Mutant CyaA Polypeptides for Safe Intracellular Delivery
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Solution Overview
Problem
Current polypeptides derived from adenylate cyclase protein CyaA, used for delivering molecules into cells, pose toxicity risks due to their cytolytic activity, limiting their therapeutic applications, especially for targeting CD11b/CD18 receptor-expressing cells.
Innovation Solution
Design of mutant polypeptides with specific substitutions (E570Q and K860R) that retain the ability to bind and translocate the N-terminal adenylate cyclase domain into cells while significantly reducing pore-forming activity, thereby minimizing cytotoxicity and enhancing safety for use as proteinaceous vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If native CyaA toxin is used for delivering molecules into cells, then translocating activity is achieved, but cytolytic activity and cell lysis occur
Solution Approach 1:
The invention extracts and removes the harmful pore-forming domain from the CyaA toxin structure, retaining only the beneficial translocating domain. This is achieved by using truncated versions of CyaA that lack the C-terminal pore-forming region, thereby eliminating cytolytic activity while preserving the ability to translocate molecules into cells.
Solution Approach 2:
The invention converts the harmful full-length CyaA toxin into a beneficial delivery vehicle by modifying its structure. The truncated CyaA derivatives maintain the translocating function but have their harmful pore-forming capability removed, effectively converting a toxic substance into a safe therapeutic delivery system.
2Object-affected harmful factors
If mutant polypeptides with reduced pore-forming activity are designed, then cytotoxicity is minimized, but binding and translocating activity must be retained
Solution Approach 1:
The invention applies local quality modification by specifically altering the C-terminal region of CyaA to reduce pore-forming activity while keeping the N-terminal translocating domain intact. This localized modification ensures that only the harmful portion is affected, while the beneficial translocating function remains preserved.
Solution Approach 2:
The invention changes the structural parameters of CyaA by truncating the polypeptide chain to remove the pore-forming domain. This parameter change (reducing molecular length and removing specific domains) directly reduces cytotoxicity while maintaining the translocating capability through the preserved N-terminal region.
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 mutant polypeptides effectively deliver molecules into CD11b/CD18-expressing cells with reduced cytolytic activity, making them suitable for therapeutic and prophylactic applications, including immunotherapy, without causing significant cell lysis or toxicity.
Implementation Method 1
binding to the CD11b/CD18 receptor
Implementation Method 2
translocating its N-terminal domain into the cell
Implementation Method 3
catalyzes uncontrolled conversion of cellular ATP to cAMP
Implementation Method 4
pore-forming activity which is reduced or suppressed
Data Source
Figure 1A~1B
Figure 1C
Figure 2A~2B
AI summary
The invention relates to mutant CyaA/E570Q+K860 polypeptides suitable for use as proteinaceous vectors for delivering one or more molecules of interest into a cell, in particular into a cell expressing the CD11b receptor. The invention further relates to polypeptide derivatives suitable for eliciting an immune response in a host. The invention is more particularly directed to polypeptides derived from an adenylate cyclase protein (CyaA) either under the form of a toxin or of a toxoid, which are mutant polypeptides. Said mutant polypeptides are capable of retaining the binding activity of native CyaA to a target cell and preferably of also retaining the translocating activity of native CyaA through its N-terminal domain into target cells and furthermore have a pore-forming activity which is reduced or suppressed as compared to that of the native CyaA toxin.