Recombinant Clostridial Neurotoxins with Random Coil Domain
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Solution Overview
Problem
Current methods for producing clostridial neurotoxins are cumbersome and time-consuming, with fermentation processes requiring strict containment due to the toxins' high toxicity, and result in variable neurotoxin activation and immune reactions, limiting their clinical application due to short duration of effect and immune response.
Innovation Solution
Recombinant clostridial neurotoxins with a random coil domain are produced by cloning a nucleic acid sequence encoding this domain into a parental clostridial neurotoxin gene and expressing it in host cells, enhancing the neurotoxin's duration of effect by modifying its subcellular localization and degradation resistance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If fermentation processes are used to produce clostridial neurotoxins, then the toxins can be produced, but the process is cumbersome and time-consuming with strict containment requirements
Solution Approach 1:
The patent replaces the traditional fermentation process (biological system) with recombinant DNA technology and host cell expression systems (molecular biology system). This substitution eliminates the need for strict containment facilities while maintaining toxin production capability, directly resolving the contradiction between productivity and containment complexity.
Solution Approach 2:
The patent uses recombinant DNA technology to copy and replicate the neurotoxin gene in host cells (such as Chinese hamster ovary cells or insect cells). This copying approach allows unlimited production capacity without the containment requirements of fermentation processes, improving productivity while reducing device complexity.
2Reliability
If traditional clostridial neurotoxin production methods are used, then toxins can be obtained, but variable neurotoxin activation and immune reactions occur
Solution Approach 1:
The patent modifies specific local regions of the neurotoxin protein structure through site-directed mutagenesis. By changing amino acid residues at specific locations (such as the light chain protease domain), the patent achieves consistent neurotoxin activation and reduced immune reactions without affecting the overall toxin function, resolving the reliability issue.
Solution Approach 2:
The patent changes molecular parameters of the neurotoxin by introducing specific amino acid mutations. These parameter changes in the protein sequence alter the toxin's activation characteristics and immune properties, achieving consistent activation and reduced harmful immune reactions while maintaining therapeutic efficacy.
3Duration of action of moving object
If conventional clostridial neurotoxins are used, then therapeutic effect is achieved, but duration of effect is short
Solution Approach 1:
The patent performs preliminary structural modification of the neurotoxin by introducing protease-resistant mutations before administration. This preliminary action extends the toxin's half-life and duration of effect, reducing the need for frequent administrations and improving both duration of action and productivity.
Solution Approach 2:
The patent creates a composite protein structure by fusing the neurotoxin with protease-resistant sequences or modifying the toxin's proteolytic stability. This composite approach enhances the toxin's resistance to degradation, extending its duration of effect and reducing administration frequency.
Data Source
AI summary
This invention relates to novel recombinant clostridial neurotoxins exhibiting increased duration of effect and to methods for the manufacture of such recombinant clostridial neurotoxins. These novel recombinant clostridial neurotoxins comprise a random coil domain, and the methods comprise the steps of inserting a nucleic acid sequence coding for a random coil domain into a nucleic acid sequence coding for a parental clostridial neurotoxin and expression of the recombinant nucleic acid sequence comprising the random coil domain-coding sequence in a host cell. The invention further relates to novel recombinant single-chain precursor clostridial neurotoxins used in such methods, nucleic acid sequences encoding such recombinant single-chain precursor clostridial neurotoxins, and pharmaceutical compositions comprising the recombinant clostridial neurotoxin with increased duration of effect.