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Nucleic acid molecules capable of inducing RNA interference with intracellular penetration and uses thereof

A nucleic acid molecule and molecular technology, applied in the direction of DNA / RNA fragments, medical preparations containing active ingredients, organic active ingredients, etc., can solve the problems of inducing toxicity, delivering siRNA efficiently, and weakening the delivery efficiency of siRNA

Active Publication Date: 2018-03-02
OLIX PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Nucleic acid molecules capable of inducing RNA interference with intracellular penetration and uses thereof
  • Nucleic acid molecules capable of inducing RNA interference with intracellular penetration and uses thereof
  • Nucleic acid molecules capable of inducing RNA interference with intracellular penetration and uses thereof

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, in order to ensure a highly efficient RNAi-inducing double-stranded nucleic acid molecule targeting CTGF, design 50 kinds of target sequences for CTGF 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 level was measured b...

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 100 mm petri dish, Dulbecco's modified Eagle's protein supplemented with 10% fetal bovine serum (Gibco), 100 μg / ml penicillin / streptomycin HeLa cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (Gibco).

[0110] Cholesterol modification (C...

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 structure RNAi-inducing nucleic acid molecule with intracellular penetration ability and its use, more specifically, relates to a RNAi-inducing nucleic acid molecule with excellent target gene inhibition efficiency and without additional cell carrier and intracellular penetration ability Nucleic acid molecule with novel structure and method for inhibiting expression of target gene using the same, said nucleic acid having at least one nucleotide phosphate skeleton contained in double-stranded nucleic acid molecule that induces RNAi is replaced by phosphorothioate or dithiophosphate A structure in which a lipophilic compound is substituted and combined. The nucleic acid molecular structure of the present invention introduces cholesterol modification and phosphorothioate modification at the same time, thereby maintaining excellent gene suppression efficiency and having intracellular penetration ability without additional cell carriers, and can fully induce RNAi The amount can be delivered to the actual target site, which can solve the problem of in vivo delivery in the previous technology. Therefore, the nucleic acid molecule of the present invention can be effectively used in the treatment of cancer or viral infection using siRNA instead of the conventional siRNA molecule, and thus is useful in the treatment of cancer or viral infection.

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