Alphavirus Replicons Using IRES for Cap-Independent Translation
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Solution Overview
Problem
Current alphavirus vectors lack control over the expression of heterologous nucleic acid sequences at the level of protein translation, leading to inefficient protein expression due to cap-dependent translation inhibition during viral infections, which limits the regulation of protein synthesis and vector performance.
Innovation Solution
Development of recombinant alphavirus replicon and helper vectors that utilize an internal ribosome entry sequence (IRES) element to direct cap-independent translation, allowing for controlled expression of heterologous nucleic acid sequences by positioning the IRES downstream of the alphavirus subgenomic promoter and upstream of the coding sequence, thereby bypassing cap-dependent translation mechanisms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cap-dependent translation is used for protein expression, then translation efficiency is high under normal conditions, but protein synthesis is inhibited during viral infections due to eIF2 phosphorylation and eIF4F loss
Solution Approach 1:
The patent introduces an IRES element as an intermediary mechanism between the mRNA and ribosome. This IRES element acts as a mediator that enables cap-independent translation initiation, allowing the system to bypass the compromised cap-dependent pathway during viral infections and maintain protein expression under stress conditions
Solution Approach 2:
The patent changes the translation initiation parameter from cap-dependent to cap-independent by incorporating the IRES element. This parameter change allows the translation system to function differently under viral infection conditions, where eIF2 phosphorylation and eIF4F loss would normally inhibit cap-dependent translation
2Productivity
If alphavirus vectors are used for protein expression, then vector replication is efficient, but control over heterologous nucleic acid expression at the translation level is lacking
Solution Approach 1:
The patent segments the alphavirus vector into distinct functional components: the alphavirus genome for replication, the IRES element for translation initiation, and the heterologous nucleic acid for protein expression. This segmentation allows independent optimization of each function while maintaining overall system performance
Solution Approach 2:
The IRES element serves multiple functions: it enables cap-independent translation, provides stress-condition stability, and allows controlled expression of heterologous proteins. This multi-functionality addresses the lack of translation control capability while maintaining vector replication efficiency
3Productivity
If heterologous nucleic acid sequences are expressed using conventional alphavirus vectors, then protein production occurs, but expression levels are reduced due to cap-dependent translation inhibition during infection
Solution Approach 1:
The patent converts the harmful effect of cap-dependent translation inhibition during viral infections into a benefit by using the IRES element. The IRES element exploits the stress-condition environment (where cap-dependent translation is inhibited) to achieve stable protein expression, turning the harmful viral infection effect into a favorable condition for maintaining protein production
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 approach enables regulated and efficient expression of heterologous proteins by minimizing the reliance on cap-dependent translation, reducing the negative impact of viral infections on protein synthesis and enhancing the performance of alphavirus vectors by maintaining protein expression levels even under stress conditions.
Implementation Method 1
IRES elements bypass cap-dependent translation inhibition; thus the translation directed by an IRES element is termed 'cap-independent.'
Data Source
AI summary
The present invention provides a recombinant nucleic acid comprising: a first nucleic acid sequence encoding a 5′ alphavirus replication recognition sequence; at least one second nucleic acid sequence encoding an alphavirus nonstructural protein; at least one alphavirus subgenomic promoter; at least one IRES element; at least one heterologous nucleic acid; and a third nucleic acid encoding a 3′ alphavirus replication recognition sequence. Further provided are methods of making alphavirus particles comprising a recombinant nucleic acid of this invention and methods of using the compositions of this invention. Also provided is a recombinant helper nucleic acid comprising: a first nucleic acid sequence encoding a 5′ alphavirus replication recognition sequence; an alphavirus subgenomic promoter; an IRES element; a second nucleic acid encoding an alphavirus structural protein; and a third nucleic acid encoding a 3′ alphavirus replication recognition sequence.