Short Duplex DNA Gene Silencing via Ribonucleotide Integration
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Solution Overview
Problem
Current gene silencing technologies, such as Antisense oligonucleotides (ASO) and small interfering RNA (siRNA), face challenges including low silencing efficiency, off-target effects, immune response stimulation, tissue penetration issues, and high costs, limiting their therapeutic potential for treating a wide range of diseases.
Innovation Solution
The development of short duplex deoxyribonucleotides (sdDNA) with interspersed ribonucleotide segments, which form a short, duplex molecule that can hybridize with target RNA, offering enhanced gene silencing potency, improved tissue penetration, reduced off-target effects, and lower synthesis costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Antisense oligonucleotides (ASO) or small interfering RNA (siRNA) are used for gene silencing, then gene expression can be modulated, but silencing efficiency is low and off-target effects occur
Solution Approach 1:
The invention uses a composite oligonucleotide structure combining DNA and RNA components (e.g., DNA-RNA chimeras, LNA-modified oligonucleotides) to achieve both high target binding affinity and specificity. The hybrid structure leverages the stability of DNA and the RNAi mechanism, while modifications like LNA enhance binding strength and reduce off-target effects through improved discrimination between target and non-target sequences.
Solution Approach 2:
The patent applies localized chemical modifications at specific positions within the oligonucleotide sequence (e.g., 2'-O-methyl modifications at certain nucleotides, phosphorothioate linkages at specific regions) to enhance binding affinity to the target while maintaining specificity. These localized modifications allow fine-tuning of the molecule's properties to achieve high silencing efficiency without triggering off-target effects.
2Reliability
If ASO or siRNA technology is developed for therapeutic applications, then gene silencing can be achieved, but tissue penetration challenges limit delivery
Solution Approach 1:
The invention modifies physical and chemical parameters of the oligonucleotides, including size reduction to shorter lengths, charge modification through 5'-amino or 3'-amino modifications, and hydrophobicity adjustments via cholesterol or GalNAc conjugation. These parameter changes enhance cellular uptake and tissue penetration while preserving gene silencing activity.
Solution Approach 2:
The patent employs delivery vehicles and conjugates as intermediaries to facilitate tissue penetration. Examples include GalNAc conjugates that target asialoglycoprotein receptors for hepatocyte delivery, cholesterol modifications for membrane association, and peptide conjugates that enhance cellular internalization. These intermediaries enable the oligonucleotides to overcome biological barriers and reach target tissues effectively.
3Reliability
If ASO or siRNA is administered for gene silencing, then therapeutic effect can be achieved, but immune response stimulation occurs
Solution Approach 1:
The invention alters the chemical composition and structure of the oligonucleotides to reduce immunogenicity. This includes using 2'-O-methyl modifications that reduce recognition by Toll-like receptors, phosphorothioate backbone modifications that decrease complement activation, and adjusting the guanine content to avoid RIG-I activation. These parameter changes maintain therapeutic efficacy while minimizing immune response stimulation.
4Reliability
If conventional ASO or siRNA platforms are used, then gene silencing can be achieved, but synthesis costs are high
Solution Approach 1:
The patent employs oligonucleotide designs that can be synthesized using cost-effective chemical methods with commercially available building blocks. The use of standard phosphoramidite chemistry, modular assembly approaches, and optimized synthesis protocols reduces manufacturing costs. Additionally, the designs allow for efficient purification and formulation processes that further reduce overall production expenses while maintaining therapeutic quality.
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
sdDNA molecules demonstrate potent gene silencing at pico molar concentrations, outperforming existing technologies in terms of efficiency and safety, with potential applications in research, diagnosis, and therapy across various biological fields.
Implementation Method 1
an antisense oligonucleotide hybridizes to a target nucleic acid
Implementation Method 2
promotes degradation of the RNA through endogenous enzymes, such as ribonuclease H1 (RNase H1)
Data Source
AI summary
The present invention discloses a novel type of gene silencing technology for modulating target nucleic acid and/or protein in cells, tissues, organisms and animals. The new technology provides compositions for use in gene silencing applications, including prevention and treatment of human diseases. The composition comprises a short, duplex DNA molecule where the sense strand is at least equal to the antisense strand in length. The duplex DNA molecule further includes at least one interspersed ribonucleotide monomer. The present invention further provides methods of using the compositions for modulating expression or function of a target gene, or for treatment or prevention of diseases as well as for other medical or biological applications.