Recombinant Diphtheria Toxin Platform for Polypeptide Delivery
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Solution Overview
Problem
Current protein delivery platforms face challenges in achieving targeted and efficient delivery of larger macromolecules, such as peptides and proteins, into cells due to nonspecific interactions with membranes, limiting their therapeutic potential.
Innovation Solution
A recombinant molecule comprising a cargo polypeptide, a diphtheria toxin enzymatic fragment (DTA), and a diphtheria toxin translocation fragment (DTB) is used to facilitate the delivery of diverse proteins into cells, leveraging the receptor-binding and translocation properties of native DTB to direct foreign proteins into the cytosol.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cell-penetrating peptides, lipid-based molecules, nanoparticles, or other protein delivery platforms are used to deliver proteins into cells, then protein delivery capability is achieved to varying degrees, but cell-selectivity and efficiency-of-delivery remain poor due to nonspecific interactions with membranes
Solution Approach 1:
The invention segments the diphtheria toxin into two functional domains: DTB (receptor-binding and translocation domain) and DTA (enzymatic domain). The DTB domain is responsible for specific cell recognition and membrane translocation, while the DTA domain provides the enzymatic function. This segmentation allows the delivery platform to achieve both high cell-selectivity (through DTB's specific receptor binding) and high delivery efficiency (through the coordinated translocation-enzymatic action), resolving the contradiction between these two parameters.
Solution Approach 2:
The invention uses the DTB domain as an intermediary that mediates between the cargo protein and the target cell membrane. The DTB domain specifically binds to cell surface receptors and facilitates the translocation of the cargo-DTA complex into the cell, enabling selective and efficient delivery without nonspecific membrane interactions. This intermediary mechanism resolves the contradiction by providing a specific recognition pathway that enhances both selectivity and efficiency.
2Adaptability or versatility
If larger macromolecules such as peptides and proteins are used as therapeutics, then therapeutic potential is increased, but penetration into cells is generally excluded due to size
Solution Approach 1:
The invention merges the cargo protein (therapeutic macromolecule) with the DTB domain (translocation facilitator) to create a fusion protein complex. This merging allows the large therapeutic protein to overcome its size-related penetration barrier by utilizing the DTB domain's ability to bind receptors and facilitate translocation. The cargo protein retains its therapeutic potential while gaining the penetration capability of the DTB domain, resolving the contradiction between these two parameters.
Solution Approach 2:
The DTB domain acts as an intermediary that enables large therapeutic macromolecules to penetrate cell membranes. Instead of relying on the macromolecule's own penetration capability (which is limited by size), the DTB domain mediates the translocation process by binding to cell surface receptors and facilitating entry. This intermediary mechanism allows large proteins to achieve cell penetration while maintaining their therapeutic potential.
3Productivity
If nonspecific protein delivery platforms are used, then delivery capability is achieved, but target cell specificity is lost
Solution Approach 1:
The invention applies local quality by endowing the DTB domain with specific receptor-binding properties that are localized to particular cell types. The DTB domain contains specific amino acid sequences that recognize and bind to cell surface receptors on target cells, providing local specificity at the cell-membrane interface. This localized specific interaction ensures that delivery capability is achieved only in target cells, resolving the contradiction between delivery capability and target cell specificity.
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 platform effectively delivers a range of proteins, including therapeutic and genome-modifying proteins, across various sizes and stabilities, demonstrating enhanced target cell specificity and translocation efficiency, overcoming limitations of existing systems.
Implementation Method 1
leveraging the receptor-binding and translocation properties of native DTB to direct foreign proteins into the cytosol
Data Source
AI summary
There is a need for delivery platforms with robust capacity that offer the possibility to deliver diverse protein-based therapeutics into specific cells. Described herein is a platform for delivering cargo polypeptides into cells, which is based on a recombinant molecule comprising: a cargo polypeptide, a diphtheria toxin enzymatic fragment (DTA), and a diphtheria toxin translocation fragment (DTB). The platform has been employed to deliver diverse cargo into cells, including those having low or high molecular weights. A hyper-stable cargo polypeptide has been delivered, as well as proteins of therapeutic significance (e.g, MecP2, SMN, FMRP, PNP, alpha-amylase, and RRSP). The platform is also useful for delivering genome-modifying proteins, such as the CRISPR protein, Cas9. Associated nucleic acids, pharmaceutical compositions, methods, uses, and kits are also described, including those of therapeutic significance aimed at treating diseases or disorders caused by enzyme or protein deficiency.