Circular RNA Stability via AAV Backsplicing

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Solution Overview

Problem

Current methods for expressing proteins from RNA molecules face challenges due to the instability and short half-life of linear RNA forms, which limits long-term gene expression in applications like gene delivery.

Innovation Solution

Development of covalently closed circular RNA (circRNA) molecules that include a gene of interest, intronic elements for backsplicing, an internal ribosome entry site (IRES) for translation, and regulatory regions in the UTRs, packaged within an AAV genome and capsid for efficient tissue-specific expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Stability of the object's composition

If linear RNA molecules are used for gene expression, then the expression system is simple, but the RNA stability is poor and half-life is short

Engineering Contradiction:

ImproveRNA stabilityVSAvoidRNA structure complexity

Core Design Contradiction:

Stability of the object's compositionVSDevice complexity

Solution Approach 1:

The patent applies circularization of linear RNA to create covalently closed circular RNA molecules. This curvature transformation eliminates the free 5' and 3' ends that are susceptible to degradation, thereby significantly enhancing RNA stability and half-life while enabling sustained protein expression in gene delivery applications

Inventive Principle:

Principle #14Spheroidality (Curvature)

2Stability of the object's composition

If circular RNA molecules are designed with intronic elements for backsplicing, then the RNA stability is enhanced, but the manufacturing complexity increases

Engineering Contradiction:

ImprovecircRNA stabilityVSAvoidcircRNA production ease

Core Design Contradiction:

Stability of the object's compositionVSEase of manufacture

Solution Approach 1:

The patent utilizes the cell's own splicing machinery to perform backsplicing of intronic elements containing inverted repeats. The intronic sequences self-assemble into circular structures through backsplicing mediated by cellular enzymes, eliminating the need for complex external circularization procedures and simplifying the manufacturing process while maintaining high stability

Inventive Principle:

Principle #25Self-service

3Duration of action of moving object

If circRNA is used for long-term gene expression, then the duration of action is prolonged, but the translation efficiency may be reduced due to lack of ribosome association

Engineering Contradiction:

Improvegene expression durationVSAvoidtranslation efficiency

Core Design Contradiction:

Duration of action of moving objectVSProductivity

Solution Approach 1:

The patent incorporates IRES (internal ribosome entry site) sequences within the circular RNA structure. The IRES element provides an alternative translation initiation mechanism that does not require the 5' cap structure, enabling efficient protein translation from the circular RNA template and resolving the conflict between prolonged expression duration and translation efficiency

Inventive Principle:

Principle #6Universality (Multi-functionality)

Data Source

PatentUS20240141379A1Methods and compositions for circular RNA molecules

Publication Date: 2024.05.02 THE UNIV OF NORTH CAROLINA AT CHAPEL HILL

AI summary

This invention is directed to AAV compositions for circular RNA expression and methods of expressing covalently closed, circular RNA.