Guide RNA 5′-Phosphate Removal for RNP Delivery
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Solution Overview
Problem
The delivery of ribonucleoprotein complexes, such as those containing Cas9 or Cpf1 proteins and guide RNA, often induces immune responses and cytotoxicity due to the presence of 5′-terminal phosphate groups, leading to off-target effects and reduced efficacy.
Innovation Solution
Removing the 5′-terminal phosphate groups from guide RNA before forming ribonucleoprotein complexes with Cas9 or Cpf1 proteins, thereby reducing immune response induction and cytotoxicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If guide RNA with 5′-terminal phosphate groups is used for RNP delivery, then the guide RNA can be directly synthesized by in-vitro transcription, but immune response and cytotoxicity are induced
Solution Approach 1:
The patent removes the harmful 5′-terminal phosphate groups from the guide RNA molecule through enzymatic treatment with alkaline phosphatase. This extraction of the problematic phosphate groups eliminates the immune response and cytotoxicity while preserving the guide RNA's functional capability to direct RGEN to target sequences.
Solution Approach 2:
The patent modifies the chemical structure of guide RNA by changing the 5′-terminal phosphate parameter to a 5′-hydroxyl group. This parameter change fundamentally alters the interaction with host immune sensors, transforming the RNA from immunogenic to non-immunogenic while maintaining its guide function.
2Ease of manufacture
If guide RNA with 5′-terminal phosphate groups is used for RNP delivery, then synthesis is straightforward, but cytotoxicity increases
Solution Approach 1:
The patent removes the harmful 5′-terminal phosphate groups from the guide RNA molecule through enzymatic treatment with alkaline phosphatase. This extraction of the problematic phosphate groups eliminates the immune response and cytotoxicity while preserving the guide RNA's functional capability to direct RGEN to target sequences.
Solution Approach 2:
The patent modifies the chemical structure of guide RNA by changing the 5′-terminal phosphate parameter to a 5′-hydroxyl group. This parameter change fundamentally alters the interaction with host immune sensors, transforming the RNA from immunogenic to non-immunogenic while maintaining its guide function.
3Object-affected harmful factors
If 5′-terminal phosphate groups are removed from guide RNA, then immune response and cytotoxicity are reduced, but an additional treatment step is required
Solution Approach 1:
The patent removes the harmful 5′-terminal phosphate groups from the guide RNA molecule through enzymatic treatment with alkaline phosphatase. This extraction of the problematic phosphate groups eliminates the immune response and cytotoxicity while preserving the guide RNA's functional capability to direct RGEN to target sequences.
Solution Approach 2:
The patent introduces alkaline phosphatase as an intermediary enzyme to facilitate the removal of 5′-terminal phosphate groups. This intermediary tool enables the transformation of guide RNA from immunogenic to non-immunogenic form through a well-established enzymatic mechanism, adding minimal complexity to the workflow.
4Speed
If guide RNA with 5′-terminal phosphate groups is used, then rapid action is achieved, but off-target effects increase
Solution Approach 1:
The patent removes the harmful 5′-terminal phosphate groups from the guide RNA molecule through enzymatic treatment with alkaline phosphatase. This extraction of the problematic phosphate groups eliminates the immune response and cytotoxicity while preserving the guide RNA's functional capability to direct RGEN to target sequences.
Solution Approach 2:
The patent modifies the chemical structure of guide RNA by changing the 5′-terminal phosphate parameter to a 5′-hydroxyl group. This parameter change fundamentally alters the interaction with host immune sensors, transforming the RNA from immunogenic to non-immunogenic while maintaining its guide function.
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 approach significantly decreases immune response and cytotoxicity, maintaining on-target activity while enhancing cell viability and genome editing efficiency.
Implementation Method 1
The 5′-terminal phosphate groups can be removed by treating the guide RNA with a phosphatase, such as calf intestinal alkaline phosphatase (CIP) or shrimp alkaline phosphatase (SAP).
Data Source
AI summary
Provided are a composition for ribonucleoprotein delivery, comprising a guide RNA free of 5′-terminal phosphates, and a method for ribonucleoprotein delivery, using the same.