Concatemeric RNA Molecules for Stable Gene Silencing

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Solution Overview

Problem

Current RNA interference (RNAi) therapies, specifically siRNAs, face challenges due to instability, low valence, and high toxicity, making efficient delivery difficult, and concatemeric siRNAs, while more stable, still trigger immune responses and cytotoxicity at therapeutic doses.

Innovation Solution

Development of concatemeric RNA molecules with enhanced Dicer processing efficiency and silencing potency, generated through rolling circle transcription, which include repeat units with specific nucleotide sequences and enzyme-specific cleavage sites, improving stability and delivery efficacy.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Stability of the object's composition

If concatemeric siRNA is used to improve stability, then stability is improved, but immune responses and cytotoxicity increase at therapeutic doses

Engineering Contradiction:

ImprovestabilityVSAvoidimmune responses and cytotoxicity

Core Design Contradiction:

Stability of the object's compositionVSObject-affected harmful factors

Solution Approach 1:

The concatemeric RNA molecule is divided into multiple repeat units, each containing a silencing sequence and a spacer sequence. This segmentation allows the molecule to maintain stability through repetition while the spacer sequences reduce immune recognition and cytotoxicity by preventing continuous immunostimulatory signaling.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Different regions of the concatemeric RNA molecule have different functions: the silencing sequences provide gene silencing activity, while the spacer sequences specifically reduce immune responses and cytotoxicity. This local differentiation of function allows simultaneous achievement of stability, reduced toxicity, and therapeutic efficacy.

Inventive Principle:

Principle #3Local quality

2Reliability

If siRNA is used for gene silencing, then gene silencing activity is achieved, but delivery efficiency is reduced due to instability and toxicity

Engineering Contradiction:

Improvegene silencing activityVSAvoiddelivery efficiency

Core Design Contradiction:

ReliabilityVSEase of operation

Solution Approach 1:

The siRNA is organized into multiple repeat units within a single concatemeric molecule, increasing the effective valence and molecular weight without requiring multiple separate delivery events. This improves delivery efficiency while maintaining gene silencing activity.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The concatemeric RNA combines multiple functional elements (silencing sequences, spacer sequences, repeat units) into a single composite molecule that simultaneously provides stability, reduced toxicity, and enhanced delivery efficiency compared to simple siRNA.

Inventive Principle:

Principle #40Composite materials

3Productivity

If higher doses of csiRNA are used to overcome cytotoxicity, then gene silencing efficacy is improved, but toxicity increases

Engineering Contradiction:

Improvegene silencing efficacyVSAvoidtoxicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The spacer sequences in specific locations within the repeat units reduce immune recognition and cytotoxicity, allowing higher effective doses of the silencing sequences to be delivered without proportional increases in toxicity.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The immunostimulatory properties of the repeat units are converted into beneficial sustained gene silencing activity, while the spacer sequences prevent harmful immune responses and cytotoxicity even at higher therapeutic doses.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

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 concatemeric RNA molecules exhibit superior silencing activity and prolonged efficacy, overcoming the delivery challenges of siRNAs by enhancing stability and reducing toxicity, with increased knockdown efficiency and sustained activity in cancer cells.

Implementation Method 1

transcribing said DNA template by rolling circle transcription (RCT) or rolling circle amplification (RCA) under appropriate conditions to yield a plurality of concatemeric RNA molecules

Methodology Applied

Scientific EffectRolling circle transcription: