P-Protein Mutations for Temperature-Controlled RNA Virus Elimination
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Solution Overview
Problem
Existing negative-strand RNA viruses or virus vectors, such as Sendai virus, lack effective methods to eliminate them from infected cells while maintaining infection and gene expression, posing safety concerns.
Innovation Solution
A temperature-sensitive negative-strand RNA virus or virus vector with specific amino acid mutations in the P protein, optionally combined with mutations in the L protein, allows for controlled elimination by adjusting culture temperature.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
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 safety deteriorates due to inability to eliminate viruses from infected cells
Solution Approach 1:
The patent applies parameter changes by introducing temperature-sensitive mutations in the P protein (D433, R434, K437 positions) that alter the virus's stability parameter. At permissive temperatures (34-37°C), the virus maintains normal function for gene transfer. At non-permissive temperatures (39-42°C), the mutations cause viral protein dysfunction and virus elimination, thus changing the virus's stability parameter based on temperature conditions.
Solution Approach 2:
The patent implements dynamics by creating a temperature-controlled system where the virus's functional state can be dynamically switched. The P protein mutations enable the virus to transition between a stable, infectious state at lower temperatures and an unstable, non-infectious state at higher temperatures, allowing dynamic control over virus persistence in infected cells.
2Object-affected harmful factors
If temperature-sensitive mutants with P protein mutations are constructed to eliminate viruses, then virus elimination capability is improved, but gene expression efficiency deteriorates due to reduced viral stability
Solution Approach 1:
The patent applies periodic action through a two-stage temperature protocol. First, cells are infected and maintained at permissive temperature (34-37°C) to allow efficient gene transfer and initial gene expression. Then, temperature is shifted to non-permissive range (39-42°C) to eliminate the virus. This periodic temperature change enables both high gene expression efficiency during the first stage and effective virus elimination during the second stage.
Solution Approach 2:
The patent implements preliminary action by maintaining permissive temperature conditions during the initial infection and gene expression phase, allowing the virus to efficiently perform its gene transfer function before the elimination phase begins. This preliminary period of stable viral function ensures high gene expression efficiency before temperature shift triggers virus elimination.
3Object-affected harmful factors
If multiple amino acid substitutions are introduced in the P protein to enhance temperature sensitivity, then virus elimination effectiveness is improved, but viral replication capability deteriorates
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions at positions D433, R434, and K437 in the P protein. These substitutions change the thermal stability parameter of the viral proteins. At permissive temperatures, the mutated proteins remain functional and support viral replication. At non-permissive temperatures, the same mutations cause protein misfolding or instability, leading to effective virus elimination without requiring complete loss of replication capability.
Data Source
AI summary
The present invention provides a temperature-sensitive negative-strand RNA virus or virus vector and an RNA genome thereof. According to the invention, provided is a negative-strand RNA virus or virus vector having a negative-strand RNA genome wherein the phosphoprotein (P protein) on the RNA genome has an amino acid mutation(s) corresponding to a substitution(s) in an amino acid(s) of the P protein corresponding to one or more or all of D433, R434, and K437 and optionally further has an amino acid mutation corresponding to a further amino acid substitution in an amino acid of the P protein corresponding to L511.