Targeted RNA Circularization for Cell-Specific Therapeutic Expression

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Solution Overview

Problem

Existing RNA-based therapeutic delivery methods face challenges in stability, delivery capacity, and specificity, particularly for cell- or tissue-specific expression, with conventional gene therapy risking deleterious effects like endogenous gene disruption.

Innovation Solution

A linear RNA polynucleotide design comprising 5' and 3' homology arms and self-splicing introns that hybridize with target RNA to circularize and express payload sequences specifically in target cells or tissues, using self-splicing introns and optional spacers and riboregulators for controlled expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Duration of action of stationary object

If DNA is integrated into the genome for long-lasting expression, then expression duration is improved, but harmful effects such as endogenous gene disruption occur

Engineering Contradiction:

Improveexpression durationVSAvoidendogenous gene disruption

Core Design Contradiction:

Duration of action of stationary objectVSObject-generated harmful factors

Solution Approach 1:

The DNA construct is segmented into separate functional modules: homology arms for targeting, self-splicing introns for circularization, and payload sequences for therapeutic expression. This segmentation allows the system to achieve long-lasting expression through circular RNA formation without integrating into the host genome, thereby avoiding endogenous gene disruption.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Self-splicing introns serve as intermediary elements that facilitate circularization of the RNA transcript without requiring genomic integration. The introns mediate the formation of circular RNA structures that provide stable, long-term expression while remaining extrachromosomal, thus avoiding the harmful effects of DNA integration.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Object-generated harmful factors

If RNA-based delivery is used to avoid genomic integration, then harmful effects are reduced, but stability and delivery capacity deteriorate

Engineering Contradiction:

Improvegenomic integration risksVSAvoidRNA stability

Core Design Contradiction:

Object-generated harmful factorsVSStability of the object's composition

Solution Approach 1:

The RNA construct is pre-designed with homology arms and self-splicing intron sequences that enable automatic circularization upon entry into the target cell. This preliminary configuration ensures that the RNA rapidly converts to a stable circular form, improving stability and persistence without requiring genomic integration.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The system transitions from a linear RNA structure to a dynamic circular structure through self-splicing intron-mediated circularization. This dynamic transformation enhances the stability and delivery capacity of the RNA payload while maintaining the advantage of avoiding genomic integration.

Inventive Principle:

Principle #15Dynamics

3Quantity of substance

If conventional RNA delivery is used for broad expression, then delivery capacity is improved, but specificity deteriorates

Engineering Contradiction:

Improvedelivery capacityVSAvoidcell-type specificity

Core Design Contradiction:

Quantity of substanceVSManufacturing precision

Solution Approach 1:

The homology arms are designed with specific sequence complementarity to target RNA molecules unique to particular cell types or tissues. This local specificity in sequence design enables the RNA construct to selectively circularize and express the payload only in the intended target cells, achieving high cell-type specificity while maintaining delivery capacity.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The homology arms function as complementary copies or mirrors of specific target RNA sequences. By designing homology arms that are complementary to cell-type-specific RNA targets, the system achieves selective binding and circularization only in the intended cell types, thereby improving specificity without compromising delivery capacity.

Inventive Principle:

Principle #26Copying

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

Facilitates targeted, stable, and cell-specific delivery and expression of therapeutic proteins, reducing off-target effects and enhancing therapeutic efficacy by ensuring proteins are produced only where needed.

Implementation Method 1

binding of the homology arms to the target RNA allows splicing/ligation

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

the 5′ homology arm and the 3′ homology are complementary to target sequences in a target RNA

Methodology Applied

Scientific EffectBase pairing:

Implementation Method 3

a 3′ self-splicing intron fragment containing a 3′ splice site dinucleotide, (vi) a 5′ self-splicing intron fragment containing a 5′ splice site dinucleotide

Methodology Applied

Scientific EffectSelf-splicing: