Minimal ARRDC1 Constructs for High-Capacity ARMM Cargo Delivery
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Solution Overview
Problem
Current delivery systems for molecular cargos such as small molecules, proteins, and nucleic acids face limitations in safety and efficiency, often triggering immune responses and being unable to package large cargos effectively.
Innovation Solution
Utilizing minimal ARRDC1 proteins, which are shorter than full-length ARRDC1, to drive the formation of ARMMs that can package and deliver molecular cargos, including Cas9 proteins and guide RNAs, through direct fusion or protein-protein interactions, and targeting specific tissues via antibodies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If full-length ARRDC1 protein is used to drive ARMM budding, then the budding function is achieved, but the cargo packaging capacity is limited due to the protein occupying significant space within the vesicle
Solution Approach 1:
The patent extracts only the essential functional domains from full-length ARRDC1 (arrestin domain, PSAP/PTAP motif, and PPXY motifs) to create a minimal ARRDC1 protein. This extraction removes non-essential regions while preserving the budding function, thereby reducing the protein's spatial occupation within ARMMs and increasing cargo packaging capacity.
Solution Approach 2:
The patent segments the ARRDC1 protein into its critical functional components (arrestin domain, PSAP/PTAP motif, PPXY motifs) and reconstructs a minimal version. This segmentation allows identification and retention of only those regions necessary for budding activity, optimizing the protein's efficiency as a cargo delivery vehicle.
2Productivity
If viral delivery systems are used to deliver molecular cargos, then delivery efficiency is achieved, but strong immune responses are triggered
Solution Approach 1:
The patent converts the harmful immune response triggered by viral vectors into a benefit by using an endogenous cellular pathway (ARRDC1-mediated budding) that the immune system does not recognize as foreign. This approach maintains high delivery efficiency while avoiding the inflammatory responses associated with viral delivery systems.
Solution Approach 2:
The patent uses ARRDC1 and ARMMs as intermediary carriers to transfer molecular cargos into target cells. Unlike viral vectors that directly infect cells and trigger immunity, this intermediary system uses a natural cellular budding mechanism, thereby achieving delivery without activating the immune response.
3Quantity of substance
If current delivery systems are used to deliver large molecular cargos, then delivery is attempted, but packaging capacity is insufficient
Solution Approach 1:
The patent changes the parameter of protein size by creating a minimal ARRDC1 version that is smaller than full-length ARRDC1. This parameter change increases the available volume within ARMMs for cargo packaging, enabling the system to accommodate larger molecular cargos such as Cas9 proteins and guide RNAs.
Data Source
AI summary
Disclosed herein are minimal arrestin domain containing protein 1 (ARRDC1) constructs, which drive the formation of ARRDC1-mediated microvesicles (ARMMs). These vesicles can be harnessed to package and deliver a variety of molecular cargos such as small molecules, nucleic acids, and proteins. An example of such cargo is the genome editor Cas9.