Single-Plasmid HAdV-4 Vector for Rapid Recombinant Packaging
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The high level of pre-existing immunity to adenovirus type 5 vectors worldwide hampers the efficacy of adenovirus type 5 vectored vaccines, while adenovirus type 4 vectors offer lower immunity and higher safety, necessitating a stable and efficient packaging system for recombinant human adenovirus type 4.
Innovation Solution
A replication-competent human adenovirus type 4 vector system with an E3-deleted genome and a single-plasmid design, allowing exogenous gene insertion between the packaging signal and E1 region, utilizing PacI and PmeI restriction enzyme sites, and incorporating mCMV promoter and SV40 polyadenylic acid tail sequences for controlled gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If adenovirus type 5 vector system is used, then vaccine development and gene therapy can be achieved, but pre-existing immunity significantly reduces efficacy
Solution Approach 1:
The patent changes the virus type parameter from adenovirus type 5 to adenovirus type 4, which has significantly lower pre-existing immunity levels in the population. This parameter change resolves the contradiction by maintaining vaccine efficacy while avoiding the harmful effect of pre-existing immunity that plagues adenovirus type 5 vectors.
2Adaptability or versatility
If multi-plasmid system is used for adenovirus packaging, then gene insertion is possible, but packaging efficiency and speed are reduced
Solution Approach 1:
The patent merges the gene insertion site directly into the adenovirus type 4 backbone plasmid, creating a single-plasmid system. This combines the functions of separate vector and insert plasmids into one construct, eliminating the need for complex multi-plasmid co-transfection while maintaining gene insertion capability and significantly improving packaging efficiency and speed.
Solution Approach 2:
The patent strategically segments the plasmid structure by placing the exogenous gene insertion site between the packaging signal and E1 region, creating distinct functional domains within the single plasmid. This segmentation allows efficient packaging while maintaining versatility for different gene insertions.
3Duration of action of moving object
If E3 region is retained in adenovirus genome, then virus replication is maintained, but immune evasion capability is reduced
Solution Approach 1:
The patent extracts and removes the E3 region from the adenovirus type 4 genome in the vector construction. This extraction eliminates the immune evasion functions that would interfere with vaccine efficacy while preserving the replication-deficient nature of the vector, which is desirable for safety in therapeutic applications.
Data Source
AI summary
A single plasmid vector system for packaging recombinant human adenovirus type 4. The vector system contains an E3 region-deleted human adenovirus type 4 (HAdV-4 or Ad4) genome, a vector sequence for amplifying plasmids in bacteria, a pBR322 replication origin, a kanamycin resistance gene, and a replication control sequence; and an exogenous gene embedding site is located behind a packaging signal of the human adenovirus type 4 and in front of an E1 region. The present invention further provides a method for packaging the recombinant human adenovirus type 4 by the single plasmid vector system and an application in vaccine and drug preparation. The vector system can be used for rapidly and efficiently preparing a human adenovirus type 4 vector recombinant virus for stably expressing an exogenous gene, and has a good application prospect in the fields of preparation of a diagnostic kit, a vaccine, a gene therapy kit and/or a tumor therapy drug, etc.