LNA-Modified Antisense Oligonucleotides for FoxP3 Inhibition
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Solution Overview
Problem
Current compounds, such as cET and FANA-modified antisense oligonucleotides and CD25 antibodies, require high doses and prolonged administration to achieve significant knockdown of FoxP3 expression, leading to potential toxicity and incomplete inhibition of immunosuppressive Tregs, highlighting the need for more potent and efficient FoxP3 inhibitors.
Innovation Solution
Development of antisense oligonucleotides modified with bridged nucleic acids like LNA, 2'Fluoro, 2'O-Methyl, or 2'O-Methoxy nucleotides that specifically target FoxP3 mRNA or pre-mRNA, achieving 40-99% reduction in FoxP3 expression at lower doses and faster onset, and combining with other therapeutic agents to enhance immune responses.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If cET or FANA-modified antisense oligonucleotides are used to inhibit FoxP3 expression, then Treg immunosuppressive function is reduced, but high doses and prolonged administration are required leading to potential toxicity
Solution Approach 1:
The patent applies parameter changes by modifying the chemical structure of antisense oligonucleotides through LNA (locked nucleic acid) incorporation. This structural modification changes the binding affinity and stability parameters of the oligonucleotide-FoxP3 mRNA complex, enabling significantly higher inhibition efficacy (40-99% knockdown) at much lower doses compared to conventional cET or FANA modifications, thereby reducing toxicity while maintaining or improving reliability of FoxP3 suppression.
Solution Approach 2:
The invention uses composite materials by combining LNA-modified nucleotides with phosphorothioate backbone modifications in the antisense oligonucleotide structure. This composite approach integrates multiple functional properties: LNA provides enhanced binding affinity and specificity to FoxP3 mRNA, while phosphorothioate modifications improve nuclease resistance and cellular uptake. The synergistic effect achieves potent FoxP3 inhibition at low doses, resolving the contradiction between efficacy and toxicity.
2Reliability
If conventional antisense oligonucleotides are administered to achieve FoxP3 knockdown, then Treg suppression is reduced, but prolonged administration is required leading to incomplete inhibition
Solution Approach 1:
The patent employs parameter changes through LNA modification to alter the kinetic parameters of oligonucleotide-mRNA interaction. The LNA-containing antisense oligonucleotides exhibit faster binding kinetics and higher thermal stability to FoxP3 mRNA, achieving 40-99% knockdown within a short administration period. This eliminates the need for prolonged administration required by conventional oligonucleotides, simultaneously improving knockdown efficiency and reducing treatment duration.
3Reliability
If high doses of antisense oligonucleotides are used to achieve significant FoxP3 knockdown, then Treg immunosuppressive function is inhibited, but potential toxicity increases
Solution Approach 1:
The invention applies parameter changes by incorporating LNA modifications that dramatically increase the binding affinity (lowering Kd values) and thermal stability of antisense oligonucleotides to FoxP3 mRNA. This allows achieving significant FoxP3 knockdown (40-99%) at very low nanomolar concentrations, eliminating the need for high dose administration and thereby preventing toxicity while maintaining reliable inhibition of Treg immunosuppressive function.
Solution Approach 2:
The patent uses a simplified molecular copying approach where LNA-modified oligonucleotides create more stable and specific copies of the complementary sequence to FoxP3 mRNA. The enhanced structural rigidity and hybridization stability of LNA allow the antisense oligonucleotides to more effectively bind and block FoxP3 translation, achieving potent inhibition at low concentrations without the toxicity associated with high dose conventional oligonucleotide administration.
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 modified antisense oligonucleotides effectively inhibit FoxP3 expression in a dose-dependent manner, reducing immunosuppressive Treg function, thereby enhancing immune responses against tumors and infections, and can be used in various cancers and chronic infectious diseases.
Implementation Method 1
hybridizing with a nucleic acid sequence of Foxp3 resulting in a reduction of FoxP3, FoxP3 mRNA, FoxP3 pre-mRNA
Implementation Method 2
at least one of the nucleotides comprises a modification selected from the group consisting of a bridged nucleic acid such as LNA, ENA, a 2′Fluoro modified nucleotide, a 2 O-Methyl modified nucleotide
Data Source
AI summary
The present invention refers to an oligonucleotide comprising 12 to 25 nucleotides, wherein at least one of the nucleotides comprises a modification selected from the group consisting of a bridged nucleic acid such as LNA, ENA, a 2′Fluoro modified nucleotide, a 2 O-Methyl modified nucleotide, a 2 O-Methoxy modified nucleotide, a FANA and a combination thereof. The oligonucleotide hybridizes with a nucleic acid sequence of Foxp3 of SEQ ID NO.1 and/or of SEQ ID NO.2 resulting in a reduction of the expression of FoxP3 mRNA, FoxP3 pre-mRNA or a combination thereof. The invention is further directed to a pharmaceutical composition comprising an oligonucleotide of the present invention and to the oligonucleotide and pharmaceutical composition, respectively for use in a method of preventing and/or treating a disorder, where FoxP3 imbalance is involved.