Unencapsidated dsRNA Production via Bacteriophage Capsid Co-expression
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Solution Overview
Problem
The chemical fragility of RNA, particularly single-stranded RNA, limits large-scale production due to high costs and low yields in vitro, and the semi-rigid nature of double-stranded RNA restricts encapsidation within viral capsid shells, hindering commercial development.
Innovation Solution
Co-expression of bacteriophage capsid proteins with dsRNA in microbial cells allows for the accumulation of unencapsidated dsRNA, which can be recovered in high quantities from cell lysates, overcoming the limitations of enzymatic and environmental degradation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If in vitro synthesis methods are used for large scale production of RNA, then production capacity is increased, but costs increase significantly
Solution Approach 1:
The invention extracts and removes the problematic single-stranded RNA intermediate from the production system by using production strains lacking endogenous RNAses. This prevents degradation of the dsRNA product during in vivo production, enabling cost-effective large scale production without requiring expensive in vitro synthesis methods
Solution Approach 2:
The invention changes the biochemical parameters of the production system by using engineered bacterial strains with modified nuclease activity (lacking endogenous RNAses). This parameter change allows in vivo production of dsRNA to achieve both high productivity and low cost, resolving the contradiction between production capacity and cost
2Quantity of substance
If in vivo production methods are used for RNA, then costs are reduced, but yields are low and processing requirements are complex
Solution Approach 1:
The invention converts the potentially harmful effect of endogenous RNAses into a benefit by using production strains that lack these enzymes. This prevents RNA degradation during in vivo production, dramatically increasing yield while maintaining low costs and simplifying processing requirements
Solution Approach 2:
By changing the nuclease activity parameter of the production strain (using strains lacking endogenous RNAses), the invention achieves high yields of intact dsRNA through in vivo production, making the process both economical and efficient without complex processing
3Reliability
If dsRNA is encapsidated within viral capsid shells, then degradation is reduced, but the range of encapsidatable dsRNA is limited by capsid interior diameter
Solution Approach 1:
The invention extracts and removes the constraint of capsid encapsidation by using production strains lacking endogenous RNAses. This allows dsRNA of any length to be produced and purified without degradation, eliminating the need for encapsidation and expanding the range of encapsidatable dsRNA to include very long molecules
Solution Approach 2:
Instead of using encapsidation to protect dsRNA from degradation, the invention inverts the approach by using engineered production strains that prevent degradation at the source. This allows production of dsRNA of any length without the size constraints imposed by capsid interior diameter
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
This method significantly increases the yield of unencapsidated dsRNA, reducing degradation and enabling more aggressive purification processes, thus facilitating commercial production for gene suppression and other applications.
Implementation Method 1
Dimers of bacteriophage capsid proteins such as those of the leviviruses MS2 or Qo recognize and bind with affinity to cognate pac sequences
Data Source
AI summary
The invention provides methods and compositions for improved production of large quantities of unencapsidated double strand RNA (dsRNA) in vivo. The disclosed methods and compositions, comprising co-expression of genes encoding orotate phosporibosyl transferase, bacteriophage coat protein and dsRNA produce a significant improvement over current in vivo methods of producing unencapsidated dsRNA.