RNA-interference-inducing nucleic acid molecule able to penetrate into cells, and use therefor

A technology of nucleic acid molecules and penetrating ability, applied in DNA/RNA fragments, medical preparations containing active ingredients, organic active ingredients, etc., can solve the problems of induced toxicity, weakened siRNA delivery efficiency, inability to deliver siRNA efficiently, etc. question

Active Publication Date: 2015-07-01
OLIX PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in most cases, in vivo (in vivo) cannot deliver siRNA as efficiently as in vitro (in vitro), due to the interaction with various proteins existing in the living body (interaction), so there is a change in the delivery efficiency of siRNA. Weak problem (Bolcato-Bellemin AL, Bonnet ME, Creusat G, et al. Sticky overhangs enhance siRNA-mediated gene silencing. Proceedings of the National Academy of Sciences of the United States of America 2007;104:16050-16055)
In addition, depending on the composition of the delivery vehicle, it accumulates in a high concentration in specific organs such as the liver or lung regardless of the site of the disease, so there is also a problem of inducing toxicity

Method used

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  • RNA-interference-inducing nucleic acid molecule able to penetrate into cells, and use therefor
  • RNA-interference-inducing nucleic acid molecule able to penetrate into cells, and use therefor
  • RNA-interference-inducing nucleic acid molecule able to penetrate into cells, and use therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Screening of RNAi-inducing double-stranded nucleic acid molecules using CTGF as a target

[0078] Before introducing various chemical modifications for an effective self-delivery structure, after designing 50 kinds of target sequences for CTGF in order to ensure a highly efficient RNAi-inducing double-stranded nucleic acid molecule targeting CTGF Perform screening.

[0079] In order to compare the CTGF gene suppression efficiency of lasiRNA and conventional RNAi-inducing constructs, siRNA, asiRNA, and lasiRNA constructs targeting each nucleotide sequence were synthesized as shown in Tables 1 to 3. Tables 1 to 3 are the base sequence information of siRNA, asiRNA, and lasiRNA structures of 24 sequences targeting CTGF (large text: RNA, small text: DNA). In order to test (test) the CTGF mRNA expression inhibitory effect of each base sequence and structure, each structure was transfected (transfection) with HaCaT (ATCC) at 10 nM, and the CTGF mRNA expression leve...

Embodiment 2

[0106] Example 2: Production of Nucleic Acid Molecules of the Present Invention and Measurement of Intracellular Absorption Rate

[0107] 2-1: Effect of cholesterol modification

[0108] First, in order to confirm the impact of cholesterol modification on the delivery of lasiRNA, the sense strand of lasiRNA, that is, the 5' end of the second strand, is labeled with cy3, and the presence or absence of cholesterol is determined with a fluorescence microscope. The resulting uptake difference. That is, the cy3-labeled lasiRNA or chol-lasiRNA structure was incubated (incubated) at 1uM in HeLa cells, and observed with a fluorescence microscope after 3 hours to compare the degree of intracellular transmission.

[0109] First, in a 100mm Petri dish (Petri dish) supplemented with 10% fetal bovine serum (Gibco company), 100 μg / ml penicillin (penicillin) / streptomycin (streptomycin) Dulbecco modified Eagle HeLa cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (Gibco). ...

Embodiment 3

[0119] Example 3: Determination of CTGF Expression Inhibition Efficiency

[0120] Using the results of the internalization test of Cy3-labeled lasiRNA in Example 2, it can be confirmed that by directly introducing cholesterol (Cholesterol) and PS modification (PS modification) into the lasiRNA structure, no delivery vehicle or additional Reagents that also enable efficient intracellular delivery of lasiRNA. However, when various chemical modifications are introduced into siRNA, it is known that the activity of the siRNA is reduced to some extent or the activity of the siRNA is drastically reduced depending on the modification. After transfection (transfection) of lasiRNA with various structures into HeLa cells, the expression (expression) change of CTGF mRNA was measured, thereby determining the influence of each modification (modification) on the gene expression inhibition of lasiRNA.

[0121] In order to confirm the effect of PS modification (PS modification) on the gene in...

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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.

Description

technical field [0001] The present invention relates to a novel structure of RNAi-inducing nucleic acid molecule with intracellular penetration ability and its application, more specifically, relates to an RNAi-inducing nucleic acid molecule with excellent target gene inhibition efficiency and intracellular penetration without additional cell carrier Nucleic acid molecule of novel structure of ability and method for inhibiting expression of target gene using the nucleic acid molecule, said nucleic acid molecule having at least one nucleotide backbone contained in a double-stranded nucleic acid molecule that induces RNAi being surrounded by phosphorothioate (Phosphorothioate ) or phosphorodithioate (phosphorodithioate) substituted and combined with a lipophilic compound (lipophilic compound) structure. Background technique [0002] RNA interference (RNA interference: RNAi) is a very specific mechanism that can effectively inhibit gene expression. This mechanism introduces dou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63A61K48/00
CPCC12N15/87C12N2310/3515C12N2310/315C12N15/63C12N15/113C12N15/111C12N2320/32C12N2310/313C12N2310/14A61P1/16A61P11/00A61P13/12A61P17/00A61P17/02A61P19/02A61P19/10A61P25/00A61P35/00A61P43/00A61P9/00A61P9/10A61P9/12A61P3/10A61K31/7088
Inventor 弘善宇
Owner OLIX PHARMA
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