Outer Membrane Vesicles With Reduced Endogenous Protein Load
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Solution Overview
Problem
Current methods for producing Outer Membrane Vesicles (OMVs) from Gram-negative bacteria are limited by low yields and contamination with endogenous proteins, which can interfere with the immune response to heterologous antigens, making it difficult to develop effective vaccines.
Innovation Solution
Genetically modify Gram-negative bacteria to delete specific genes encoding OMV proteins, enhancing vesicle production and reducing endogenous protein content, while expressing heterologous antigens on the OMVs, using techniques like CRISPR/Cas-based methods to inactivate genes such as ompA and others, and purifying OMVs to enhance immunogenicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional OMV production methods are used, then OMVs are produced, but yields are low and endogenous proteins contaminate the preparation
Solution Approach 1:
The patent extracts and removes harmful endogenous proteins from the OMV preparation by genetically deleting specific genes (ompA, fhuA, malE, lamB, tsx, phoE, fepA, yghJ) that encode these proteins. This extraction principle directly addresses the contamination issue while preserving the OMV structure and heterologous antigens.
Solution Approach 2:
The patent changes the genetic parameters of the bacterial strain by introducing specific gene deletions. This modifies the composition of OMVs produced, reducing endogenous protein content while maintaining or enhancing OMV yield and immunogenicity against heterologous antigens.
2Reliability
If endogenous proteins are present in OMVs, then OMVs are produced, but immune response to heterologous antigens is interfered with
Solution Approach 1:
By genetically deleting genes encoding endogenous proteins that interfere with immune responses, the patent extracts these harmful components from the OMV preparation, thereby improving the reliability and specificity of the immune response to heterologous antigens.
Solution Approach 2:
The patent applies local quality modification by selectively removing specific endogenous proteins (through targeted gene deletions) while preserving other components of the OMV that are beneficial for immune response, such as heterologous antigens and essential structural proteins.
3Object-generated harmful factors
If gene deletion is performed to reduce endogenous proteins, then endogenous protein content decreases, but bacterial genome is modified
Solution Approach 1:
The patent segments the genome modification process into specific, targeted gene deletions rather than random or comprehensive genome reduction. Each deleted gene (ompA, fhuA, malE, lamB, tsx, phoE, fepA, yghJ) is selected based on its contribution to endogenous protein content, allowing systematic reduction of harmful proteins while maintaining essential bacterial functions.
Data Source
AI summary
The invention relates to gram-negative bacteria carrying gene-inactivating mutations that cause deletion of proteins belonging to the OMV proteome, to Outer Membrane Vesicles (OMVs) produced by these bacteria and immunogenic compositions thereof.