ARRDC1-Mediated Microvesicles for Cas9 Delivery
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Solution Overview
Problem
Current methods for delivering proteins, such as Cas9, into cells face challenges including poor permeability and target specificity, with viral delivery systems risking immune responses and toxicity.
Innovation Solution
Utilizing ARRDC1-mediated microvesicles (ARMMs) to load and deliver Cas9 proteins or variants, which are derived from an endogenous budding pathway, reducing immune response and allowing specific targeting through tissue-specific markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If viral delivery systems are used to deliver Cas9 proteins, then delivery efficiency is improved, but immune responses and toxicity increase
Solution Approach 1:
The patent uses ARRDC1-mediated microvesicles as an intermediary delivery vehicle between the Cas9 protein and target cells. These microvesicles are derived from endogenous plasma membrane budding, serving as a natural mediator that avoids viral triggers while maintaining efficient delivery capability, thus resolving the contradiction between delivery efficiency and immune response
Solution Approach 2:
Instead of using viral vectors that replicate and may cause toxicity, the patent employs a non-viral copying approach using microvesicles that transfer Cas9 proteins through plasma membrane budding. This copying mechanism achieves delivery without the harmful replication and immune activation associated with viral systems
2Device complexity
If protein transduction is used to deliver Cas9 proteins, then delivery method simplicity is improved, but permeability and target specificity are poor
Solution Approach 1:
The patent introduces ARRDC1-mediated microvesicles as an intermediary that enhances both permeability and target specificity while maintaining relative simplicity. The microvesicles naturally fuse with target cell membranes (improving permeability) and can be directed to specific tissues (improving target specificity), overcoming the limitations of direct protein transduction
Solution Approach 2:
The microvesicle system utilizes the cell's own plasma membrane budding machinery to deliver Cas9 proteins, making the delivery system self-service rather than requiring external viral vectors or complex transduction mechanisms. This self-service approach improves reliability while keeping the system relatively simple
3Object-affected harmful factors
If endogenous budding pathway is used to produce ARMMs, then immune response is reduced, but delivery targeting capability must be enhanced
Solution Approach 1:
The patent applies local quality by incorporating tissue-specific markers or ligands on the surface of ARRDC1-mediated microvesicles. This allows different regions or surfaces of the microvesicle to have different functions: the core maintains endogenous budding properties (low immune response) while the surface provides specific targeting capability through localized marker expression
Solution Approach 2:
The ARRDC1-mediated microvesicle system achieves universality by combining multiple functions in a single delivery platform: it maintains endogenous budding characteristics to avoid immune responses while simultaneously providing targeting capability through customizable surface markers, thus serving both safety and specificity requirements
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
ARMMs enable efficient and targeted delivery of Cas9 proteins into cells, avoiding immune responses and improving the specificity and safety of protein-based therapeutics.
Implementation Method 1
ARMMs are microvesicles that are distinct from exosomes and which, like budding viruses, are produced by direct plasma membrane budding (DPMB)
Implementation Method 2
DPMB is driven by a specific interaction of TSG101 with a tetrapeptide PSAP (SEQ ID NO: 74) motif of the arrestin-domain-containing protein ARRDC1 accessory protein
Data Source
AI summary
Methods, systems, compositions and strategies for the delivery of WW domain-containing fusion proteins into cells in vivo, ex vivo, or in vitro via ARMMs are provided. Methods, systems, compositions and strategies for the delivery of Cas9 proteins and/or Cas9 variants into cells in vivo, ex vivo, or in vitro via fusion to ARMM associated proteins (e.g., ARRDC1 or TSG101) are also provided.