RNA-interference-inducing nucleic acid molecule able to penetrate into cells, and use therefor
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Publication Number
US-10883105-B2
Publication Date
2021-01-05
Expiration Date
Abstract
The present invention relates to a novel, RNAi-inducing nucleic acid molecule having cell penetrating ability and the use thereof, and more particularly, to a novel, RNAi-inducing double-stranded nucleic acid molecule, which has a replacement of the phosphate backbone of at least one nucleotide with phosphorothioate or phosphorodithioate, and has a lipophilic compound conjugated thereto, and thus has high target gene-silencing efficiency while having the ability to penetrate cells without needing a separate intracellular delivery vehicle, and to a method of silencing a target gene using the nucleic acid molecule. The nucleic acid structure according to the present invention has both cholesterol modification and phosphorothioate modification introduced therein, and thus has high gene silencing efficiency while having the ability to penetrate cells without needing a separate intracellular delivery vehicle. Thus, it can be delivered into an actual target area in an amount sufficient for induction of RNAi, and thus can overcome the in vivo delivery problem occurring in the prior art. Therefore, the nucleic acid molecule according to the invention can effectively substitute for conventional siRNA molecules to treat cancer or viral infections.
Core Innovation
The invention relates to an RNAi-inducing double-stranded nucleic acid (lasiRNA) for inhibiting expression of a connective tissue growth factor (CTGF) gene in a cell. The RNAi-inducing nucleic acid molecule includes a first strand and a second strand that form a double-stranded region and a single-stranded region, with the 5′ end of the first strand and the 3′ end of the second strand forming a blunt end. The first strand includes at least one phosphorothioate (PS) or phosphorodithioate (PS2) bond and at least one 2′-O-alkyl modified nucleotide, while the second strand includes at least one PS or PS2 bond, at least one 2′-O-alkyl modified nucleotide, and a cholesterol moiety at the 3′ end.
A key problem addressed is that siRNA and related nucleic acids require enhanced cell penetration and effective intracellular delivery to achieve CTGF gene silencing. The invention positions a cholesterol moiety conjugated to the nucleic acid as a contributor to cell penetration, while the phosphate backbone substitution using PS or PS2 provides additional enhancement. The document states that cholesterol alone is insufficient, and that optimizing the amount of PS affects uptake and silencing, with uptake increasing as PS increases but silencing decreasing if PS is too high.
The invention further defines design parameters of the lasiRNA, including first and second strand lengths and a long single-stranded overhang, and reports that longer antisense overhang yields higher silencing. The document also reports that PS2 generally reduces silencing compared with PS. In a therapeutic context, the method treats CTGF-associated diseases or disorders including hypertrophic scar and keloid, with in vivo examples describing substantial CTGF silencing and persistence after intradermal administration in a rat model.
Claims Coverage
The independent claim defines a method for inhibiting CTGF gene expression using a specific RNAi-inducing nucleic acid structure with sequence (SEQ ID NOs) and defined chemical modifications, and further limits the therapeutic use to CTGF-associated diseases or disorders of hypertrophic scar or keloid. The claim set includes dependent refinements that constrain the number and positions of phosphorothioate bonds, the pattern of 2′-O-alkyl modifications on the first strand, additional cholesterol linkage details, and an additional nucleotide-derivative constraint in the single-stranded region.
Method for inhibiting CTGF gene expression using a cholesterol-modified PS/PS2 lasiRNA
A method comprising contacting a cell with a pharmaceutical composition comprising an RNAi-inducing nucleic acid molecule having a first strand consisting of the nucleotide sequence of SEQ ID NO: 154 and comprising at least one phosphorothioate or phosphorodithioate bond and at least one 2′-O-alkyl modified nucleotide, and a second strand consisting of the nucleotide sequence of SEQ ID NO: 153 and comprising at least one phosphorothioate or phosphorodithioate bond, at least one 2′-O-alkyl modified nucleotide, and a cholesterol moiety at the 3′ end thereof; wherein the second strand binds to the first strand such that the first strand has a double-stranded region to which the second strand binds and a single-stranded region to which the second strand does not bind, and wherein the 5′ end of the first strand and the 3′ end of the second strand form a blunt end; and treating a CTGF-associated disease or disorder that is a hypertrophic scar or keloid.
Phosphorothioate-bond constrained first strand for CTGF silencing
The method wherein the first strand has between 7 and 12 phosphorothioate bonds.
Phosphorothioate-bond positions closest to the 3′ end of the first strand
The method wherein the 7 to 12 phosphorothioate bonds are 7 to 12 internucleotide bonds positioned closest to the 3′ end of the first strand of the nucleic acid molecule.
Positional 2′-O-Me modification pattern on the first strand
The method wherein the first strand contains 2′-O-Me modified nucleotides at positions 1, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31 of SEQ ID NO: 154.
Cholesterol attachment via a phosphorothioate bond at the 3′ end of the second strand
The method wherein the cholesterol moiety is covalently attached to the 3′ end of a second strand via a phosphorothioate bond.
Deoxyadenosine derivative with a phenyl group in the first-strand single-stranded region
The method wherein at least one nucleotide in the single-stranded region of the first strand is a deoxyadenosine derivative having a phenyl group.
Across the independent and dependent claims, the inventive coverage centers on a CTGF gene-inhibiting lasiRNA that contains a cholesterol moiety at the 3′ end of the second strand together with phosphorothioate/phosphorodithioate bonds and 2′-O-alkyl (including specified 2′-O-Me) modifications, using a defined double-stranded/single-stranded binding geometry with blunt ends, and restricting the use to hypertrophic scar or keloid; dependent claims further narrow the phosphorothioate bond count and position, the positional 2′-O-Me pattern, the cholesterol linkage, and a deoxyadenosine-phenyl derivative in the single-stranded region.
Stated Advantages
Enhanced cell penetration / intracellular delivery of the RNAi-inducing nucleic acid by using PS/PS2 backbone substitution and a cholesterol moiety.
CTGF mRNA knockdown in cells using PS-optimized chol-lasiRNAs.
Substantial CTGF silencing in vivo with persistence for at least about 5 days after intradermal administration.
Effective silencing at low doses as reported by IC50.
Documented Applications
Treatment of CTGF-associated diseases or disorders including hypertrophic scar and keloid.
In vivo CTGF silencing in a rat skin intradermal injection setting.
Use in CTGF-associated applications referenced as cancer or viral infections [only as suggested in the provided content].
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