G-Quadruplex ASO Conjugates for Nuclease Resistance

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Solution Overview

Problem

Current antisense oligonucleotide (ASO) therapies face challenges in delivering ASOs to specific target organs and tissues with minimal off-site accumulation and in releasing them into the correct cellular compartment, such as the cytoplasm or nucleus, due to limitations in nuclease resistance and compatibility with RNase H cleavage.

Innovation Solution

The development of novel G-quadruplex-containing conjugates, where an antisense oligonucleotide (ASO) is heterologously combined with a G-quadruplex structure, enhanced with chemical modifications and linked via a modified linker, allowing for targeted delivery and enhanced nuclease resistance while maintaining compatibility with RNase H degradation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If phosphorothioate (PS) backbone modification is applied uniformly across ASO compounds, then nuclease resistance and serum albumin binding are improved, but the complexity of chemical modification increases

Engineering Contradiction:

Improvenuclease resistanceVSAvoidchemical modification complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The ASO is divided into three distinct segments: a 5' cap region with PS-modified nucleotides for nuclease resistance and albumin binding, a central gap region with unmodified DNA nucleotides for RNase H cleavage activity, and a 3' tail region with PS-modified nucleotides for stability. This segmentation allows each region to perform its specific function optimally without compromising the others.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Different chemical modifications are applied to different regions of the ASO based on their specific functional requirements. The 5' and 3' ends receive PS modification for protection and binding, while the central region remains unmodified to maintain RNase H compatibility. This local differentiation of chemical properties optimizes overall performance.

Inventive Principle:

Principle #3Local quality

2Reliability

If bridged nucleic acid (BNA) analogues such as LNA are incorporated into ASO, then binding affinity with target RNA is enhanced, but compatibility with RNase H cleavage is reduced

Engineering Contradiction:

Improvebinding affinityVSAvoidRNase H cleavage compatibility

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The ASO is segmented into regions with different nucleotide types: BNA analogues (LNA or cEt) are placed in the 5' and/or 3' cap regions to provide high binding affinity and stability, while the central gap region contains unmodified DNA nucleotides that are compatible with RNase H cleavage. This spatial segregation resolves the conflict between affinity enhancement and enzyme compatibility.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

BNA analogues are locally applied only where high binding affinity is needed (the cap regions flanking the target site), while the central region maintains DNA characteristics necessary for RNase H recognition and cleavage. This localized application of different nucleotide properties optimizes both binding and cleavage functions.

Inventive Principle:

Principle #3Local quality

3Object-affected harmful factors

If ASO is designed for targeted delivery to specific organs and tissues, then off-site accumulation is reduced, but the complexity of delivery system increases

Engineering Contradiction:

Improveoff-site accumulationVSAvoiddelivery system complexity

Core Design Contradiction:

Object-affected harmful factorsVSDevice complexity

Solution Approach 1:

GalNAc (N-acetylgalactosamine) moieties are attached to the 5' end of the ASO, serving as a molecular intermediary that specifically binds to asialoglycoprotein receptors on hepatocytes. This intermediary enables targeted delivery to the liver without requiring complex delivery systems, as the GalNAc-ASO conjugate is actively transported into liver cells via receptor-mediated endocytosis.

Inventive Principle:

Principle #24Intermediary (Mediator)

4Duration of action of stationary object

If chemical modifications are applied to improve nuclease resistance, then half-life in circulation is extended, but compatibility with biological degradation pathways is reduced

Engineering Contradiction:

Improvecirculation half-lifeVSAvoidbiological degradation compatibility

Core Design Contradiction:

Duration of action of stationary objectVSAdaptability or versatility

Solution Approach 1:

The ASO is segmented into protected regions (5' and 3' caps with PS modification) that resist nuclease degradation and extend circulation half-life, and a vulnerable central region (gap with unmodified DNA) that remains accessible to RNase H for targeted cleavage of the RNA transcript. This segmentation ensures both stability in circulation and efficacy at the target site.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Chemical modifications are applied locally only where needed for stability (the terminal cap regions), while the central region maintains natural DNA properties to enable biological degradation via RNase H. This localized modification strategy balances protection with controlled degradability.

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

This approach improves the stability, delivery, and efficacy of ASOs by enhancing nuclease resistance and cellular uptake, allowing for effective gene knockdown with reduced off-target effects and improved therapeutic index.

Implementation Method 1

Methodology Applied

Scientific EffectG-quadruplex structure:

Implementation Method 2

Antisense technology utilizes the complementary hybridization of an antisense oligonucleotide (ASO) to modulate the amount, activity, or function of the target RNA

Methodology Applied

Scientific EffectComplementary hybridization:

Implementation Method 3

This issue was adequately addressed by the 'gapmer' design, which places these nucleotide analogues at the two flanking ends of a middle gap that is made up of DNA units. In this manner, an ASO gapmer is bestowed with improved nuclease resistance and binding affinity for the target RNA, yet still allows for RNase H cleavage of the bound RNA partner within the gap

Methodology Applied

Scientific EffectRNase H cleavage: Enzyme

Data Source

PatentUS11390867B2G-quadruplex-containing antisense oligonucleotides

Publication Date: 2022.07.19 NANYANG TECH UNIV

AI summary

The present invention relates to conjugates comprising (a) an antisense oligonucleotide (ASO) and (b) at least one G-quadruplex structure, wherein the ASO and the at least one G-quadruplex structure are heterologous to each other. The G-quadruplex of the conjugate may be further conjugated with ligands or functional moieties for addressable delivery and enhanced properties including potency and therapeutic index. Further encompassed are such conjugates for use as a medicament, a method of modulating the stability, translation, splicing, cleavage, or activity of a target nucleic acid molecule using such conjugates, and use of G-quadruplex in stabilizing antisense oligonucleotides.