Split Viral Vector for Directed Evolution
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Solution Overview
Problem
Existing methods of directed evolution, particularly those using mutator polymerases and attenuated RNA viruses, suffer from low and non-dynamic mutation rates, leading to insufficient mutagenic diversity and genomic instability, which hampers the effective evolution of gene products with desirable characteristics.
Innovation Solution
The use of a split, non-competent viral vector system based on mutator RNA viruses, where the gene of interest is stably and recombinantly integrated into the viral vector, allowing for serial passaging and the accumulation of mutations in an indel and recombination-averse manner.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If mutator polymerases are used for directed evolution, then mutation rate is increased, but mutation rate remains low and non-dynamic
Solution Approach 1:
The patent implements dynamic mutation rates by introducing a conditional mutator polymerase system where the mutation rate can be adjusted based on experimental conditions. The mutator polymerase expression is controlled by inducible promoters or regulatory elements that allow the system to transition between low and high mutation rates, enabling adaptation to different evolutionary stages and selection pressures.
2Productivity
If attenuated RNA viruses are used with inserted gene of interest, then viral propagation is enabled, but genomic instability and viral recombination occur
Solution Approach 1:
The patent segments the viral genome into multiple components, with the gene of interest inserted into a non-essential region or as a separate complementing component. This segmentation isolates the gene of interest from critical viral genomic elements, reducing the risk of recombination while maintaining viral propagation capability through complementation or split-genome systems.
Solution Approach 2:
The patent introduces intermediary elements such as self-cleaving peptides (2A peptides), internal ribosome entry sites (IRES), or complementing RNA components that mediate the expression of the gene of interest without requiring its integration into the essential viral genome. These intermediaries allow viral propagation while protecting the gene of interest from recombination events.
3Adaptability or versatility
If split viral vector systems are used, then gene of interest integration is achieved, but system integrity is lost before variants can be fixed
Solution Approach 1:
The patent employs preliminary stabilization measures by designing the split viral vector system with pre-configured complementary components that ensure stable maintenance of the gene of interest during serial passaging. The system includes pre-established regulatory elements, packaging signals, and complementation mechanisms that prevent system degradation before beneficial variants can accumulate and become fixed in the population.
Data Source
AI summary
The present invention provides nucleic acids sequences, viral particles, viruses, vectors systems, host cells, kits, apparatus, and methods of evolution of a gene product of a gene of interest. The nucleic acid sequences of the invention have been developed primarily for use in evolution of biomolecules of interest. Using a split, non-competent viral vector, the gene of interest can be stably and recombinantly integrated into the viral vector via in-frame insertion of the open reading frame of the gene of interest with an aspect of the native but attenuated viral genome. This configuration of the viral genome, leveraging a split viral vector and an aspect of a viral factor that has been shown to robustly interact with the split viral vector, enables the serial passaging of the recombinant viral particle in an indel and recombination-averse manner.