Differentially Modified Oligonucleotide Strands for Gene Silencing
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Solution Overview
Problem
Current RNA interference (RNAi) technologies face challenges in enhancing the efficiency and specificity of gene silencing, particularly in modulating gene expression through the RNA interference pathway, with existing oligonucleotides often experiencing off-target effects and limited stability.
Innovation Solution
The development of oligomeric compounds with modified sugar moieties, such as 2'-OCH3 and 2'-F nucleosides, in both the antisense and sense strands, along with phosphorothioate internucleoside linkages, to enhance hybridization efficiency and specificity, and the incorporation of capping groups and overhang regions to improve nuclease resistance and cellular uptake.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild type nucleic acids are used, then the structure is simple and easy to manufacture, but the stability and specificity are insufficient
Solution Approach 1:
The patent applies local quality by introducing specific modified nucleosides (2'-OCH3 and 2'-F) at particular positions within the oligomeric strands rather than uniform modification throughout. The antisense strand contains 2'-OCH3 modified nucleosides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 and 2'-F modified nucleosides at other positions, while the sense strand has 2'-OCH3 modified nucleosides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100. This localized strategic modification enhances stability and specificity without requiring complete structural redesign of the entire oligomeric compound.
Solution Approach 2:
The patent employs composite materials by combining different types of modified nucleosides (2'-OCH3 and 2'-F) within the same oligomeric strands, and by incorporating phosphorothioate internucleoside linkages at specific positions. This creates a composite structure that integrates multiple functional elements: the 2'-OCH3 modifications provide stability, the 2'-F modifications enhance specificity, and the phosphorothioate linkages improve nuclease resistance. This composite approach allows the oligomeric compounds to achieve multiple performance improvements simultaneously.
2Productivity
If modified oligomeric compounds are used to enhance specificity and stability, then gene silencing efficiency improves, but off-target effects and limited stability persist
Solution Approach 1:
The patent applies parameter changes by systematically varying the positions and types of modified nucleosides within the oligomeric compounds. The antisense strand uses 2'-OCH3 modified nucleosides at even positions (2, 4, 6, ..., 100) and 2'-F modified nucleosides at odd positions (1, 3, 5, ..., 99), while the sense strand uses 2'-OCH3 modified nucleosides at even positions (2, 4, 6, ..., 100). These parameter variations optimize the balance between gene silencing efficiency and specificity by tuning the molecular recognition properties of the oligomeric compounds.
Solution Approach 2:
The patent applies segmentation by dividing the oligomeric strands into regions with different modification patterns. The antisense and sense strands are segmented into blocks of modified nucleosides separated by unmodified regions, creating a modular structure. This segmentation allows different regions to perform specialized functions: modified regions enhance stability and specificity, while unmodified regions maintain hybridization capability and reduce off-target effects.
3Reliability
If phosphorothioate internucleoside linkages are incorporated, then nuclease resistance increases, but manufacturing complexity increases
Solution Approach 1:
The patent applies local quality by incorporating phosphorothioate internucleoside linkages at specific positions within the oligomeric compounds rather than throughout the entire structure. The linkages are placed at positions where they provide maximum nuclease resistance while minimizing impact on hybridization and cellular uptake. This localized incorporation reduces manufacturing complexity compared to complete phosphorothioate modification.
Solution Approach 2:
The patent applies partial action by incorporating phosphorothioate linkages at a subset of positions rather than at every internucleoside linkage. This partial modification provides sufficient nuclease resistance for in vivo stability while keeping the manufacturing process more feasible than complete modification. The selective placement of phosphorothioate linkages at critical positions achieves the necessary protection without excessive manufacturing complexity.
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
These modifications lead to increased stability, enhanced specificity, and improved loading of the antisense strand into the RISC complex, resulting in more efficient gene silencing with reduced off-target effects and increased nuclease resistance.
Implementation Method 1
The compositions can hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA
Implementation Method 2
The compositions can include one or more 3'-capping groups, 5'-capping groups, 5'-phosphate moieties, 3'-linked conjugate groups, non hybridizing 3'-overhang regions and a non hybridizing 3'-overhang regions that are linked to a conjugate group. The compositions can further include varying numbers of phosphorothioate internucleoside linkages that enhance the in vivo activity.
Data Source
AI summary
The present invention provides double stranded compositions wherein the first strand is modified to have a particular motif and the second strand is modified a selected motif. The motifs are defined by positioning of differentially modified nucleosides wherein at least the sugar moieties are different. More particularly, the present compositions comprise an antisense strand that is modified to have a positional/full motif and the sense strand is modified to have an alternating motif, a hemimer motif, a blockmer motif, a gapped motif, a positional motif, a positional/full motif or a fully modified motif. Each strand further comprises one or more phosphorothioate internucleoside linkage. The compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes. In preferred embodiments the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA. The present invention also provides methods for modulating gene expression.