Engineered Arenavirus Vectors for Genetic Stability
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Solution Overview
Problem
Current replicating viral vector systems, such as arenaviruses, face challenges in achieving high production yields and genetic stability, particularly when tri-segmented designs like r3LCMVrev and artLCMVrev exhibit poor growth and instability, respectively, due to issues with segment recombination and promoter control, which affects their suitability for industrial production and safety.
Innovation Solution
Engineered arenavirus particles with specific nucleotide sequences and genomic segments are designed to encode functional fragments of GP, NP, L, or Z proteins, separated across multiple mRNA transcripts, utilizing buffer peptides and optimized signal peptides to enhance genetic stability and transgene expression, while maintaining high titers and immune response elicitation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If arenavirus ORFs are separated over two or more mRNA transcripts to improve genetic stability, then genetic stability is improved, but device complexity increases
Solution Approach 1:
The arenavirus genome is divided into multiple separate mRNA transcripts, with each transcript containing specific open reading frames (ORFs). This segmentation prevents recombination between ORFs on the same transcript, thereby improving genetic stability while managing complexity through organized separation of viral components.
2Stability of the object's composition
If tri-segmented arenavirus designs are used to improve attenuation and safety, then genetic stability is improved, but productivity decreases due to poor growth
Solution Approach 1:
The patent applies local quality by assigning specific functional characteristics to different segments. The first mRNA transcript contains ORFs optimized for structural proteins, while the second transcript contains ORFs for non-structural proteins. This localized optimization allows each segment to be fine-tuned for both stability and growth performance.
Solution Approach 2:
The patent modifies parameters such as the 5' untranslated region (UTR) sequences and promoter elements in each mRNA transcript to optimize both genetic stability and viral growth. By adjusting these parameters locally in each segment rather than globally, the virus achieves improved stability without sacrificing productivity.
3Device complexity
If NP and GP ORFs are positioned next to 5' UTR to simplify genome structure, then device complexity is reduced, but transgene expression levels decrease
Solution Approach 1:
Instead of relying solely on the traditional 5' UTR positioning for gene expression control, the patent introduces a second dimension of regulation through the 3' UTR sequences and intergenic regions. This allows NP and GP ORFs to be positioned in configurations that simplify genome structure while maintaining high expression levels through optimized 3' end elements.
Data Source
AI summary
The present application relates to arenavirus particles containing a genome engineered such that an arenaviral open reading frame (“ORF”) is sequestered into two or more functional fragments and these fragments are expressed from two or more viral mRNA transcripts. The arenavirus particles described herein are genetically stable and provide high-level transgene expression. In certain embodiments, the arenavirus particles are tri-segmented. In particular, described herein is a nucleotide sequence comprising one or more ORFs comprising a nucleotide sequence encoding a functional fragment of arenavirus GP, NP, L or Z. Also described herein is an arenavirus particle containing a genome engineered such that an arenaviral ORF is sequestered into two or more functional fragments and these fragments are expressed from two or more viral mRNA transcripts. Also described herein is an arenavirus genomic or antigenomic segment engineered such that the transcription thereof results in one or more mRNA transcripts comprising a nucleotide sequence encoding a functional fragment of arenavirus GP, NP, L or Z. The arenavirus particles described herein may be suitable for vaccines and/or treatment of diseases and/or for the use in immunotherapies.