Poxvirus Vector Reconstitution from Modular DNA Fragments

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Solution Overview

Problem

Current methods for generating recombinant Modified Vaccinia Ankara (MVA) vectors are laborious and limited by the restricted host cell tropism and assembly deficiencies, necessitating the development of alternative vectors for research, prophylactic, and therapeutic uses.

Innovation Solution

A method involving transfection of host cells with multiple DNA fragments, each containing partial poxvirus genomic sequences, which assemble via homologous recombination to form a full-length poxvirus genome, facilitated by helper viruses like Fowl pox virus, allowing for the insertion of heterologous DNA sequences such as antigens or other heterologous gene sequences.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If traditional methods are used to generate recombinant MVA vectors, then the process is limited by restricted host cell tropism and assembly deficiencies, but the current licensed vectors are available for use

Engineering Contradiction:

Improvehost cell tropismVSAvoidvector generation process

Core Design Contradiction:

Adaptability or versatilityVSEase of manufacture

Solution Approach 1:

The MVA genome is divided into multiple DNA fragments that can be independently synthesized and then assembled in host cells. This segmentation allows the genome to be constructed in a modular fashion, overcoming the limitations of traditional single-step transfection methods and enabling assembly in mammalian cells that previously could not support MVA replication.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

DNA fragments containing MVA genome sequences are pre-synthesized and prepared before transfection into host cells. This preliminary preparation of genomic segments allows for controlled assembly and expression in mammalian cells, bypassing the need for traditional cell culture-based attenuation processes.

Inventive Principle:

Principle #10Preliminary action

2Adaptability or versatility

If multiple DNA fragments are transfected to assemble full-length poxvirus genome, then heterologous sequences can be inserted, but the process becomes more complex

Engineering Contradiction:

Improveinsertion of heterologous sequencesVSAvoidtransfection process

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

Helper viruses or plasmid vectors serve as intermediaries to facilitate the assembly of multiple DNA fragments into a complete poxvirus genome. These intermediary elements simplify the complex process of genome reconstruction by providing templates and necessary viral proteins, making it easier to insert heterologous sequences while maintaining genome integrity.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The transfected DNA fragments contain all necessary information and sequences required for self-assembly into a complete poxvirus genome. The system utilizes the host cell's natural repair and recombination mechanisms to automatically assemble the fragments without requiring extensive external intervention, thereby reducing operational complexity.

Inventive Principle:

Principle #25Self-service

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach enables efficient reconstitution of poxvirus vectors, including MVA, in mammalian cells, facilitating the expression of antigens and heterologous sequences, and supports the generation of multiantigenic vaccine vectors with improved stability and immunogenicity.

Implementation Method 1

infected the host cell with a helper virus before, during, or after the transfection of the one or more DNA fragments to initiate the transcription of the one or more DNA fragments

Methodology Applied

Scientific EffectTranscription:

Implementation Method 2

two or more DNA fragments are co-transfected into the host cell, each DNA fragment comprises a partial sequence of the poxvirus genome such that the two or more DNA fragments are assembled sequentially by homologous recombination

Methodology Applied

Scientific EffectHomologous recombination:

Data Source

PatentUS20250215456A1Poxvirus-based vectors produced by natural or synthetic DNA and uses thereof

Publication Date: 2025.07.03 CITY OF HOPE

AI summary

Disclosed are methods of producing poxvirus-based vectors or recombinant poxvirus-based vectors from naturally derived, chemically synthesized DNA fragments, or a combination of naturally derived and chemically synthesized DNA fragments. One or more DNA sequences encoding one or more antigens, subunits or fragments thereof or other heterologous gene sequences are inserted in one or more poxvirus insertion sites in one or more DNA fragments. The methods include transfecting a host cell with one or more circular or linear DNA fragments such that a poxvirus or recombinant poxvirus is reconstituted in the host cell, the reconstituted poxvirus or recombinant poxvirus comprising the genome of a desired poxvirus. Also disclosed are poxviruses or recombinant poxviruses produced by the technology and uses thereof.