Double Trans-Splicing Molecule for Precise Exon Replacement
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current RNA repair strategies, such as classical gene therapy, often fail to precisely correct dominant mutations in genetic disorders due to the loss of regulatory sequences, whereas trans-splicing technologies have limitations in efficiently replacing exonic sequences within target mRNAs, particularly for conditions like Duchenne muscular dystrophy.
Innovation Solution
Development of a double trans-splicing molecule that contains both 3′ and 5′ splice regions, along with target binding domains, to facilitate concomitant 3′ and 5′ trans-splicing reactions, enabling the precise replacement of mutated exons with normal sequences within the dystrophin gene, thereby preserving regulatory elements and achieving high efficiency in correcting genetic defects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If classical gene therapy is used to correct genetic mutations, then genetic defects can be addressed, but regulatory sequences are lost and precision in correcting dominant mutations is poor
Solution Approach 1:
The invention extracts only the necessary coding sequences (exons) for correction while leaving the regulatory sequences (introns, 5' UTR, 3' UTR) intact in the target pre-mRNA. The trans-splicing molecule delivers only the corrected exon sequences to replace mutated ones, thereby avoiding loss of regulatory elements that would occur with complete gene replacement approaches.
Solution Approach 2:
The invention applies local quality by targeting specific mutated exons for correction while preserving the rest of the pre-mRNA structure including regulatory sequences. The trans-splicing molecule is designed with specific target binding domains that recognize and bind to flanking intronic sequences adjacent to the mutated exon, enabling localized correction without affecting other parts of the gene.
2Manufacturing precision
If trans-splicing technology is used to replace exonic sequences, then precision in exon replacement can be achieved, but efficiency in replacing exonic sequences within target mRNAs is limited
Solution Approach 1:
The invention merges multiple functional elements into a single trans-splicing molecule: target binding domains (antisense sequences) that recognize flanking intronic regions, splice donor/acceptor sites for trans-splicing reactions, and the corrected exon sequence to be inserted. This integrated design enables simultaneous achievement of precise targeting and efficient replacement in a single molecular construct.
Solution Approach 2:
The trans-splicing molecule acts as an intermediary that facilitates the replacement of mutated exons by providing both the corrected sequence and the necessary splice sites. It mediates the interaction between the spliceosome machinery and the target pre-mRNA, enabling efficient trans-splicing reactions that replace mutated exons with corrected sequences at high precision and improved efficiency.
3Device complexity
If simple trans-splicing is used to correct mutations, then the process is simpler, but it cannot replace whole nucleotide sequences such as exonic sequences while preserving tissue specificity
Solution Approach 1:
The invention segments the trans-splicing molecule into distinct functional modules: 5' target binding domain, 5' splice donor site, corrected exon sequence, 3' splice acceptor site, and 3' target binding domain. This segmentation allows the molecule to perform complex functions (replacing entire exons while preserving tissue specificity) while maintaining a relatively simple overall structure that can be designed and synthesized straightforwardly.
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 double trans-splicing approach achieves an efficiency of up to 53% in repairing transcripts, with no non-specific end products, effectively addressing the limitations of existing RNA repair strategies by precisely correcting genetic mutations while maintaining tissue specificity and regulatory sequences.
Implementation Method 1
The compositions of the invention provide methods and compositions for generating novel nucleic acid molecules through targeted spliceosome mediated simple or double trans-splicing
Implementation Method 2
Introns are removed from pre-mRNAs in a precise process called cis-splicing. Splicing takes place as a coordinated interaction of several small nuclear ribonucleoprotein particles (snRNPs) and many protein factors that assemble to form an enzymatic complex known as the spliceosome
Data Source
AI summary
The present invention provided methods and compositions for generating novel nucleic acid molecules through targeted spliceosome mediated simple or double trans-splicing. The compositions of the invention include pre-trans-splicing molecules (PTMs) designed to interact with a target precursor messenger RNA molecule (target pre-mRNA) and to mediate a simple or double trans-splicing reaction resulting in the generation of a novel chimeric RNA molecule (Chimeric RNA).