Nucleic Acid Vector Self-Destruction via Nuclease Cleavage
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Solution Overview
Problem
Current strategies for controlling the release and self-destruction of genetically engineered bacteria in environmental settings are inadequate, particularly in therapeutic, cosmetic, or industrial applications, as they rely on external stimuli or are prone to uncontrolled dissemination due to environmental factors or horizontal gene transfer.
Innovation Solution
A nucleic acid vector system that encodes a DNA modifying enzyme, such as a nuclease, which is expressed in bacteria to cleave and inactivate itself after ensuring the expression of a transgene, allowing for controlled self-destruction independent of external signals, thereby preventing uncontrolled release.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a nucleic acid vector is introduced into a bacterial population for therapeutic or industrial application, then the desired genetic function is achieved, but the vector may persist and disseminate uncontrolled in the environment
Solution Approach 1:
The patent incorporates a nuclease gene and target sequence into the nucleic acid vector before introduction into the bacterial population. The vector is designed to express the nuclease at a predetermined time, which then cleaves the target sequence on the same vector, causing self-destruction. This preliminary design ensures automatic containment without requiring external intervention.
Solution Approach 2:
The nucleic acid vector contains all necessary components (nuclease gene, target sequence, and regulatory elements) to carry out its own destruction. The vector expresses the nuclease that recognizes and cleaves its own target sequence, enabling self-service containment without relying on external containment signals or host bacterium characteristics.
2Reliability
If a kill switch system is engineered to sense environmental cues and produce toxins, then bacterial containment is achieved, but the system requires tight control and robust sensing that is challenging to achieve
Solution Approach 1:
The patent extracts the sensing and control requirements from the containment system by using constitutive expression of the nuclease under a strong promoter. Instead of requiring complex environmental sensing and signal transduction pathways, the system simply relies on the inherent ability of the strong promoter to drive continuous transcription, greatly simplifying the control mechanism while maintaining reliability.
Solution Approach 2:
The nucleic acid vector is designed as a temporary, disposable element that performs its function (introducing the desired genetic circuit) and then automatically destroys itself through the expressed nuclease. This eliminates the need for long-term containment mechanisms or complex control systems, as the vector is meant to be short-lived by design.
3Reliability
If an auxotrophic strain is used for containment by requiring a specific metabolite, then bacterial control is achieved, but the system fails if the metabolite becomes available in the environment or is acquired by horizontal gene transfer
Solution Approach 1:
The containment mechanism is self-contained within the nucleic acid vector itself through the inclusion of the nuclease and target sequence. The vector does not rely on host bacterium characteristics or environmental metabolites for containment. Instead, the vector autonomously expresses the nuclease that destroys the target sequence on the same vector, making the containment mechanism independent of environmental adaptability.
4Duration of action of moving object
If the nucleic acid vector expresses the nuclease immediately, then self-destruction is triggered, but the transgene expression duration is insufficient to achieve the desired effect
Solution Approach 1:
The patent design the vector with the nuclease gene and target sequence already present before introduction. The strong promoter is prepared to drive immediate and continuous expression upon introduction, ensuring that the nuclease is produced in sufficient quantities during the desired time window. This preliminary design allows the transgene to be expressed for the necessary duration before self-destruction occurs.
Solution Approach 2:
The patent uses a strong promoter to dramatically increase the transcription rate and nuclease expression level. This parameter change ensures that the nuclease is produced in high quantities quickly, maintaining the vector's function for the desired duration and then efficiently triggering self-destruction when the nuclease concentration reaches a threshold.
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 ensures controlled self-destruction of the nucleic acid vector, maintaining the desired activity duration and preventing environmental dissemination, enhancing safety and efficacy in targeted applications.
Implementation Method 1
a nucleic acid sequence or vector comprising a gene encoding a DNA modifying enzyme, e.g. a nuclease, which can be expressed in a target bacterial cell, wherein the DNA modifying enzyme when expressed from the nucleic acid sequence or vector modifies said nucleic acid sequence or vector at one or multiple locations in the nucleic acid sequence
Data Source
AI summary
The invention relates to methods, kits, and compositions for reducing the level of or eliminating a nucleic acid vector in situ. The invention encompasses compositions and methods for selectively eradicating nucleic acid vectors in the microbiota using packaged phagemids. The microbiota can be intestinal and the packaged phagemids can be administered orally. The phagemid encodes a nuclease or other enzyme that genetically modifies the nucleic acid vector so that the nucleic acid vector can be inactivated or eliminated.