Phosphorodiamidate Linkage Antisense Oligonucleotides for Stability and Uptake
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Solution Overview
Problem
Current antisense oligonucleotides face challenges in achieving effective protein expression inhibition due to limitations in nuclease resistance, acid stability, and cellular uptake, particularly in systemic delivery, where the polyanionic nature hinders lipid membrane crossing.
Innovation Solution
Development of antisense oligonucleotides with N3′→P5′ phosphorodiamidate linkages in the backbone, which act as effective RNase H substrates for mRNA degradation and steric blockers, enhancing acid stability and cellular uptake by reducing charge and improving membrane permeability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional phosphodiester backbone is used in antisense oligonucleotides, then the oligonucleotides can bind to target mRNA, but they exhibit poor acid stability and are degraded by nucleases
Solution Approach 1:
The patent modifies the backbone chemistry by changing the phosphodiester linkage to a phosphorodiamidate linkage, which alters the chemical parameters of the oligonucleotide backbone. This chemical modification provides both enhanced acid stability and nuclease resistance simultaneously, resolving the contradiction between these two stability parameters.
Solution Approach 2:
The patent creates a composite backbone structure combining phosphorodiamidate linkages with the oligonucleotide sequence. This composite chemical structure integrates the benefits of both acid stability and nuclease resistance into a single molecular framework, allowing the oligonucleotide to maintain integrity in acidic environments while resisting enzymatic degradation.
2Reliability
If antisense oligonucleotides are used to inhibit protein expression, then target mRNA can be degraded, but cellular uptake is limited due to polyanionic nature
Solution Approach 1:
The patent modifies the electrical and chemical parameters of the oligonucleotide backbone by introducing phosphorodiamidate linkages, which change the charge distribution and reduce the overall polyanionic character. This parameter change decreases electrostatic repulsion with cell membranes, thereby enhancing cellular uptake while maintaining the ability to inhibit protein expression through mRNA degradation.
3Reliability
If phosphorodiamidate linkages are introduced to improve stability, then acid stability increases, but synthesis complexity increases
Solution Approach 1:
The patent employs phosphoramidite intermediaries as building blocks in the synthesis process. These pre-formed phosphorodiamidate-containing nucleoside phosphoramidites serve as mediators that can be incorporated into the growing oligonucleotide chain through standard phosphoramidite coupling chemistry, simplifying the overall synthesis process despite the advanced backbone chemistry.
Data Source
AI summary
This invention relates to antisense oligonucleotides comprising at least one N3′→P5′ phosphorodiamidate linkage (NPN) in the backbone, useful for modulating gene expression involved in the pathogenesis of a disease. Compounds useful as building blocks of said antisense oligonucleotides and methods of preparing building block compounds including NPN linkages are provided.