High-efficiency nanoparticle-type double-helical oligo-RNA structure and method for preparing same
Inventors
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Assignees
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BioneerBioneer is a biotechnology company specializing in molecular biology, offering solutions in nucleic acid synthesis, molecular diagnostics, protein production, and laboratory automation. The company's expertise covers oligonucleotide chemistry, gene synthesis, sample preparation, molecular diagnostics, and analytical services such as mass spectrometry and sequencing, serving clinical, research, and industrial sectors globally.
Bioneer is a biotechnology company specializing in molecular biology, offering solutions in nucleic acid synthesis, molecular diagnostics, protein production, and laboratory automation. The company's expertise covers oligonucleotide chemistry, gene synthesis, sample preparation, molecular diagnostics, and analytical services such as mass spectrometry and sequencing, serving clinical, research, and industrial sectors globally.
Publication Number
US-9326941-B2
Publication Date
2016-05-03
Expiration Date
Abstract
Provided are a double-stranded oligo RNA structure and a method of preparing the same, and more specifically, a double-stranded oligo RNA structure in which a polymer compound is covalently bound to a double-stranded oligo RNA in order to improve stability in vivo and a cell delivery efficiency of the double-stranded oligo RNA, and a method of preparing the same.The double-stranded oligo RNA structure having the optimized structure according to the present invention may not inhibit functions of the double-stranded oligo RNA, but effectively improve stability and cell membrane permeability of the double-stranded oligo RNA, such that the double-stranded oligo RNA may be delivered into the cell even at a low concentration dosage thereof to be significantly used as a tool for treatment of cancer, infectious diseases, and the like, as well as a new delivery system of the double-stranded oligo RNA.
Core Innovation
The invention provides a double-stranded oligo RNA structure in which a chemical material is bound to an RNA duplex. One of two moieties (A and B) is a hydrophilic material and the other is a hydrophobic material, and the sense strand (S) and the antisense strand (AS) form the double-stranded oligo RNA. Covalent bonds or linker-mediated covalent bonds (X and Y) bind the chemical material to the RNA ends.
The disclosed structure enables nanoparticle self-assembly and stability by combining hydrophobic and hydrophilic moieties linked to the dsRNA ends. Strand-end positioning is addressed by distinguishing the sense strand (S) and antisense strand (AS) and by using 5′ phosphorylation and/or 5′ phosphate variants associated with the antisense 5′ end (pAS), including variants with up to three 5′ phosphate groups. The arrangement is presented as a way to maximize RNAi function and enhance RNAi gene expression inhibition.
A method is provided for preparing the double-stranded oligo RNA structure by synthesizing RNA single strands, covalently binding hydrophilic and hydrophobic materials at defined strand ends, separating from a solid support, and forming the ds structure by annealing a complementary RNA single strand. The document further describes targeting examples including survivin mRNA, with survivin-targeting duplex constructs used to show improved mRNA inhibition at low concentration and increased efficacy. Optional ligand attachment on the hydrophilic end for receptor-mediated internalization is also described along with therapeutic and diagnostic use as pharmaceutical compositions, including lyophilized formulation.
Claims Coverage
Independent claim coverage includes 3 independent claims. The inventive features center on an amphipathic dsRNA with hydrophilic and hydrophobic moieties bound through covalent or linker-mediated covalent bonds, and on end-specific covalent attachment steps followed by annealing with a complementary RNA strand that may include 5′ phosphate features.
Amphipathic double-stranded oligo RNA with covalent hydrophilic and hydrophobic binding
A double-stranded oligo RNA structure in which a chemical material represented by formula 1 is bound, wherein one of A and B is a hydrophilic material and the other is a hydrophobic material, X and Y are simple covalent bond or linker-mediated covalent bond, S is a sense strand, and AS is an antisense strand.
Solid-support synthesis and annealing to form ds oligo RNA with 5′ covalent hydrophobic binding
A method of preparing a double-stranded oligo RNA structure comprising synthesizing an RNA single strand on a solid support containing a hydrophilic material bound thereto; preparing an RNA-polymer structure by covalently binding a hydrophobic material to a 5′ end of the RNA containing the hydrophilic material; separating from the solid support; and forming the double-stranded oligo RNA structure by annealing the RNA-polymer structure and an RNA single strand of a complementary sequence.
Solid-support synthesis with covalent hydrophilic attachment and 3′ hydrophobic covalent binding followed by annealing
A method of preparing a double-stranded oligo RNA structure comprising synthesizing an RNA single strand on a solid support containing a functional group bound thereto; covalently binding a hydrophilic material to the material obtained by step (1); separating from the solid support; forming the RNA-polymer structure by covalently binding a hydrophobic material through a functional group bound to a 3′ end of the material obtained by step (3); and forming the double-stranded oligo RNA structure by annealing the RNA-polymer structure and an RNA single strand of a complementary sequence.
Overall, the claim set defines an amphipathic ds oligo RNA architecture with hydrophilic/hydrophobic moieties connected via covalent or linker-mediated covalent bonds at dsRNA ends, and it covers preparation by end-specific covalent attachment using solid-support synthesis and subsequent annealing to a complementary RNA single strand.
Stated Advantages
Improved RNAi gene expression inhibition (enhanced mRNA inhibition) including improved mRNA inhibition at low concentration and increased efficacy in survivin-targeting constructs.
Enhanced stability and cell membrane permeability via self-assembly into stable nanoparticles.
Documented Applications
Therapeutic and diagnostic use, including cancer and infectious disease treatment, using SAMiRNA/pharmaceutical compositions and methods of administering SAMiRNA.
Targeting survivin mRNA for RNAi gene expression inhibition using survivin-targeting dsRNA constructs.
Optional ligand-bound SAMiRNA variants for receptor-mediated internalization (endocytosis) are described.
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