Recombinant Bacteria T3SS Delivery for Targeted Protein Translocation
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Solution Overview
Problem
Existing engineered microorganisms for drug delivery and immunotherapy face challenges such as undesirable immune responses and inefficient targeted delivery due to leaky expression and lack of replication, leading to short-lived presence in the host system.
Innovation Solution
Development of recombinant gram-negative bacterial strains equipped with a Type III protein secretion system, inducible genetic regulatory circuits, and synthetic adhesins for targeted delivery of cargo proteins to eukaryotic cells, ensuring tight control over expression and adhesion to specific target cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If attenuated pathogenic bacteria expressing T3SS are used for delivery, then cargo proteins can be injected into target cells, but undesirable immune responses are triggered
Solution Approach 1:
The patent extracts the harmful pathogenicity attributes from the bacterial delivery system while retaining the useful T3SS delivery machinery. Specifically, it uses replication-defective bacteria where the T3SS and cargo delivery functions are separated from the pathogenic replication capability, allowing delivery without triggering harmful immune responses associated with active pathogenic bacteria.
Solution Approach 2:
The patent segments the bacterial system into functional modules: replication-defective bacterial chassis providing structural framework, T3SS machinery providing injection capability, and inducible regulatory circuits providing controlled expression. This segmentation allows each component to be optimized independently, separating delivery function from pathogenicity.
2Object-generated harmful factors
If replication-defective bacteria or minicells are used to avoid immune responses, then harmful immune reactions are reduced, but the bacteria are easily eliminated from the host system due to lack of replication
Solution Approach 1:
The patent applies preliminary action through pre-establishment of inducible regulatory circuits and synthetic adhesins in the replication-defective bacteria before administration. The regulatory circuits are pre-configured to control T3SS expression timing, and adhesins are pre-formed to ensure targeted binding, allowing the bacteria to achieve their therapeutic effect quickly before being eliminated, maximizing their functional presence in the host.
3Object-generated harmful factors
If nonpathogenic microorganisms are engineered to assemble delivery systems, then pathogenesis is avoided, but leaky expression of the delivery system occurs leading to leaky delivery of effector proteins
Solution Approach 1:
The patent implements feedback control through inducible regulatory circuits that monitor and control T3SS expression. These circuits use regulatory proteins that respond to environmental signals or internal states to activate or repress T3SS transcription, ensuring precise timing and preventing leaky expression. The system provides feedback loops where the regulatory proteins bind to operator sequences to modulate gene expression levels.
Solution Approach 2:
The patent introduces dynamic control mechanisms where the regulatory circuits can switch between active and repressed states based on environmental conditions. The synthetic adhesins and T3SS components are dynamically regulated rather than constitutively expressed, allowing the system to adapt its delivery capability based on host environment and target cell presence.
4Object-generated harmful factors
If nonpathogenic microorganisms are used for delivery, then pathogenesis is avoided, but targeted delivery of effectors to specific cells is not achieved
Solution Approach 1:
The patent applies local quality by engineering synthetic adhesins with specific binding affinities for particular host cell surface molecules. Different adhesin variants can be deployed to target different cell types or tissues, providing localized delivery capability. The regulatory circuits are also localized in their expression control, activating T3SS specifically when and where needed based on local environmental signals.
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 precise and controlled delivery of cargo proteins to target cells, reducing unwanted immune responses and improving the persistence and efficacy of therapeutic agents for diseases like cancer and inflammatory diseases.
Implementation Method 1
The major uses of such programmed biomachines have been drug delivery to the cytosol of cells/organisms... a type Ill protein secretion system (T3SS)... capable of adhering specifically the target cell and subsequently injecting the cargo protein into said cell
Implementation Method 2
at least one synthetic adhesin (SA) driving adhesion of the recombinant bacterial strain to a target cell, wherein the strain is capable of adhering specifically the target cell
Data Source
AI summary
The invention relates to a recombinant bacterium and uses thereof. In particular, it relates to the use of recombinant bacteria to translocate cargo proteins into the cytosol of target cells. Said recombinant bacteria comprises a T3 secretion system under the control of a genetic regulatory circuit and a targeting module which allow the recombinant bacteria to target specific cells, adhere to these and inject their cargo into the cytosol of the target cells.