Multiple PMO-CPP Conjugates with Cleavable Linkers
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Solution Overview
Problem
Antisense oligonucleotides (AONs), particularly phosphorodiamidate morpholino oligonucleotides (PMOs), face challenges with poor uptake in skeletal and cardiac muscle cells, leading to ineffective gene modulation, and exhibit increased toxicity when conjugated with cationic cell-penetrating peptides (CPPs).
Innovation Solution
Development of multiple PMO-CPP conjugates with cleavable linkers, incorporating glutamic acid and peptidase cleavage sites, such as Phenylalanine-Lysine, to reduce steric hindrance and toxicity, allowing for enhanced exon skipping efficiency with a safer margin by releasing PMOs from CPPs within cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PMOs are conjugated with cationic cell-penetrating peptides (CPPs) to enhance uptake into skeletal and cardiac muscle cells, then cellular uptake is improved, but toxicity increases
Solution Approach 1:
The invention divides the single PMO-CPP conjugate into multiple separate PMO moieties attached to the CPP. This segmentation allows the CPP to maintain its cell-penetrating function while distributing the PMO load, reducing steric hindrance and toxicity. The multiple PMOs work collectively to achieve the desired gene modulation effect without the adverse effects of a single large conjugate.
Solution Approach 2:
The invention changes the structural parameters of the conjugate by attaching multiple smaller PMO moieties instead of one large PMO. This parameter change in the architecture of the conjugate (from 1:1 to multi:1 ratio) improves cellular uptake efficiency while reducing toxicity by minimizing steric hindrance and optimizing the CPP-PMO interaction.
2Object-affected harmful factors
If multiple PMO moieties are attached to CPP to reduce steric hindrance and toxicity, then safety margin is improved, but conjugate complexity increases
Solution Approach 1:
The conjugate structure is segmented into multiple identical or similar PMO-CPP units rather than one complex single unit. This segmentation approach, while increasing the number of components, actually simplifies the design by using repeating modular units that can be synthesized and characterized more easily than a single complex conjugate.
3Reliability
If single PMO-CPP conjugates are used to achieve exon skipping, then gene modulation is achieved, but the safety margin is reduced due to high toxicity
Solution Approach 1:
The single PMO-CPP conjugate is segmented into multiple PMO moieties attached to the CPP. This segmentation maintains the exon skipping efficacy by preserving the functional PMO units while reducing toxicity through decreased steric hindrance and optimized cellular uptake, thereby improving the safety margin.
Solution Approach 2:
The invention changes the stoichiometric parameters of the conjugate from a 1:1 ratio to a multi:1 ratio of PMO to CPP. This parameter change optimizes the balance between efficacy (maintaining sufficient PMO for exon skipping) and safety (reducing toxicity through better distribution and reduced steric hindrance).
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 multiple PMO-CPP conjugates demonstrate improved exon skipping efficacy and safety, achieving a higher safety margin and efficacy compared to single PMO-CPP constructs, with reduced toxicity and increased efficiency in gene modulation, including in skeletal and cardiac muscle cells.
Implementation Method 1
incorporating glutamic acid and peptidase cleavage sites, such as Phenylalanine-Lysine
Data Source
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AI summary
The present disclosure relates to antisense oligonucleotides (AONs), such as phosphorodiamidate morpholino oligonucleotides (PMOs). The present disclosure further relates to the conjugation of multiple PMOs to cationic cell penetrating peptides (CPPs) to enhance the uptake of PMOs into skeletal and cardiac muscle cells.