Reducing Agrobacterium Endotoxin via LPS Genetic Modification
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Solution Overview
Problem
Current methods for eliminating lipopolysaccharide (LPS) contamination from Agrobacterium-derived biologics are expensive and labor-intensive, inadequate for many pharmaceuticals, and pose risks due to endotoxicity, necessitating a solution for reducing LPS toxicity in plant-made biologics.
Innovation Solution
Genetically modifying Agrobacterium by deleting or disrupting genes involved in LPS synthesis, such as lpxXL and acpXL, and introducing exogenous nucleic acid encoding Lipid A 4′-phosphatase (LpxF) from Francisella tularensis to produce LPS variants with reduced toxicity and improved immunogenicity, while maintaining the ability to mediate T-DNA transfer and tumor-inducing gene delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If current purification processes are used to eliminate endotoxin from plant-made biologics, then LPS toxicity is reduced, but the process becomes expensive and labor-intensive
Solution Approach 1:
The patent applies preliminary action by genetically modifying the Agrobacterium strain before biologic production to reduce LPS toxicity at the source. The modified strain possesses altered LPS structure that is inherently less toxic, eliminating the need for complex downstream purification processes. This preemptive modification resolves the contradiction by addressing the harmful factor before it requires expensive removal.
Solution Approach 2:
The patent converts the harmful LPS produced by Agrobacterium into a beneficial form by generating LPS variants with reduced endotoxicity. The modified Agrobacterium produces LPS that retains immunostimulatory properties while having significantly reduced toxicity, thus converting a harmful byproduct into a safer component that does not require extensive purification.
2Object-affected harmful factors
If LPS is completely removed from Agrobacterium-derived biologics, then toxicity is eliminated, but purification becomes more complex and costly
Solution Approach 1:
The patent applies parameter changes by modifying the chemical structure of LPS through genetic engineering of the Agrobacterium strain. The altered LPS variants have different physical and chemical properties that reduce endotoxicity while maintaining functionality. This structural parameter modification eliminates the need for complex purification processes designed to remove toxic LPS.
3Reliability
If Agrobacterium is genetically modified to reduce LPS toxicity, then drug safety is improved, but the modification process adds complexity
Solution Approach 1:
The patent applies the taking out principle by extracting and removing specific genes responsible for producing highly toxic LPS forms from the Agrobacterium genome. By deleting these specific genetic elements, the strain produces LPS variants with reduced toxicity. This targeted gene removal achieves improved drug safety through a relatively straightforward genetic modification approach.
Data Source
AI summary
The present invention relates to the fields of genetically modified Agrobacterium strains, vaccine adjuvants, and generally molecular biology and immunology. Provided herein are modified Agrobacterium strains that produce lipopolysaccharide (LPS) having reduced toxicity or detoxified lipopolysaccharide, and methods of obtaining such strains for plant-based production of biologies. Also provided herein are uses of reduced or detoxified LPS as adjuvants suitable for clinical use.