Acceptor Splice Module for AAV Split-Gene Trans-Splicing
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Solution Overview
Problem
Existing gene therapy methods, particularly those using adeno-associated viral (AAV) vectors, face limitations due to packaging capacity and inefficient reconstitution of split coding sequences, as current rAAV dual vector systems rely on concatemerization and homologous recombination rather than mRNA splicing in trans, leading to low efficiency.
Innovation Solution
Development of a pre-mRNA trans-splicing molecule with a strong acceptor splice region, comprising a pyrimidine tract and a specific acceptor splice site, to facilitate efficient mRNA splicing and reconstitution of split coding sequences in trans, using AAV vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If rAAV dual vector systems use concatemerization and homologous recombination for reconstitution, then packaging capacity is reduced, but reconstitution efficiency remains low
Solution Approach 1:
The patent replaces the mechanical/genetic reconstitution mechanisms (concatemerization and homologous recombination) with an RNA splicing mechanism. By introducing acceptor splice sites and pyrimidine tracts into the split coding sequences, the system uses the cellular spliceosome machinery to join RNA transcripts, achieving high-efficiency reconstitution without relying on inefficient DNA-level recombination processes.
2Productivity
If split coding sequences are designed for trans-splicing, then reconstitution efficiency is improved, but sequence design complexity increases
Solution Approach 1:
The patent incorporates acceptor splice sites and pyrimidine tracts during the initial design of split coding sequences. These splicing elements are built into the sequence architecture from the outset, allowing the cellular machinery to automatically perform the joining function without requiring complex post-transcriptional manipulation or selection processes.
Solution Approach 2:
The patent optimizes specific sequence parameters including the composition of pyrimidine tracts (rich in C and T residues), the spacing between splice elements, and the consensus sequences of acceptor splice sites. These parameter optimizations enhance splicing efficiency while maintaining manageable sequence design complexity through adherence to established splicing motifs.
3Productivity
If strong acceptor splice sites are used, then mRNA splicing efficiency is enhanced, but sequence length increases
Solution Approach 1:
The patent extracts and utilizes only the essential splicing elements (acceptor splice site consensus sequence and pyrimidine tract) required for efficient splicing, omitting non-essential sequence portions. This focused approach incorporates strong splicing signals while minimizing the added sequence length impact.
Solution Approach 2:
The patent incorporates pyrimidine tracts with at least 60% pyrimidine bases (C and T), which may be longer than the absolute minimum required, to ensure strong splicing signals. This partial excess in pyrimidine content compensates for potential variability in splicing efficiency while keeping the overall sequence addition manageable.
Data Source
AI summary
The present invention relates to an acceptor splice region, as well as uses and applications thereof.