Modified Arterivirus Replicons for Tunable Multigene Expression
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Solution Overview
Problem
Existing methods are inadequate for efficiently expressing multiple heterologous polypeptides in heterologous expression systems, including multigenic expression systems for simultaneously producing multiple heterologous polypeptides in cells and/or host organisms.
Innovation Solution
Development of a nucleic acid molecule encoding a modified arterivirus genome or replicon RNA, devoid of certain sequences like ORF2a and ORF2b, with optimized subgenomic promoters and transcriptional termination signals, allowing for tunable expression of multiple heterologous genes in recombinant cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional viral expression vectors are used, then single gene expression is achieved, but simultaneous production of multiple heterologous polypeptides is limited
Solution Approach 1:
The arterivirus genome is segmented into multiple expression cassettes, each containing a subgenomic promoter and heterologous gene. This segmentation allows independent expression of multiple genes simultaneously, resolving the contradiction between single-gene simplicity and multi-gene functionality.
Solution Approach 2:
The modified arterivirus replicon system serves multiple functions: it can express multiple heterologous genes simultaneously, produce subgenomic RNAs, and facilitate complex protein interactions. This multi-functionality enables both high productivity and versatility in a single system.
2Adaptability or versatility
If complex multigenic expression systems are developed, then multiple heterologous polypeptides can be produced, but system complexity increases
Solution Approach 1:
Multiple expression cassettes are merged into a single arterivirus replicon genome, allowing coordinated expression of multiple genes from one integrated system. This reduces the complexity of managing separate expression systems while maintaining multi-gene capacity.
Solution Approach 2:
The subgenomic promoters within the replicon system allow dynamic and tunable expression levels for each heterologous gene. This dynamic control simplifies optimization compared to static complex systems, as expression levels can be adjusted independently for each gene cassette.
3Productivity
If existing viral vectors are modified for gene expression, then heterologous protein production is improved, but efficient simultaneous production of multiple polypeptides remains inadequate
Solution Approach 1:
Each expression cassette within the replicon is optimized with specific subgenomic promoters and structural elements tailored to its heterologous gene. This local optimization ensures consistent and reliable expression for each gene while maintaining overall system productivity for multiple polypeptides.
Data Source
AI summary
The present disclosure generally relates to viral-based expression systems suitable for the production of molecule of interests in recombinant host cells. The disclosure particularly relates to nucleic acid constructs, such as expression vectors, containing a modified arterivirus genome or replicon RNA in which at least some of its original viral sequence has been deleted. Also included in the disclosure are viral-based expression vectors including one or more expression cassettes encoding heterologous polypeptides. In some embodiments, the expression cassettes are configured and positioned at defined locations on the viral genome so as to enable expression of the heterologous polypeptides in a tunable manner.