Non-Replicating Adenovirus Vector Construction via Genome Segmentation

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Solution Overview

Problem

Existing adenoviral vectors face challenges such as large nucleotide sequences making in vitro manipulation difficult, potential for unchecked replication, and recombination with mammalian cell lines leading to infectious replicating viruses.

Innovation Solution

Development of non-replicating adenovirus expression vectors with mutations that render them replication incompetent, carrying a nucleotide expression cassette for therapeutic gene expression, and utilizing a protocol for efficient production in bacteria and a mammalian cell line to prevent contamination.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If adenoviral vectors are used for gene therapy, then therapeutic gene expression is achieved, but the risk of unchecked replication and recombination with mammalian cell lines increases

Engineering Contradiction:

Improvesafety of gene therapyVSAvoidreplication and recombination risk

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent removes the E1 and E3 genes from the adenoviral vector genome. The E1 gene deletion prevents replication by eliminating essential viral functions, while E3 deletion removes genes involved in immune evasion and apoptosis inhibition. This extraction of harmful genetic elements eliminates the risk of unchecked replication and recombination while preserving the vector's ability to deliver therapeutic genes.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent converts the potential harm of replication-competent vectors into a benefit by deliberately creating replication-incompetent vectors. The mutations that prevent replication are framed as safety features rather than deficiencies, allowing the vector to deliver therapeutic genes without the risk of causing infectious disease or uncontrolled viral replication in patients.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

2Manufacturing precision

If adenoviral vectors with large nucleotide sequences are manipulated in vitro, then complete viral genomes are obtained, but manipulation difficulty and error rate increase

Engineering Contradiction:

Improveaccuracy of vector constructionVSAvoidcomplexity of in vitro manipulation

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent divides the adenoviral genome into smaller manageable segments by deleting the E1 and E3 regions. This segmentation allows the vector to be constructed from smaller DNA fragments that are easier to manipulate in vitro, reducing the complexity of cloning and assembly while maintaining the essential functions needed for gene delivery.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

By extracting and removing the E1 and E3 gene regions from the adenoviral genome, the patent reduces the total nucleotide sequence length from approximately 36 kb to a more manageable size. This extraction simplifies in vitro manipulation, reduces the risk of recombination errors, and makes the vector construction process more reliable while still preserving the capacity to deliver therapeutic genes.

Inventive Principle:

Principle #2Taking out (Extraction)

3Productivity

If common mammalian cell lines are used to produce adenovirus, then production efficiency is high, but endogenous adenoviral genes can recombine with mutant vectors to create infectious viruses

Engineering Contradiction:

Improveproduction efficiencyVSAvoidrecombination and contamination risk

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent extracts and removes the E1 and E3 genes from the adenoviral vector, creating a replication-incompetent configuration that cannot recombine with endogenous adenoviral sequences in mammalian cell lines. This extraction eliminates the risk of generating infectious replicating viruses during production while maintaining the ability to produce high titers of vector particles using standard cell lines like HEK293.

Inventive Principle:

Principle #2Taking out (Extraction)

Data Source

PatentUS20250137013A1Adenoviral expression vector and methods and cell lines for production

Publication Date: 2025.05.01 LUNG BIOTECH PBC

AI summary

An adenovirus expression vector is provided. The adenovirus expression vector may include: a) one or more mutations that render the adenovirus replication incompetent and b) at least one nucleotide sequence encoding a protein or an RNA is provided. A method of synthesizing an adenovirus vector is also provided. The synthesis may include: a) producing a plurality of overlapping adenovirus sub-fragments, each sub-fragment comprising a portion of the full genome of the adenovirus; b) circularizing the sub-fragments to form plasmid structures; and c) assembling the circularized sub-fragments into a linear structure, wherein the vector comprises a combination of two or more sub-fragments. A mammalian cell line configured to replicate adenoviral vectors, wherein the cell line comprises nucleotide sequences expressing E1A and E1B gene products but is devoid of other adenovirus sequences is also provided.