Single-Stranded Loop Oligonucleotides for Simplified RNAi Manufacturing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current RNAi agent manufacturing processes for double-stranded siRNAs are complex, time-consuming, expensive, and environmentally unsustainable, while overlooking process-related challenges that affect stability and potency.
Innovation Solution
A single-stranded oligonucleotide design with intra-strand duplexed regions and chemical modifications, allowing for simplified synthesis and purification, while maintaining or enhancing RNAi efficacy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If double-stranded siRNA is synthesized through separate sense and antisense strands with multistep purification and annealing, then RNAi potency is achieved, but manufacturing complexity and time increase significantly
Solution Approach 1:
The patent segments the siRNA into two complementary oligonucleotide strands (Z1 and Z2) within a single continuous sequence, allowing them to be synthesized together as one piece rather than separately. This segmentation enables the strands to self-anneal into the required duplex structure after transcription, eliminating multiple purification and annealing steps while maintaining RNAi potency.
Solution Approach 2:
The patent merges the synthesis of sense and antisense strands into a single transcription process by designing a continuous single-stranded oligonucleotide sequence containing both complementary strands. This combining approach allows both strands to be produced simultaneously in one reaction, followed by in vitro transcription to generate the functional siRNA duplex, thereby simplifying the manufacturing process.
2Manufacturing precision
If multiple purification steps are performed on single strands before annealing, then product purity is improved, but manufacturing time and cost increase
Solution Approach 1:
The patent performs preliminary design of the single-stranded oligonucleotide sequence to inherently contain both complementary strands (Z1 and Z2) with appropriate spacing and structural features. This preliminary structuring allows the strands to self-anneal correctly after transcription without requiring extensive purification steps, as the sequence design itself guides proper duplex formation.
Solution Approach 2:
The complementary strands Z1 and Z2 within the single-stranded oligonucleotide are designed to self-anneal into the functional siRNA duplex structure after in vitro transcription. This self-service mechanism eliminates the need for external annealing protocols and multiple purification steps, as the molecules automatically form the correct structure through intramolecular base pairing.
3Duration of action of stationary object
If chemical modifications are incorporated to improve stability, then RNAi agent durability is enhanced, but synthesis complexity increases
Solution Approach 1:
The patent applies chemical modifications locally at specific positions within the oligonucleotide sequence rather than uniformly throughout. For example, modified nucleotides are placed at the 5' ends of strands Z1 and Z2 to enhance stability and facilitate DICER processing, while leaving other regions unmodified or differently modified. This localized approach improves stability without requiring complex modification of the entire sequence.
Solution Approach 2:
The patent utilizes various chemical modification parameters including 2'-O-methyl, 2'-fluoro, LNA, and phosphorothioate modifications at different positions and combinations. By adjusting these chemical parameters at specific locations, the patent optimizes both stability and biological activity while maintaining compatibility with standard synthesis methodologies.
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 single-stranded oligonucleotide design simplifies manufacturing and purification processes, preserving or improving RNAi agent stability and potency, addressing environmental sustainability concerns.
Implementation Method 1
Z1 and Z2 are capable of forming an intra-strand duplexed region comprising 3 or more consecutive base pairs
Data Source
AI summary
One aspect of the present invention relates to a single-stranded oligonucleotide having a having a sequence represented by formula (I): (5′-Z1-3′)-Q1-L-Q2-(5′-Z2-3′) (I). In formula (I), Z1 is a first oligonucleotide, comprising 15-100 optionally modified nucleotides that is substantially complementary to a target gene; Z2 is a second oligonucleotide, comprising 15-100 optionally modified nucleotides that is substantially complementary to Z1; and Z1 and Z2 are capable of forming an intra-strand duplexed region comprising 3 or more consecutive base pairs. L is a linking group. Q1 and Q2 each independently represent 0 to 12 optionally modified nucleotides. At least one nucleotide in formula (I) is a modified nucleotide. Other aspects of the invention relate to a pharmaceutical composition and a method for inhibiting the expression of one or more target gene in a subject.