pri-miRNA AIL Mutations for Precise microRNA Expression Control

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Solution Overview

Problem

Conventional methods for controlling microRNA expression, such as knocking-out or knocking-down target miRNA sequences, suffer from issues like production of unexpected miRNA sequences, off-target gene expression, and difficulty in controlling expression levels, and cannot increase miRNA expression.

Innovation Solution

Introduce mutations in the asymmetric internal loop (AIL) region of primary microRNA (pri-miRNA) to modulate the size of the AIL, controlling the activity of Microprocessor to produce single or double cut precursor microRNAs (pre-miRNAs), thereby modulating miRNA expression levels.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If conventional methods (knocking-out or knocking-down) are used to control miRNA expression, then miRNA expression levels can be reduced, but off-target gene expression and unexpected miRNA sequences are produced

Engineering Contradiction:

ImprovemiRNA expression levelsVSAvoidoff-target gene expression

Core Design Contradiction:

Quantity of substanceVSObject-generated harmful factors

Solution Approach 1:

The invention changes the structural parameters of pri-miRNA by introducing mutations in the AIL region to modulate its size. This parameter change directs the Microprocessor to produce single cut pre-miRNA instead of double cut pre-miRNA, thereby controlling miRNA expression levels without producing off-target effects or unexpected sequences

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention segments the miRNA processing pathway by interfering with the first cleavage step performed by Microprocessor. By modifying the AIL region, the process is divided into single cut and double cut pathways, allowing selective control over which pathway is activated to avoid harmful off-target effects

Inventive Principle:

Principle #1Segmentation

2Quantity of substance

If conventional methods are used to control miRNA expression, then expression levels can be reduced, but difficulty in controlling precise expression levels occurs

Engineering Contradiction:

ImprovemiRNA expression levelsVSAvoidcontrol precision of expression levels

Core Design Contradiction:

Quantity of substanceVSManufacturing precision

Solution Approach 1:

The invention enables precise control of miRNA expression levels by changing the AIL region size parameter. By introducing specific mutations (insertions or deletions) in the AIL region, the processing efficiency is precisely modulated, allowing fine-tuned control over single cut pre-miRNA production and corresponding miRNA expression levels

Inventive Principle:

Principle #35Parameter changes

3Quantity of substance

If conventional methods are used, then miRNA expression can be reduced, but it is not possible to increase miRNA expression

Engineering Contradiction:

ImprovemiRNA expression levelsVSAvoidability to increase or decrease expression

Core Design Contradiction:

Quantity of substanceVSAdaptability or versatility

Solution Approach 1:

The invention inverts the conventional approach by instead of reducing pre-miRNA or mature miRNA levels, it increases single cut pre-miRNA production through AIL region modification. This inverted strategy at the processing stage enables both upregulation and downregulation of final miRNA expression levels, providing bidirectional control

Inventive Principle:

Principle #13The other way round (Inversion)

Solution Approach 2:

The invention provides versatile control by changing the AIL region size parameter to different values. By introducing various mutations (different insertion or deletion sizes), the processing efficiency can be adjusted to achieve either increased or decreased miRNA expression levels as needed

Inventive Principle:

Principle #35Parameter changes

4Quantity of substance

If gene editing is used to control miRNA expression, then expression can be modulated, but complete knock-out occurs which may affect normal cellular functions

Engineering Contradiction:

ImprovemiRNA expression levelsVSAvoidnormal cellular functions

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The invention applies partial action by interfering only with the first cleavage step of miRNA processing while leaving the second cleavage step intact. By modulating single cut pre-miRNA production through AIL region changes, it achieves partial control over miRNA expression without complete knock-out, preserving normal cellular functions that depend on miRNAs

Inventive Principle:

Principle #16Partial or excessive action

Data Source

PatentUS12534725B2Method for controlling microRNA expression

Publication Date: 2026.01.27 THE HONG KONG UNIV OF SCI & TECH

AI summary

Disclosed are methods of producing single cut precursor microRNA (pre-miRNA) in a host cell from a primary microRNA (pri-miRNA). Also disclosed are method of increasing production levels of single cut precursor microRNA (pre-miRNA) and decreasing production levels of double cut precursor microRNA (pre-miRNA) in a host cell from a primary microRNA (pri-miRNA); a method of decreasing production levels of single cut precursor microRNA (pre-miRNA) and increasing production of double cut precursor microRNA (pre-miRNA) in a host cell from a primary microRNA (pri-miRNA); a method of modulating expression levels of microRNA (miRNA) in a host cell; a method of modulating expression levels of microRNA (miRNA) in a subject; a method of treating a disease in a subject. Also disclosed herein is a genetically modified primary microRNA (pri-miRNA).