Non-Replicative DNA Vector for Microbiome Modulation
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Solution Overview
Problem
Existing methods for delivering DNA payloads to bacterial populations face challenges in containing the exogenous DNA within target bacteria, leading to potential spread and replication, and require antibiotic resistance markers for selection, which is undesirable.
Innovation Solution
Development of a non-replicative DNA vector using a conditional origin of replication based on phage-inducible chromosomal islands (PICIs) that is packaged into phage-derived particles, allowing efficient delivery and expression without replication or the need for antibiotic resistance markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If exogenous DNA is delivered to bacterial populations, then gene expression and therapeutic effect are achieved, but DNA replication and spread to other bacteria occur
Solution Approach 1:
The patent extracts the replication capability from the DNA payload by using a non-replicative vector system. The vector is designed to deliver the therapeutic gene without the ability to replicate autonomously in the target bacteria, thereby separating the gene delivery function from the replication function that causes spread.
Solution Approach 2:
The patent uses a conditional origin of replication system as an intermediary mechanism. The origin of replication is engineered to be non-functional under the specific conditions present in the target bacterial population, allowing the DNA to be delivered and expressed without replicating. This conditional system acts as a mediator that permits delivery while preventing unwanted replication and spread.
2Stability of the object's composition
If antibiotic resistance markers are used for selection, then stable maintenance of DNA payload is achieved, but antibiotic resistance spread and safety concerns arise
Solution Approach 1:
The patent extracts and removes the antibiotic resistance selection marker from the DNA payload. Instead of using traditional antibiotic resistance genes for selection and maintenance, the system relies on the non-replicative nature of the vector and conditional replication control to maintain the therapeutic gene without requiring antibiotic resistance markers.
Solution Approach 2:
The patent employs a disposable, non-replicative DNA vector that delivers the therapeutic gene temporarily without establishing long-term stable maintenance through replication. The vector performs its delivery function and then naturally degrades or is lost, eliminating the need for persistent antibiotic resistance markers while still achieving the therapeutic effect.
3Object-generated harmful factors
If DNA payload is delivered without replication capability, then DNA spread is prevented, but gene expression level and duration are reduced
Solution Approach 1:
The patent changes the parameters of the origin of replication to create a conditional system that is non-functional under the specific physiological conditions of the target bacteria. By modifying the replication control parameters (temperature sensitivity, chemical induction, or conditional promoters), the system allows high-level gene expression during the delivery phase while preventing replication under normal physiological conditions, thereby extending the effective duration without spread.
Data Source
AI summary
The present invention concerns nucleic acids of interest for modulating the microbiome of a host, to vectors encoding the nucleic acids and to methods for in vivo modulating the microbiome of a subject by delivering the nucleic acid of interest.