miRNA Expression Construct Spacer Optimization

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Solution Overview

Problem

Current miRNA-based therapeutics face limitations such as short-lived siRNAs, toxicity from shRNAs, and weak knockdown activity of synthetic miRNAs, which hinder their therapeutic potential for long-term gene correction and immune cell function in cancer and viral infections.

Innovation Solution

A miRNA expression construct comprising a promoter element, a spacer of at least 50 nucleotides in length, and a miRNA hairpin, optimized for efficient processing and knockdown, is developed. This construct includes specific sequences and structures to enhance target gene silencing, particularly for immune checkpoint genes, and can be delivered via viral vectors for sustained expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If synthetic miRNAs are used to mimic natural pathway, then therapeutic potential is improved, but knockdown activity is weak compared to shRNAs

Engineering Contradiction:

Improvetherapeutic potentialVSAvoidknockdown activity

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent modifies the miRNA hairpin structure by changing parameters such as stem length, loop size, and sequence composition to optimize processing by DROSHA and DICER, thereby enhancing mature miRNA production and knockdown activity while maintaining therapeutic safety

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The construct combines multiple elements including promoter, spacer, hairpin, and processing signals into a composite expression unit that synergistically enhances both the stability of miRNA and its knockdown efficiency, resolving the contradiction between therapeutic potential and knockdown activity

Inventive Principle:

Principle #40Composite materials

2Productivity

If shRNAs are used to bypass DROSHA processing, then knockdown efficiency is improved, but toxicity increases due to obstruction of natural miRNA pathway

Engineering Contradiction:

Improveknockdown efficiencyVSAvoidtoxicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent converts the potential harm of high knockdown efficiency (which causes toxicity) into a benefit by using miRNA that naturally processes through DROSHA/DICER, achieving sufficient knockdown without overwhelming the cytoplasm with dsRNA, thus eliminating toxicity while maintaining effectiveness

Inventive Principle:

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

Solution Approach 2:

The miRNA construct uses transient expression through viral vectors, allowing the miRNA to be produced and function temporarily without requiring permanent integration or accumulation, thereby avoiding long-term toxicity while maintaining adequate knockdown during the therapeutic window

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

3Productivity

If siRNAs are used for gene correction, then short-term effect is achieved, but long-term expression is insufficient

Engineering Contradiction:

Improveshort-term knockdownVSAvoidlong-term expression

Core Design Contradiction:

ProductivityVSDuration of action of stationary object

Solution Approach 1:

The patent employs viral vectors to deliver the miRNA construct before the actual knockdown is needed, allowing for pre-processing and accumulation of mature miRNA in the cell, which then provides sustained knockdown effect over an extended period rather than immediate short-term action

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The construct enables continuous production of mature miRNA through persistent viral expression, ensuring uninterrupted knockdown activity over time, thereby transitioning from short-term siRNA effect to long-term sustained gene correction

Inventive Principle:

Principle #20Continuity of useful action

4Productivity

If spacer sequence is added to enhance knockdown efficiency, then knockdown efficiency is improved, but construct complexity increases

Engineering Contradiction:

Improveknockdown efficiencyVSAvoidconstruct architecture

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent introduces a spacer sequence specifically at the 5' end of the hairpin where it is most effective for enhancing DROSHA processing and mature miRNA production, rather than adding spacers throughout the entire construct, thereby improving knockdown efficiency with minimal increase in overall construct complexity

Inventive Principle:

Principle #3Local quality

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 miRNA expression construct achieves enhanced knockdown efficiency and sustained expression of target genes, improving therapeutic potential for cancer and viral infection treatments by optimizing spacer length and sequence, and promoting effective immune cell function.

Implementation Method 1

Structural elements of the hairpin provide a signal for processing by DROSHA and DICER, leading to formation of a ̃20-23 bp mature miRNA duplex

Methodology Applied

Scientific EffectRNA processing:

Implementation Method 2

The functional strand of mature miRNA duplex is incorporated into the RISC complex, which facilitates target mRNA recognition and eventually gene knockdown

Methodology Applied

Scientific EffectRNA interference:

Implementation Method 3

The presence of a spacer appears to enhance knockdown efficiency (Stegmeier et al., 2005), however it is not known whether sequence length or other biophysical parameters of the spacer are of importance

Methodology Applied

Scientific EffectGene expression regulation:

Implementation Method 4

miRNA genes are most of the time driven by polII-dependent promoters which allow tissue specific or/and inducible expression

Methodology Applied

Scientific EffectTranscription:

Data Source

PatentUS11649455B2Micro RNA expression constructs and uses thereof

Publication Date: 2023.05.16 UNIVERSITY OF ZURICH

AI summary

The present disclosure relates to miRNA expression constructs, such as for expression of multiple miRNAs and use thereof to knockdown target gene expression. In some aspects, the expression constructs include a promoter element, a spacer sequence and a miRNA coding sequence. In some aspects, constructs provide enhanced immune cell function.