E4orf6-Inserted Adenoviral Vector to Prevent RCA Formation
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Solution Overview
Problem
Existing adenoviral vectors face safety issues due to the potential creation of replication competent adenovirus (RCA) during production, leading to adenovirus infection and inflammatory responses, and current strategies to prevent RCA are inefficient or pose safety risks.
Innovation Solution
A novel adenoviral vector is developed by inserting the E4 gene, specifically the E4orf6 gene, into the E1 gene-deleted region, with the E4 gene expressed in a forward direction and an antigen protein sequence inserted at the 5' position, minimizing RCA occurrence and maintaining adenovirus productivity and antigen expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the E1 gene is deleted to create a non-replicable adenoviral vector, then replication safety is improved, but the risk of RCA formation through homologous recombination increases
Solution Approach 1:
The E4orf6 gene, which is essential for adenovirus replication, is completely removed from the adenoviral vector. This extraction eliminates the replication capability while preventing RCA formation, as the homologous recombination cannot restore the complete E4orf6 gene function. The vector maintains non-replicable status with enhanced safety against RCA.
Solution Approach 2:
The adenoviral genome is segmented into essential and non-essential regions. The E1 and E4orf6 genes (essential for replication) are deleted, while other regions are preserved for vector function. This segmentation allows the vector to maintain packaging and delivery capabilities while eliminating replication potential and RCA risk.
2Reliability
If the E4 gene is deleted to prevent RCA, then safety is improved, but adenovirus productivity may be affected
Solution Approach 1:
The E4orf6 gene is inserted into the E1-deleted region as an intermediary element. This insertion provides a homologous sequence that can support efficient packaging and production of the viral vector without enabling replication, as the E1 gene remains deleted. The E4orf6 acts as a mediator to maintain productivity while preserving safety.
Solution Approach 2:
The genomic parameters of the adenoviral vector are changed by deleting the E4orf6 gene and inserting it into the E1-deleted region. This parameter change (gene location and presence) maintains the non-replicable state while optimizing packaging efficiency and viral production through the provided homologous sequence.
3Reliability
If homologous recombination occurs during vector production, then RCA can be formed, but preventing this reduces manufacturing efficiency
Solution Approach 1:
The E1 gene, which is required for both replication and homologous recombination events that could generate RCA, is completely extracted from the vector genome. This extraction prevents the homologous recombination pathway that would otherwise allow RCA formation, while the manufacturing process remains efficient through the use of HEK293 cells that provide complementary functions.
Solution Approach 2:
The E4orf6 gene sequence is copied and inserted into the E1-deleted region of the vector. This copying provides a homologous sequence that facilitates efficient packaging and production without enabling replication or RCA formation, as the E1 gene remains absent. The copied sequence serves as a template for efficient vector production.
Data Source
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AI summary
The present invention relates to a novel adenoviral vector not including a replication competent adenovirus. A recombinant E1/E3/E4-deleted adenoviral vector, of the present invention, in which an antigenic protein and an E4orf6 gene are inserted in an E1 gene-deleted region, has adenovirus productivity, degree of antigen expression, neutralizing antibody production amount, and T cell induction ability that are similar to those of a control group, and thus can be effectively used as a carrier for various vaccines for diseases or anti-cancer vaccines.