Chimeric Antisense Polynucleotide Design for Specificity and Potency
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Solution Overview
Problem
Current antisense oligonucleotides face challenges in achieving optimal efficiency, potency, and safety due to the trade-off between longer probes providing higher specificity and lower activity, and shorter probes offering higher potency but with lower sequence specificity and potential toxicity.
Innovation Solution
A chimeric antisense polynucleotide design that includes a central nucleotide region flanked by wing regions with low protein affinity and enhanced DNase/RNase resistance, allowing for increased length without the toxicity of shorter probes, and forming a double-stranded antisense agent for targeted gene expression modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If longer antisense oligonucleotide probes are used, then sequence specificity is improved, but activity and potency deteriorate
Solution Approach 1:
The antisense oligonucleotide is divided into distinct functional segments: a central gapmer region (5-15 nucleotides) that provides high RNA binding affinity and nuclease resistance, flanked by 5' and 3' wing regions (1-10 nucleotides each) that enhance stability and reduce toxicity. This segmentation allows each region to contribute its optimal properties without the drawbacks of uniformly long probes.
Solution Approach 2:
The oligonucleotide employs a composite structure combining different nucleotide types: gapmer nucleotides (modified sugars like LNA or BNA with phosphorothioate backbones) in the central region for high affinity and stability, and natural or modified nucleotides in the wing regions for reduced protein binding and improved pharmacokinetics. This composite approach achieves both high specificity and high activity.
2Productivity
If shorter antisense oligonucleotide probes are used, then activity and potency are improved, but sequence specificity deteriorates and toxicity increases
Solution Approach 1:
Different regions of the oligonucleotide are assigned different qualities: the central gapmer region has high binding affinity and nuclease resistance properties, while the terminal wing regions have low protein binding affinity and enhanced stability. This local differentiation allows the molecule to achieve both high potency through the active central region and high specificity through the stabilizing terminal regions.
3Object-affected harmful factors
If oligonucleotide length is increased to reduce toxicity, then sequence specificity is improved, but activity is reduced
Solution Approach 1:
The oligonucleotide is segmented into a compact central gapmer region (5-15 nucleotides) that maintains high activity, flanked by shorter wing regions (1-10 nucleotides) that provide stability and reduce toxicity. This segmentation achieves toxicity reduction without sacrificing activity by optimizing the functional contribution of each segment.
Solution Approach 2:
The molecule employs parameter changes in nucleotide composition and modification: phosphorothioate backbone modifications increase nuclease resistance and reduce toxicity, while LNA or BNA sugar modifications enhance RNA binding affinity. These parameter changes allow the oligonucleotide to achieve both low toxicity and high activity at optimized lengths.
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 chimeric antisense polynucleotide and double-stranded antisense agent effectively modify RNA transcription levels and protein levels in cells, offering improved specificity and reduced toxicity, achieving antisense effects comparable to longer probes with the efficiency of shorter ones.
Implementation Method 1
the ASO binds to a transcription product (mRNA) of the target gene, and a partial double strand is formed
Implementation Method 2
a partial DNA-RNA hetero-duplex is formed. Because this structure is recognized by RNase H, and the mRNA of the target gene is thereby decomposed
Data Source
AI summary
Chimeric single-stranded polynucleotides and double-stranded antisense agents useful for modifying the expression of a target gene by means of an antisense effect are disclosed. The chimeric single-stranded antisense polynucleotide and double-stranded antisense agents comprise a central nucleotide region flanked by a first 5′-wing region and a first 3′-wing region of modified nucleotides, which are themselves flanked by a second 5′-wing region and/or a second 3′-wing region of nucleotides that have a low affinity for proteins and/or that have higher resistance to DNase or RNase than a natural DNA or RNA and are missing in a cell when the chimeric polynucleotide delivered. The double-stranded antisense agent further comprises a complementary strand annealed to the antisense strand. The polynucleotide can be used to modify RNA transcription levels, miRNA activity, or protein levels in cells.