Phage Transduction Particles for Pure Antibacterial Payload Delivery
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Solution Overview
Problem
Existing methods for producing phage particles for antibacterial applications often result in impure compositions and uncontrollable propagation, leading to risks of acquiring undesirable foreign genes and dosing uncertainties.
Innovation Solution
The use of non-self-replicative transduction particles, comprising helper phages and plasmids with integrated helper phage genes, which require helper phages for replication, and encode CRISPR/Cas systems to target and kill bacteria, allowing for controlled delivery of antibacterial agents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional helper phage systems are used to package phagemid DNA into phage particles, then phage production is achieved, but the compositions are impure and uncontrollable propagation occurs leading to acquisition of undesirable foreign genes
Solution Approach 1:
The phage system is segmented into two separate DNA components: (i) a phagemid DNA containing the antibacterial payload and packaging signal, and (ii) a helper phage DNA providing structural proteins but lacking packaging signal. This segmentation prevents contamination of the final product with unwanted phage variants and ensures only desired particles are produced
Solution Approach 2:
A packaging signal acts as an intermediary element that directs the helper phage packaging machinery to package only the phagemid DNA into particles. This mediator ensures specific packaging of the desired DNA while excluding the helper phage DNA, achieving both purity and controllability
2Productivity
If self-replicative phage particles are used, then efficient bacterial infection and killing is achieved, but uncontrollable propagation and dosing uncertainties occur
Solution Approach 1:
The self-replication capability is extracted from the phage particle by providing all necessary replication functions through the helper phage in trans. The phagemid DNA lacks origin of replication and essential genes, so it cannot replicate autonomously. This extraction maintains killing efficiency while enabling precise dosing control
Solution Approach 2:
The helper phage provides all services needed for phagemid DNA replication and particle production, including origin of replication, structural proteins, and packaging machinery. The phagemid DNA is entirely dependent on the helper phage, ensuring controlled propagation while maintaining productivity
3Reliability
If CRISPR/Cas systems are delivered via traditional phage methods, then antibacterial action is achieved, but risk of acquiring undesirable foreign genes increases
Solution Approach 1:
The CRISPR/Cas system, which normally protects bacteria from foreign DNA, is repurposed as an antibacterial weapon by directing it against the target bacteria's genome. The same mechanism that prevents unwanted gene acquisition is used to actively destroy target bacteria, converting a protective system into a therapeutic tool
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 the production of highly pure phage compositions that effectively kill target bacteria while minimizing the risk of acquiring foreign genes and controlling dosing, suitable for medical and environmental applications.
Implementation Method 1
the antibacterial means encodes a Cas nuclease
Implementation Method 2
a DNA sequence encoding a guide RNA (eg, a single guide RNA) or comprising a CRISPR array for producing guide RNA, wherein the guide RNA is capable of targeting the genome of the target bacteria
Data Source
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AI summary
The invention relates to the production of phage and transduction particles using DNAs (eg, plasmids and helper phage, mobile genetic elements (MGEs) or plasmids with chromosomally integrated helper phage genes), as well as the phage, helper phage, kits, compositions and methods involving these. The particles are particularly useful for delivering toxic payloads into target bacteria for antibacterial action. Embodiments enable production of highly pure compositions of such particles for medical or environmental use and for containment of the particles, which may be useful for containing antibacterial action.