Influenza A Virus Artificial Intron Insertion for Expanded Coding Capacity
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Solution Overview
Problem
The existing literature lacks a clear consensus on the range or limits of viral RNA splicing across the Orthomyxoviridae family, particularly in non-splicing segments, and the effects of additional splicing on viral biology remain unclear, with existing recombinant viruses showing poor replication when splicing is introduced in certain segments.
Innovation Solution
The introduction of artificial introns into non-splicing segments of influenza A virus (IAV) genomes, specifically designed to form splice sites and include a branch site, allowing for splicing and expression of reporter proteins with minimal impact on viral fitness, and the use of these recombinant viruses in screening assays and vaccine development.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If artificial introns are introduced into non-splicing segments of influenza virus, then protein coding capacity is expanded, but viral replication efficiency deteriorates
Solution Approach 1:
The patent applies parameter changes by carefully designing the artificial intron structure with specific parameters: intron length (50-200 nucleotides), branch site position (20-50 bases downstream of 5' end), and splice site sequences (5' GU and 3' AG). By optimizing these parameters, the patent achieves a balance between expanding protein coding capacity through alternative splicing and maintaining viral replication efficiency, resolving the technical contradiction between versatility and productivity.
2Adaptability or versatility
If splicing is introduced in non-splicing segments, then genetic space is expanded, but viral fitness deteriorates
Solution Approach 1:
The patent applies local quality by introducing splicing capability only in specific non-splicing segments (such as segments 3, 4, 5, or 6) while leaving other segments unchanged. The artificial intron is precisely positioned within the segment to enable alternative splicing that expands genetic space without disrupting essential viral functions, thereby maintaining viral fitness while achieving genetic expansion.
Solution Approach 2:
The patent uses partial action by implementing splicing in only certain non-splicing segments rather than all segments. This selective approach allows the virus to expand its genetic space through alternative splicing in specific locations while avoiding the fitness costs that would result from introducing splicing throughout the entire genome.
3Adaptability or versatility
If multiple proteins are produced from single segment, then coding capacity increases, but splicing regulation complexity increases
Solution Approach 1:
The patent applies segmentation by dividing the viral genome into distinct segments with specific splicing characteristics. By placing artificial introns in specific segments and using segment-specific splicing regulation, the patent enables multiple proteins to be produced from single segments without creating a single complex regulatory system, thereby increasing coding capacity while managing splicing regulation complexity through modular segmental organization.
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 recombinant viruses with artificial introns demonstrate robust immune responses and stability, enabling the expansion of protein coding capacity and potential for superior vaccine platforms with minimal disruption to viral replication and disease causation.
Implementation Method 1
Nuclear replication enables viral access to another tool for diversifying their encoded proteins: the host cell splicing machinery, which can allow distinct proteins to be produced from a single transcript.
Data Source
AI summary
The present invention provides recombinant viral segments comprising an artificial intron, DNA constructs encoding these viral segments, and recombinant viruses comprising these viral segments. Also provided are methods of making and using the recombinant viruses described herein.