ADAM10-Knockout HEK293 Exosomes for Stable CD24 Loading
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Solution Overview
Problem
Existing methods for loading CD24, ApoE, and MyD88 inhibitor polypeptides into exosomes face challenges such as limited efficacy, high production costs, short elimination half-life, and safety risks, making them unsuitable for effective treatment of severe inflammatory diseases.
Innovation Solution
Loading dimeric CD24 and/or ApoE proteins, and MyD88 inhibitor polypeptides into exosomes from HEK293 cells with ADAM10 gene knockout using CRISPR gene editing and specific plasmid constructs, enhancing their efficacy and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CD24 is loaded into exosomes through overexpression in HEK293 cells, then exosomes containing CD24 can be collected and purified for inflammatory treatment, but the efficacy has no obvious advantages compared to CD24-Fc fusion proteins and the production process is more complex with higher cost
Solution Approach 1:
The patent extracts and removes the ADAM10 protease from the system by knocking out its gene in HEK293 cells. This prevents ADAM10 from cleaving CD24, thereby extracting the problematic degradation step from the production process and preserving full-length functional CD24 in the exosomes.
Solution Approach 2:
The patent changes the genetic parameter of the HEK293 cells by knocking out the ADAM10 gene. This parameter change fundamentally alters the cellular environment to prevent CD24 degradation, enabling stable production of functional CD24-loaded exosomes with enhanced efficacy.
2Reliability
If MyD88 inhibitor polypeptides are used to inhibit innate immune responses, then they can effectively block inflammatory signaling, but they have short elimination half-life and require cell membrane penetration which is difficult to achieve
Solution Approach 1:
The patent uses exosomes as an intermediary carrier to deliver MyD88 inhibitor polypeptides. The exosomes protect the polypeptides from degradation, facilitate their delivery to target cells, and prolong their circulation half-life, thereby solving the delivery and stability problems of MyD88 inhibitors.
3Reliability
If full-length ApoE protein is administered to treat severe inflammatory diseases, then it can play a regulatory and ameliorative role, but it usually exists in the form of lipoprotein and free ApoE protein exhibits weak efficacy and poor in vivo stability
Solution Approach 1:
The patent uses exosomes as an intermediary vehicle to deliver ApoE protein. The exosomal carrier protects ApoE from degradation in the complex in vivo environment, maintains its structural integrity, and enhances its stability and efficacy compared to free ApoE protein administration.
4Ease of operation
If Fusion proteins are formed by MyD88 inhibitor polypeptides and membrane-penetrating peptides to enable cell entry, then they can penetrate cell membranes, but they lack targeting property and pose safety risks due to extensive systemic immunosuppression
Solution Approach 1:
The patent uses exosomes as a natural intermediary that inherently possesses cell membrane fusion capabilities. This eliminates the need for artificial membrane-penetrating peptide fusions, providing targeted delivery to immune cells while maintaining safety and avoiding systemic immunosuppression side effects.
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 method achieves a 1000-fold enhancement in efficacy compared to free CD24-Fc fusion proteins, improves drug safety, and prolongs the duration of inflammation suppression, making exosomes a promising focus for drug development in immune-regulating therapies.
Implementation Method 1
knocking out ADAM10 gene in HEK293 cells
Implementation Method 2
Extracellular vesicles (EVs) have a vesicular structure and are secreted during cell growth. They often carry intracellular proteins and nucleic acids during their formation
Implementation Method 3
loading dimeric CD24 and/or ApoE proteins, and MyD88 inhibitor polypeptides into exosomes... achieving over a 1000-fold enhancement in efficacy
Data Source
AI summary
The present disclosure relates to a method for loading a dimeric CD24 into an HEK293 cell exosome with ADAM10 gene knocked out. By means of loading a dimeric CD24 and/or ApoE protein into an HEK293 cell exosome with ADAM10 gene knocked out, the efficacy is improved over 1000 times compared with that of a free CD24-Fc fusion protein and ApoE protein. Meanwhile, a MyD88 inhibitor polypeptide is loaded into the exosome, so that the inhibition efficacy of the exosome on an inherent immune inflammatory response is improved. After the CD24-exosome, the ApoE-exosome, or the CD24-ApoE-exosome loaded with the described inhibitor polypeptide is phagocytosed and removed by an immune cell, the MyD88 inhibitor polypeptide can be released in the cell, the inflammation inhibition efficacy is continuously exerted, and the duration of drug's action is effectively prolonged.