles, providing precise discrimination.
3Productivity
If comprehensive sequence parameters are analyzed to identify suitable pseudoexons, then the success rate of SSO-mediated incorporation increases, but the computational and analytical workload increases
Solution Approach 1:
The patent performs preliminary computational screening of gene sequences using established algorithms (such as MaxEntScan for splice site strength prediction) to identify pseudoexons meeting predefined criteria before experimental validation. By filtering candidates in silico based on length, MaxEnt scores, and nucleotide composition, the method reduces the number of candidates requiring wet-lab testing, thereby reducing overall time and resource investment.
Solution Approach 2:
The patent defines specific parameter thresholds (pseudoexon length 30-300 nt, MaxEnt scores within defined ranges, pyrimidine content 20-80%, guanine content 10-50%, SSO binding region +9 to +39 downstream of 5' splice site) that can be rapidly evaluated using computational tools. These standardized parameters enable high-throughput screening of candidate pseudoexons, increasing the success rate of SSO-mediated incorporation while minimizing manual analysis time.
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 approach enables the modulation of gene expression by promoting the inclusion of pseudoexons, thereby inactivating or disrupting the function of disease-causing proteins, while minimizing effects on normal gene function.
Implementation Method 1
an SSO, in vivo, hybridizes to the pre-mRNA within the region +9 to +39 downstream to the 5′ splice site of said pseudoexon
Data Source
AI summary
The present invention relates to a method for identifying splice switching oligonucleotides (SSOs) able to modulate expression of a target protein in a cell by promoting incorporation of a pseudoexon into the mature mRNA upon binding to the pre-mRNA in the region +9 to +39 downstream to the 5′ splice site of said pseudoexon. The invention also relates to SSOs obtained by said method and uses thereof. In particular, the present invention relates to allele specific splice switching oligonucleotides (SSOs).