Phosphorodiamidate Morpholino Oligonucleotides for Aberrant Splicing

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Solution Overview

Problem

Hutchinson-Gilford progeria syndrome (HGPS) is characterized by premature aging due to a single-point mutation in the lamin A (LMNA) gene, leading to the production of progerin, a dominant negative mutant protein that causes nuclear defects, and existing therapies are inadequate in modulating aberrant splicing effectively.

Innovation Solution

Phosphorodiamidate morpholino oligonucleotides (PMOs) are used to target and modulate aberrant splicing of human LMNA pre-mRNA by hybridizing to specific sites, preventing the recognition by the cellular splicing machinery and reducing the expression of progerin, with specific sequences provided as SEQ ID NOs: 4 and 11.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional antisense oligonucleotides are used to target LMNA splicing, then splicing modulation is attempted, but the oligonucleotides are degraded by nucleases and fail to effectively reduce progerin expression

Engineering Contradiction:

Improvestability of oligonucleotideVSAvoidefficacy in reducing progerin

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent modifies the chemical parameters of the oligonucleotide backbone by replacing phosphodiester bonds with phosphorodiamidate morpholino structures. This chemical parameter change confers nuclease resistance while maintaining the ability to hybridize to target RNA and modulate splicing, thereby simultaneously improving stability and therapeutic efficacy

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention creates a composite oligonucleotide structure combining morpholino rings with phosphorodiamidate linkages and nucleobase attachments. This composite material integrates the structural stability of morpholino with the hybridization capability of nucleobases, achieving both nuclease resistance and functional activity

Inventive Principle:

Principle #40Composite materials

2Reliability

If RNA cleavage is permitted by using traditional antisense oligonucleotides, then oligonucleotide degradation occurs, but using SSOs with double-stranded structure eliminates RNA cleavage possibility while improving stability

Engineering Contradiction:

Improveresistance to degradationVSAvoidstructural complexity of oligonucleotide

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent extracts the phosphodiester backbone from the oligonucleotide structure and replaces it with phosphorodiamidate morpholino linkages. This extraction of the vulnerable backbone component and its replacement with a more stable alternative eliminates the susceptibility to nuclease-mediated phosphodiester bond cleavage while maintaining oligonucleotide function

Inventive Principle:

Principle #2Taking out (Extraction)

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 PMOs effectively inhibit the expression of mutant LMNA protein mRNA by correcting aberrant splicing patterns, reducing progerin accumulation and associated nuclear defects, thereby addressing the premature aging symptoms in HGPS.

Implementation Method 1

SSOs block aberrant splicing sites by hybridizing at or near the sites thereby preventing recognition by the cellular splicing machinery

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

the resulting double-stranded structure eliminates the possibility of RNA cleavage by RNase H

Methodology Applied

Scientific EffectStructural protection:

Data Source

PatentEP3399038B1Oligonucleotide analogues targeting human lmna

Publication Date: 2022.11.16 SAREPTA THERAPEUTICS INC

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AI summary

Provided are LMNA-targeted antisense oligonucleotides for reducing expression of one or more aberrantly spliced LMNA mRNA isoforms that encode progerin.