Disulfide-Stabilized Polypeptides for Neutral-pH Enzyme Stability
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Solution Overview
Problem
Recombinant polypeptides used in enzyme replacement therapy for genetic diseases have poor stability at neutral pH, leading to rapid degradation in serum and a short half-life, limiting their therapeutic effectiveness.
Innovation Solution
Introduction of non-native cysteine residues to form disulfide bridges within or between protein monomers, stabilizing proteins such as alpha-galactosidase A and palmitoyl protein thioesterase 1, enhancing their stability and half-life at neutral pH through the formation of disulfide bonds.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If recombinant wildtype polypeptides are used for enzyme replacement therapy, then the therapeutic enzyme can be delivered by intravenous infusion, but the polypeptides have poor stability at neutral pH and are quickly degraded in serum, resulting in short half-life
Solution Approach 1:
The patent introduces non-native cysteine residues at specific positions (e.g., D233C and I359C for alpha-galactosidase A) to alter the chemical structure and enable disulfide bond formation, which fundamentally changes the stability parameter of the polypeptide at neutral pH
Solution Approach 2:
The invention creates a composite protein structure by forming disulfide bonds between cysteine residues, combining intramolecular and intermolecular bonding to produce a stabilized polypeptide composition with enhanced structural integrity and resistance to degradation
2Reliability
If non-native cysteine residues are introduced to form disulfide bridges, then the protein stability and half-life are enhanced at neutral pH, but the protein structure becomes more complex
Solution Approach 1:
The patent introduces cysteine residues at specific localized positions within the protein sequence (such as D233C and I359C for alpha-galactosidase A, or A171C and A183C for PPT1) rather than uniformly throughout, achieving stabilization at key structural locations with minimal overall complexity increase
Solution Approach 2:
The invention creates discrete disulfide bond structures (such as intramolecular bonds within a single monomer or intermolecular bonds between monomers) that segment the protein into stabilized functional units, allowing the complexity to be managed through modular bonding patterns
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
The stabilized proteins exhibit significantly increased half-life and effectiveness in treating genetic disorders by reducing or slowing symptoms, particularly in lysosomal storage disorders like Fabry disease and CLN1 disease.
Implementation Method 1
the stabilized form comprises one or more non-native cysteine residues that form a disulfide bridge between non-native cysteines within the protein or between non-native cysteines of two monomers of the protein
Data Source
AI summary
Provided herein are polypeptides comprising one or more non-native cysteine residues that form a disulfide bridge between non-native cysteines within the protein or between non-native cysteines of two monomers of the protein. Such modified human polypeptides are useful in treatment of genetic diseases via enzyme replacement therapy and/or gene therapy.