CRISPR Cascade Activation for Cell-Specific In Vivo Therapeutics
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Solution Overview
Problem
Existing biotherapeutic delivery systems face challenges in achieving non-specific delivery while ensuring cell-specific functionality, leading to issues with aspecific toxic effects on unaffected cells and tissues.
Innovation Solution
A CRISPR-based cascade system using a first ribonucleoprotein complex with a combination guide nucleic acid that includes a blocked second guide nucleic acid, which upon binding to a target nucleic acid, becomes unblocked to form a second ribonucleoprotein complex, activating effector nucleic acids in a cell-specific manner.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If non-specific delivery of therapeutic components is used, then the need for complex cell-targeting systems is alleviated, but aspecific toxic effects on unaffected cells and tissues occur
Solution Approach 1:
The system divides the therapeutic delivery into two separate components: (1) a cascade system component that can be delivered non-specifically to all cells, and (2) a cell-specific marker that only exists in target cells. The cascade system includes a first ribonucleoprotein complex with a combination guide nucleic acid that couples a first guide nucleic acid (specific for the cell marker) and a blocked second guide nucleic acid. This segmentation allows simple delivery while ensuring action only in cells with the specific marker, preventing aspecific toxicity.
Solution Approach 2:
The system performs preliminary action by delivering the cascade system components to all cells in advance, but the therapeutic effect is conditional. The blocked second guide nucleic acid is designed to be unblocked only when the first ribonucleoprotein complex binds to the cell-specific marker. This preliminary delivery followed by conditional activation ensures that therapeutic components are present in all cells but only become active in target cells, resolving the contradiction between simple delivery and specific action.
2Object-affected harmful factors
If cell-specific targeting systems are used, then aspecific toxic effects are overcome, but complex cell-targeting systems are required
Solution Approach 1:
The cascade system component serves multiple functions: it can be delivered non-specifically to any cell type, it contains the machinery to detect cell-specific markers, and it can activate therapeutic effects. The combination guide nucleic acid couples detection of the cell marker with activation of the therapeutic effector nucleic acid. This multi-functionality eliminates the need for separate complex targeting systems while ensuring cell-specific action.
Solution Approach 2:
The cell-specific marker acts as an intermediary that bridges non-specific delivery and specific therapeutic action. The first guide nucleic acid is designed to be complementary to this marker, serving as the recognition element. When the marker is present, it triggers the unblocking of the second guide nucleic acid, which then activates the therapeutic effector. This intermediary mechanism allows simple delivery systems to achieve cell-specific effects without requiring complex targeting moieties.
3Ease of manufacture
If therapeutic components are activated in all cells, then delivery is simplified, but toxicity increases due to aspecific effects
Solution Approach 1:
The system implements local quality by making the therapeutic component's activity dependent on the local cellular environment. The blocked second guide nucleic acid contains a region that is complementary to the cell-specific marker. Only in cells where this marker is present does the first ribonucleoprotein complex become activated and unblock the second guide, thereby activating the therapeutic effector nucleic acid. This local activation mechanism simplifies delivery while preventing aspecific toxicity through conditional activation based on cellular characteristics.
4Object-generated harmful factors
If cell-specific activation is implemented, then toxicity is reduced, but the system complexity increases
Solution Approach 1:
The system uses a nested structure where the combination guide nucleic acid contains a first guide nucleic acid nested within it that is complementary to the cell marker. When the first guide binds to its target, it triggers a conformational change or cleavage event that unblocks the second guide nucleic acid. This nested arrangement allows two levels of specificity (marker detection and effector activation) to be integrated into a single molecular construct, reducing overall system complexity while maintaining cell-specific activation and low toxicity.
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 system enables selective activation of effector nucleic acids in target cells, reducing non-specific toxicity and enhancing therapeutic efficacy by ensuring precise cellular action.
Implementation Method 1
a first guide nucleic acid which comprises a region that binds to the first nucleic acid-guided nuclease and a region complementary to a target nucleic acid of interest
Implementation Method 2
the first nucleic acid-guided nuclease exhibits both cis- and trans-cleavage activity and upon binding of the target nucleic acid of interest to the first ribonucleoprotein complex, the first ribonucleoprotein complex becomes active initiating trans-cleavage activity thereby unblocking the blocked second guide nucleic acid portion
Implementation Method 3
the second guide nucleic acid portion when unblocked is able to form the second ribonucleoprotein complex with the second guide nucleic acid-guided nuclease
Implementation Method 4
the at least one unblocked second guide nucleic acid molecule forms the second ribonucleoprotein complex with the second nucleic acid-guided nuclease and is able to bind to and activate the effector nucleic acid
Data Source
AI summary
The present disclosure relates to compositions of matter and methods used to activate effector nucleic acids and effector targets in vivo via a CRISPR-based cascade system. The compositions and methods achieve non-specific delivery of cascade system components to cells yet the cascade system works in a cell-specific manner.