Double Mutant Factor VIII Phe309Ser Asp519Val Stability
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Solution Overview
Problem
Current treatments for hemophilia A, particularly recombinant Factor VIII proteins, face challenges in expression levels due to inefficient mRNA expression and misfolded protein interactions, leading to stability and secretion issues, with prior art focusing on single or multiple mutations that do not effectively combine stability and secretion enhancements.
Innovation Solution
A double mutant B-domain deleted Factor VIII protein with mutations at Phe309Ser and Asp519Val is expressed using an optimized expression cassette and vector system, enhancing both stability and secretion through synergistic effects, resulting in improved activity and circulation half-life.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If recombinant Factor VIII is expressed in heterologous systems, then therapeutic protein production is achieved, but expression level is low due to inefficient mRNA expression and misfolded protein interactions
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (Phe309Ser and Asp519Val) at strategically selected positions in the Factor VIII protein sequence. These parameter changes at the molecular level alter the protein's interaction properties with chaperones and the secretory pathway, thereby improving both expression level and secretion efficiency simultaneously
Solution Approach 2:
The patent applies local quality by making targeted local modifications at specific domains (A1 and A2 domains) rather than global changes. The mutations are localized to specific residues (309 and 519) that are critical for protein folding and secretion, allowing improvement of secretion efficiency without compromising overall protein structure and function
2Stability of the object's composition
If single mutations are introduced to improve stability, then protein stability is enhanced, but secretion enhancement is not achieved
Solution Approach 1:
The patent merges two separate mutation benefits into a single double mutant construct. By combining the Phe309Ser mutation (which improves stability) with the Asp519Val mutation (which improves secretion), the invention achieves synergistic effects where both stability and secretion are enhanced simultaneously, overcoming the limitation of single mutations
3Reliability
If mutations are introduced to improve secretion, then secretion efficiency is enhanced, but stability improvement is not achieved
Solution Approach 1:
The patent combines two mutations in a single construct where each mutation contributes a different benefit. The Asp519Val mutation primarily enhances secretion by improving interaction with the secretory pathway, while the Phe309Ser mutation concurrently enhances stability. This merging approach ensures both properties are improved together
4Quantity of substance
If B-domain deleted Factor VIII is produced, then molecular weight is reduced and expression is improved, but immunogenicity and stability issues persist
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (Phe309Ser and Asp519Val) in the B-domain deleted Factor VIII construct. These parameter changes address the stability issues inherent in BDD-FVIII by modifying residues that are critical for protein folding and structural integrity, thereby enhancing stability while maintaining the expression benefits of B-domain deletion
Data Source
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AI summary
The present disclosure is in the field of haemophilia therapeutics, particularly recombinant Factor VIII protein products. The present disclosure relates to a double mutant B-domain deleted Factor VIII gene having mutations at Phe309Ser and Asp519Val respectively. The disclosure also relates to the production of double mutant B-domain deleted Factor VIII protein having mutations at Phe309Ser and Asp519Val respectively. The said protein shows enhanced activity and stability and therefore is used in the management of haemophilia.