Mutated Alphavirus Replicase mRNA for Controlled Protein Expression

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Existing mRNA therapies face limitations due to structural instability, innate immunogenicity, and inefficient in vivo delivery, particularly for self-replicating mRNA systems which have high cytotoxicity and unpredictable replication, making them unsuitable for repeated administration.

Innovation Solution

A limited self-replicating mRNA molecular system is developed, featuring a mutated alphavirus replicase with specific mutations in the nsP2 region and a target protein mRNA, optimized with chemical modifications and structural elements like 5' cap structures and UTR sequences, to achieve controlled replication and reduced cytotoxicity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Duration of action of moving object

If self-replicating mRNA molecular system is used to amplify protein translation instructions, then expression duration and protein production efficiency are improved, but cytotoxicity and immune response increase

Engineering Contradiction:

Improveexpression durationVSAvoidcytotoxicity

Core Design Contradiction:

Duration of action of moving objectVSObject-affected harmful factors

Solution Approach 1:

The patent extracts and removes the viral structural protein coding sequence from the self-replicating mRNA system, retaining only the replicase function. This extraction eliminates the harmful viral structural proteins while preserving the beneficial self-replication capability, thereby reducing cytotoxicity and immune response while maintaining expression duration

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent introduces specific nucleoside modifications (such as pseudouridine, inosine, or other non-canonical nucleosides) to change the chemical parameters of the mRNA molecule. These modifications reduce innate immune recognition and cytotoxicity while preserving the self-replication function and expression duration

Inventive Principle:

Principle #35Parameter changes

2Productivity

If self-replicating mRNA molecular system is used to prolong expression, then protein production is enhanced, but unpredictability of replication and toxicity increase

Engineering Contradiction:

Improveprotein production efficiencyVSAvoidreplication predictability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent introduces a feedback control mechanism where the replicase activity is regulated by the presence of specific cellular factors or metabolic conditions. This feedback loop ensures that replication occurs only under appropriate conditions, making the replication process predictable and controllable while maintaining high protein production efficiency

Inventive Principle:

Principle #23Feedback

Solution Approach 2:

The patent makes the replicase activity dynamic and responsive to cellular conditions rather than static and autonomous. The replicase function is activated or inhibited based on cellular metabolic state, allowing predictable control of replication timing and duration while maintaining high productivity

Inventive Principle:

Principle #15Dynamics

3Object-affected harmful factors

If nucleoside modification is applied to reduce immune rejection, then innate immune response is reduced, but modification complexity and production difficulty increase

Engineering Contradiction:

Improveimmune responseVSAvoidproduction difficulty

Core Design Contradiction:

Object-affected harmful factorsVSEase of manufacture

Solution Approach 1:

The patent segments the mRNA modification process into modular steps: first synthesizing the basic mRNA sequence, then introducing specific nucleoside modifications at defined positions. This segmentation allows each modification to be optimized independently and simplifies the overall production process while maintaining effective immune evasion

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent uses chemical intermediaries or enzymatic tools that facilitate the introduction of modified nucleosides during mRNA synthesis. These intermediaries simplify the manufacturing process by providing ready-to-incorporate modified nucleotides, reducing the complexity of producing modified mRNA while maintaining effective immune response reduction

Inventive Principle:

Principle #24Intermediary (Mediator)

Data Source

PatentUS20240200042A1Limited self-replicating mRNA molecular system, producing method and use

Publication Date: 2024.06.20 ZHENHE PHARM (HANGZHOU) CO LTD

AI summary

The application relates to the technical field of biomedicine, in particular to a limited self-replicating mRNA molecular system, producing method and use. The limited self-replicating mRNA molecular system including: a first mRNA encoding a mutated alphavirus replicase; and at least one second mRNA encoding a target protein; by generating specific mutation adjustments in the nsP2 subunit of mutated replicase, this limited self-replicating mRNA molecular system can achieve limited self-replication and avoid cytotoxicity; by constructing different mRNA with mutated alphavirus replicase and different target proteins, the mutated alphavirus replicase encoded by the first mRNA can simultaneously replicate multiple different target proteins, achieving sustained expression of multiple target proteins.