Modified Neuraminidase Mutations to Prevent Lysosomal Crystallization
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Solution Overview
Problem
Existing gene therapy using human neuraminidase 1 leads to intracellular crystallization, causing cell damage and posing risks, and there is a need for a modified neuraminidase that does not crystallize and can be effectively localized within lysosomes.
Innovation Solution
Development of modified neuraminidase with specific amino acid mutations (W173N or K175S and K358N) and co-expression with cathepsin A to prevent crystallization and enhance lysosomal localization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If human neuraminidase 1 is overexpressed in cells for gene therapy, then therapeutic effect is improved, but intracellular crystallization occurs causing cell damage
Solution Approach 1:
The patent applies local quality by directing neuraminidase 1 to specific subcellular locations (lysosomes) through targeted amino acid mutations. The mutations at positions 173, 175, and 358 create local structural changes that prevent crystallization while maintaining enzymatic function, allowing high expression without harmful crystal formation in the cytoplasm
Solution Approach 2:
The patent changes the amino acid sequence parameters of neuraminidase 1 by introducing specific mutations (W173N/K175S and K358N). These parameter changes modify the protein's physical and chemical properties to eliminate crystallization tendency while preserving catalytic activity, enabling safe overexpression for therapy
2Reliability
If neuraminidase 1 is overexpressed to treat lysosomal storage diseases, then disease treatment efficacy is improved, but crystallization occurs leading to loss of enzyme functionality
Solution Approach 1:
The patent uses local quality by confining neuraminidase 1 expression to lysosomal compartments through targeted mutations. This localized approach maintains high enzyme concentration where needed for disease treatment while preventing harmful crystal aggregation in other cellular regions, preserving overall enzyme functionality
Solution Approach 2:
The patent converts the potential harm of overexpression-induced crystallization into a benefit by designing mutations that redirect aggregation-prone regions to lysosomal targeting sequences. The same structural features that could cause harmful crystallization are transformed into beneficial lysosomal localization signals, ensuring enzyme delivery to the correct destination
3Quantity of substance
If neuraminidase 1 is expressed without proper localization signals, then expression level is improved, but lysosomal co-localization is insufficient reducing therapeutic benefit
Solution Approach 1:
The patent merges two functions into a single protein construct: high-level expression capability and lysosomal targeting. By combining strong promoter-driven expression with lysosome-targeting amino acid sequences (mutations at positions 173, 175, and 358), the patent achieves both high expression levels and correct subcellular localization simultaneously
Data Source
AI summary
Provided are a modified-type neuraminidase, a gene encoding the modified-type neuraminidase, a combination of the modified-type neuraminidase and cathepsin A, a combination of the gene encoding the modified-type neuraminidase and a gene encoding cathepsin A, a vector including said genes, and a pharmaceutical composition containing same. The pharmaceutical composition can be used for the therapy of lysosomal storage disease.