Temperature-Sensitive RNA Viral Vector for Post-Infection Elimination
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Solution Overview
Problem
Existing negative-strand RNA viruses or virus vectors, such as Sendai virus, lack effective methods for controlled elimination from infected cells, posing safety concerns and limiting their application in regenerative medicine and gene transfer.
Innovation Solution
A temperature-sensitive negative-strand RNA virus or virus vector is developed with specific amino acid mutations in the P protein and optionally the L protein, allowing for controlled elimination by adjusting culture temperature, and further enhanced by fusion with bromotag for targeted degradation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional negative-strand RNA viruses or virus vectors are used for gene transfer, then gene transfer efficiency and gene expression efficiency are improved, but the ability to eliminate the virus or virus vector from infected cells is poor, posing safety concerns
Solution Approach 1:
The patent applies temperature-sensitive mutations to viral proteins (P protein with D433A/R434A/K437A mutations and L protein with N1197S/L1558I/K1796E mutations) that allow the virus to replicate efficiently at permissive temperatures (34°C) but become non-functional at restrictive temperatures (39°C). This dynamic temperature-dependent control enables the virus to be eliminated from cells by simply changing the culture temperature, resolving the contradiction between maintaining virus functionality for gene transfer and enabling virus elimination for safety.
2Adaptability or versatility
If temperature-sensitive mutants with P protein mutations are constructed to eliminate viruses, then virus elimination capability is improved, but gene transfer efficiency may be reduced
Solution Approach 1:
The patent employs a two-stage temperature control strategy: first, cells are infected and cultured at permissive temperature (34°C) to allow efficient virus replication and gene transfer; second, temperature is shifted to restrictive condition (39°C) to eliminate the virus after gene expression is achieved. This preliminary action of establishing infection under favorable conditions before switching to elimination conditions ensures both high gene transfer efficiency and effective virus clearance.
3Adaptability or versatility
If multiple amino acid mutations are introduced to increase temperature sensitivity, then virus elimination effectiveness is improved, but viral protein stability and function may be compromised
Solution Approach 1:
The patent introduces specific amino acid mutations at precisely defined positions in the viral proteins: D433A/R434A/K437A in the P protein and N1197S/L1558I/K1796E in the L protein. These localized mutations are strategically placed to confer temperature sensitivity without disrupting the overall structural stability and essential functions of the proteins. The mutations create temperature-dependent conformational changes that selectively affect viral replication machinery while preserving protein integrity at permissive temperatures.
Data Source
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AI summary
The present invention provides a temperature-sensitive negative-strand RNA virus or viral vector, and its RNA genome. According to the present invention, there is provided a negative-strand RNA virus or viral vector in which at least one of the viral proteins encoded by the genome (preferably the P protein or a temperature-sensitive P protein) is in the form of a fusion protein with a bromotag.