Tri-Segmented Arenavirus Genome Layout for Stable Transgene Expression
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Solution Overview
Problem
Existing arenaviruses with rearranged open reading frames (ORFs) tend to recombine into replication-competent bi-segmented particles, leading to attenuated growth and loss of transgene expression, posing challenges for stable genetic manipulation and vaccine development.
Innovation Solution
Engineering arenavirus genomic segments to carry ORFs in positions other than their wild-type positions, resulting in tri-segmented particles that maintain genetic stability and ensure lasting transgene expression, using arenavirus untranslated regions (UTRs) to control ORF expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If arenavirus ORFs are rearranged to create tri-segmented particles, then genetic stability and transgene expression are improved, but device complexity and manufacturing difficulty increase
Solution Approach 1:
The arenavirus genome is divided into three separate segments (L1, L2, and S) instead of the conventional two segments. This segmentation prevents recombination into replication-competent bi-segmented forms while maintaining viral functionality and enabling sustained transgene expression. Each segment contains specific ORFs in non-canonical positions, with L1 and L2 segments containing duplicated ORFs that cannot properly recombine.
2Reliability
If arenavirus ORFs are rearranged to prevent recombination, then reliability of transgene expression is improved, but ease of manufacture deteriorates
Solution Approach 1:
The invention changes the fundamental parameters of the arenavirus genome by creating tri-segmented particles with ORFs positioned in non-canonical locations. The L segments contain ORFs in alternative arrangements (e.g., L1 with NP-ORF and L-ORF in opposite orientations, L2 with duplicated ORFs), and the S segment contains GP and NP ORFs. These parameter changes ensure that recombination does not produce replication-competent viruses, thereby guaranteeing reliable and sustained transgene expression without reversion to wild-type configuration.
3Duration of action of moving object
If tri-segmented particles are engineered with ORFs in non-wild-type positions, then duration of transgene expression is extended, but productivity of viral replication is reduced
Solution Approach 1:
The tri-segmented arenavirus particles exhibit dynamic characteristics where the genome segments remain separate and cannot recombine into replication-competent forms. This dynamic separation ensures that transgene expression continues over extended periods without the virus reverting to a replication-competent state. The duplicated ORFs in L1 and L2 segments are maintained in non-functional arrangements that prevent productive recombination, thereby extending transgene expression duration while limiting viral replication productivity.
Data Source
Figure 1A~1D
Figure 2A~2C
Figure 3A~3D
AI summary
Provided herein is a modified arenavirus genomic segment, wherein the arenavirus genomic segment is engineered to carry a viral ORF in a position other than the wild-type position of the ORF. Further provided herein is an arenavirus particle comprising the arenavirus genomic segment. A vaccine and a pharmaceutical composition comprising the arenavirus particle are also provided.