WO2022225353A1 - Multifunctional nucleic acid structure for target gene modulation and uses thereof - Google Patents

Abstract

The present application relates to a multifunctional nucleic acid structure for target gene modulation and uses thereof, wherein a structure according to one example is capable of both effectively reducing the expression of a first target gene, as well as additionally modulating selective splicing, degrading mRNA of a second target gene, and/or inhibiting miRNA action.

Description

Multifunctional nucleic acid construct for target gene regulation and use thereof

Cross-Citation with Related Application(s)

This application claims the benefit of priority based on the Republic of Korea Patent Application No. 10-2021-0052010 dated April 21, 2021, and all contents disclosed in the documents of the Korean patent application are incorporated as a part of this specification.

The present application relates to multifunctional nucleic acid constructs and uses thereof for target gene regulation.

RNA interference is a sequence-specific post-transcriptional gene silencing process mediated by short interfering RNA (siRNA), etc. in animals, in which a sense strand having a sequence homologous to the mRNA of a target gene (target gene) and a sequence complementary thereto The double-stranded RNA composed of the antisense strand having an antisense strand is introduced into a cell or the like to induce mRNA degradation of the target gene, thereby suppressing the expression of the target gene.

The RNAi field is evaluated to have much greater potential than ribozymes because it can suppress the expression of practically all genes, and it can be used as a therapeutic agent for diseases that were difficult to treat with existing drugs without restrictions. Rising as a solution, RNAi therapeutics are recognized as next-generation new drug technologies, and research on nucleic acid constructs with effective RNAi activity is active.

Antisense oligonucleotide (ASO) is a single-stranded oligonucleotide DNA having a length of 19-30 nt and can inhibit protein expression through complementary binding to intracellular mRNA and mRNA degradation by RNaseH. In addition, in the case of ASO, related research is being actively conducted because it can realize corrected gene expression by inducing alternative splicing of genes in the cell nucleus.

Prior art literature

Patent Literature

(Patent Document 1) Republic of Korea Patent Publication No. 10-2007-0028363

One purpose of this application is

a stem-loop structure;

The stem-loop structure includes a stem structure having a double-stranded polynucleotide of 33 to 38 bp in length and a loop structure having a single-stranded polynucleotide of 3 to 25 nt in length,

The stem structure includes a section including an antisense region of 20 to 25 nt in length including a sequence complementary to the first target gene, and a sense region having a length of 20 to 25 nt capable of complementary binding to a section including the antisense region. Including a section including

the stem structure comprises the sense region after 13 nt from the 5' end of the stem structure,

A polynucleotide of 5 to 30 nt in length extending from the 5' end of the section including the sense region in the stem structure and a polynucleotide of 5 to 30 nt in length extending from the 3' end of the section including the antisense region in the stem structure including,

A nucleic acid construct comprising at least one characteristic selected from the group consisting of the following (1) to (4):

(1) a polynucleotide sequence extending from the 5' end of the segment containing the sense region in the stem structure, the polynucleotide sequence extending from the 3' end of the segment containing the antisense region in the stem structure, or both are ASO (antisense oligonucleotide) sequence or anti-miRNA sequence;

(2) a polynucleotide sequence extending at the 5' end of the sense region, a polynucleotide sequence extending at the 3' end of the antisense region, or both comprising chemically modified nucleotides;

(3) the stem structure comprises chemically modified nucleotides; and

(4) the loop structure comprises chemically modified nucleotides.

Another object of the present application is to provide a composition for inhibiting gene expression, including the nucleic acid construct.

Another object of the present application is cancer, proliferative disease, digestive disease, kidney disease, neurological disease, mental disease, blood and tumor disease, cardiovascular disease, respiratory disease, endocrine disease, infectious disease, musculoskeletal disease, including the nucleic acid construct , to provide a pharmaceutical composition for preventing or treating at least one disease selected from the group consisting of gynecological diseases, genitourinary diseases, skin diseases, and ophthalmic diseases.

As used herein, "RNAi (RNA interference)" or "RNA interference" refers to a biological process mediated by short interfering nucleic acid molecules to inhibit or down-regulate the expression of genes in cells, as is generally known in the art. For example, by introducing a double-stranded RNA (dsRNA) composed of a strand having a sequence homologous to the mRNA of the target gene and a strand having a sequence complementary thereto, the degradation of the target gene mRNA is induced. By doing so, it may mean a mechanism for suppressing the expression of a target gene. "Small interfering RNA (siRNA)" refers to a short double-stranded RNA (dsRNA) that mediates sequence-specific efficient gene silencing.

As used herein, the term "antisense oligo (ASO)" refers to a chemically modified deoxygenase that complementarily binds to a sequence of mRNA expressed in cells and inhibits degradation or alternative splicing of mRNA by RNase H. It refers to ribonucleotides or single-stranded oligonucleotides composed of ribonucleotides.

As used herein, “anti-miRNA” refers to a short single-stranded oligonucleotide containing a modified nucleotide complementary to a miRNA and forms a double strand with a target miRNA to decompose miRNA through RNase H or inhibit the mechanism of miRNA based on RISC. It refers to a nucleic acid that blocks the action of miRNA through

As used herein, “nucleic acid” or “polynucleotide” refers to deoxyribonucleotides, ribonucleotides or modified nucleotides in single or double-stranded form, and polymers thereof, and known nucleotide analogues or modified backbones nucleic acids containing residues or linkages. For example, nucleic acids or polynucleotides can be single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or purine and pyrimidine bases or other natural, chemically or biochemically modified , polymers comprising non-natural or derivatized nucleotide bases.

As used herein, "nucleotide" is used as it is art-recognized. Nucleotides generally include base, sugar and phosphate moieties. The base may be a natural base (standard) or a modified base well known in the art. Such bases are generally located at the 1' position of the moiety per nucleotide. Additionally, nucleotides may be unmodified or modified at sugar, phosphate and/or base moieties.

As used herein, "nucleotide" includes natural bases (standard), and modified bases well known in the art, and is used as recognized in the art. The base is generally located at the 1' position of the moiety per nucleotide. Nucleotides generally contain a base, a sugar and a phosphate group. Nucleotides may be unmodified or modified with sugar, phosphate, and/or base moieties (nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, etc.).

As used herein, “hybridizable” or “complementary” or “substantially complementary” means that a nucleic acid (e.g., RNA, DNA) is produced under appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. Non-covalently binding, i.e. Watson-Crick base pairs and/or G/U base pairs, to other nucleic acids in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid). is meant to include a sequence of nucleotides capable of forming, "annealing", or "hybridizing". Standard Watson-Crick base-pairing includes: pairing of adenine (A) with thymidine (T), pairing of adenine (A) with uracil (U), and pairing of guanine (G) with cytosine (C) Pairing of [DNA, RNA]. In addition, for hybridization between two RNA molecules (eg, dsRNA), and for hybridization of a DNA molecule and an RNA molecule: Guanine (G) can also base pair with uracil (U). For example, G/U base-pairing is partially responsible for the degeneracy (ie, redundancy) of the genetic code in the context of base-pairing of codons in mRNA with base-pairing of tRNA anti-codons.

As used herein, the term "antisense region" or "antisense strand" refers to a polynucleotide substantially or 100% complementary to all or part of a target gene (eg, a first target gene), for example, mRNA. (messenger RNA), non-mRNA RNA sequences (e.g., microRNA, piwiRNA, tRNA, rRNA, and hnRNA, siRNA, miRNA, shRNA, DsiRNA, lsiRNA, ss-siRNA, piRNA, endo-siRNA or asiRNA) or coding sequences or complementary to the non-coding DNA sequence in whole or in part. The “antisense strand” and “guide strand” may be used interchangeably. The guide strand is a single-stranded portion sequenced for the purpose of inhibiting a target, and substantially binds to an Argonaute protein, and serves to guide the Argonaute complex to recognize a target gene.

As used herein, the term "sense region" or "sense strand" refers to a polynucleotide having the same nucleic acid sequence as all or part of a target gene, and includes messenger RNA (mRNA), non-mRNA RNA sequences (eg, microRNA, piwiRNA, tRNA, rRNA and hnRNA, siRNA, miRNA, shRNA, DsiRNA, lsiRNA, ss-siRNA, piRNA, endo-siRNA or asiRNA) or a polynucleotide identical in whole or in part to a coding or non-coding DNA sequence. . The “sense strand” and “passenger strand” may be used interchangeably. The carrier strand forms a double-stranded structure with the guide strand among the nucleic acid molecules according to an embodiment, and serves as a carrier to help the guide strand bind to the agonist protein.

As used herein, “Dicer substrate nucleic acid” and “Dicer substrate RNA (ribonucleic acid)” refer to nucleic acids that are considered to be recognized and processed by Dicer in an RNA interference (RNAi) pathway.

As used herein, “chemical modification” refers to any modification of the chemical structure of a nucleotide different from that of a native nucleic acid, nucleotide, DNA, and/or RNA.

As used herein, the nucleotide at the nth position (or at the nth position) from the 5' end of the sense region (strand), antisense region (strand), or polynucleotide strand is the sense strand, antisense strand, or polynucleotide strand. It refers to the nucleotide positioned at the nth position counted from the 5' end of

Hereinafter, the present invention will be described in detail.

the work aspect

a stem-loop structure;

The stem-loop structure includes a stem structure having a double-stranded polynucleotide of 33 to 38 bp in length and a loop structure having a single-stranded polynucleotide of 3 to 25 nt in length,

The stem structure includes a section including an antisense region of 20 to 25 nt in length including a sequence complementary to the first target gene, and a sense region having a length of 20 to 25 nt capable of complementary binding to a section including the antisense region. Including a section including

the stem structure comprises the sense region after 13 nt from the 5' end of the stem structure,

A polynucleotide of 5 to 30 nt in length extending from the 5' end of the section including the sense region in the stem structure and a polynucleotide of 5 to 30 nt in length extending from the 3' end of the section including the antisense region in the stem structure including,

A nucleic acid construct comprising the characteristics of one or more (eg, one or more, two or more, three or more, or all four) selected from the group consisting of the following (1) to (4):

(1) a polynucleotide sequence extending from the 5' end of the segment containing the sense region in the stem structure, the polynucleotide sequence extending from the 3' end of the segment containing the antisense region in the stem structure, or both are ASO (antisense oligonucleotide) sequence or anti-miRNA sequence;

(2) the polynucleotide sequence extending at the 5' terminus, the polynucleotide sequence extending at the 3' terminus, or both comprise chemically modified nucleotides;

(3) the stem structure comprises chemically modified nucleotides; and

(4) the loop structure comprises chemically modified nucleotides.

In the present specification, the lower branch point of the stem structure is, in the section including the sense region, the end portion of the stem structure that continues toward the polynucleotide sequence extending from the 5' end of the section, or in the section including the antisense region, the It may refer to the terminal portion of the stem structure that extends towards the polynucleotide sequence extending from the 3' end of the segment.

In the present specification, the upper branch point of the stem structure means a terminal portion of the stem structure leading to the loop structure in the section including the sense region, or the terminal portion of the stem structure leading to the loop structure in the section including the antisense region. can

In the present specification, the 5' end of the stem structure refers to the end portion of the stem structure extending toward the polynucleotide sequence extending from the 5' end of the section in the section including the sense region among the lower branch points of the stem structure described above. can That is, the 5' end of the stem structure may refer to the 5' end of the section including the sense region of the stem structure.

In the present specification, the 3' end of the stem structure refers to the end portion of the stem structure that extends toward the polynucleotide sequence extended from the 3' end of the section in the section including the antisense region among the lower branch points of the stem structure described above. can That is, the 3' end of the stem structure may refer to the 3' end of the section including the antisense region of the stem structure.

The nucleic acid construct according to an embodiment includes a stem-loop structure, wherein the stem structure comprises an antisense region (or a section including the antisense region) comprising a sequence complementary to a first target gene, and complementary to the antisense region It may include a sense region capable of binding (or a section including the sense region).

The stem structure is 25 to 50bp, 25 to 45bp, 25 to 40bp, 25 to 39bp, 25 to 38bp, 25 to 37bp, 25 to 36bp, 25 to 35bp, 30 to 50bp, 30 to 45bp, 30 to 40bp, 30 to 39bp, 30-38bp, 30-37bp, 30-36bp, 30-35bp, 31-40bp, 31-39bp, 31-38bp, 31-37bp, 31-36bp, 31-35bp, 32-40bp, 32-39bp, 32 to 38 bp, 32 to 37 bp, 32 to 36 bp, 32 to 35 bp, 33 to 40 bp, 33 to 39 bp, 33 to 38 bp, 33 to 37 bp, 33 to 36 bp, 33 to 35 bp, 34 to 40 bp, 34 to 39 bp, 34 to 38 bp, 34 to 37 bp, 34 to 36 bp, 34 to 35 bp, such as 35 bp in length.

The stem structure may have a bubble structure due to mismatching of some sequences.

The nucleic acid construct according to an example may include one or more (one or more, two or more, or all three) features selected from the group consisting of the following (1) to (3) for a similar action to pri-miRNA. can:

(1) the stem structure is 1 to 13nt, 1 to 10nt, 1 to 8nt, 1 to 7nt, 1 to 6nt, 1 to 5nt, 2 to 13nt, 2 to 10nt, 2 to 8nt, 2 from the lower branch point of the stem structure to 7nt, 2 to 6nt, 2 to 5nt, 10nt, 9nt, 8nt, 7nt, 6nt, or 5nt mismatched GHG sequence having a bubble structure (H is a bubble forming sequence);

(2) the top branching point of the stem structure of the construct includes a 4 nt-long UGUG sequence from the last sequence of the stem structure to the loop structure; and

(3) at the lower branch point of the stem structure of the construct, a UG sequence of 2 nt in length from the last sequence of the polynucleotide strand extending from the 5' end (or 3' end) of the stem structure to the first sequence starting with the stem .

The stem structure may include a sense region 13 nt after the 5' end of the stem structure. The sense region may be 20 to 25 nt, 20 to 24 nt, 20 to 23 nt, 20 to 22 nt, 20 to 21 nt, 21 to 25 nt, 21 to 24 nt, 21 to 23 nt, 21 to 22 nt, or 21 nt, but is not limited thereto. .

In one example, the section including the sense region is 19 to 70 nt (nucleotide), 20 to 70 nt, 21 to 70 nt, 22 to 70 nt, 23 to 70 nt, 25 to 70 nt, 19 to 66 nt, 20 to 66 nt, 21 to 66 nt, 22 to 66 nt, 23 to 66 nt, 25 to 66 nt, 19 to 60 nt, 20 to 60 nt, 21 to 60 nt, 22 to 60 nt, 23 to 60 nt, 25 to 60 nt, 19 to 55 nt, 20 to 55 nt, 21 to 55 nt, 22 to 55 nt, 23 to 55 nt, 25 to 55 nt, 19 to 52 nt, 20 to 52 nt, 21 to 52 nt, 22 to 52 nt, 23 to 52 nt, 25 to 52 nt, 19 to 50 nt, 20 to 50 nt, 21 to 50 nt, 22 to 50 nt, 23 to 50 nt, 25 to 50 nt, 19 to 45 nt, 20 to 45 nt, 21 to 45 nt, 22 to 45 nt, 23 to 45 nt, 25 to 45 nt, 19 to 40 nt, 20 to 40 nt, 21 to 40 nt, 22 to 40 nt, 23 to 40 nt, 25 to 40 nt, 19 to 38 nt, 20 to 38 nt, 21 to 38 nt, 22 to 38 nt, 23 to 38 nt, 25 to 38 nt, 19 to 36 nt, 20 to 36 nt, 21 to 36 nt, 22-36 nt, 23-36 nt, 25-36 nt, 19-35 nt, 20-35 nt, 21-35 nt, 22-35 nt, 23-35 nt, 25-35 nt, 19-30 nt, 20-30 nt, 21-30 nt, 22-30 nt, 23-30 nt, 25-30 nt, 19-28 nt, 20-28 nt, 2 1 to 28 nt, 22 to 28 nt, 23 to 28 nt, 25 to 28 nt, 19 to 25 nt, 20 to 25 nt, 21 to 25 nt, 22 to 25 nt, 23 to 25 nt, or 25 nt in length It may be a single stranded polynucleotide.

In one embodiment, the section including the antisense region is 20 to 70 nt, 21 to 70 nt, 22 to 70 nt, 23 to 70 nt, 25 to 70 nt, 27 to 70 nt, 20 to 66 nt, 21 to 66 nt, 22 to 66 nt, 23 to 66 nt, 25 to 66 nt, 27 to 66 nt, 20 to 60 nt, 21 to 60 nt, 22 to 60 nt, 23 to 60 nt, 25 to 60 nt, 27 to 60 nt, 20-55 nt, 21-55 nt, 22-55 nt, 23-55 nt, 25-55 nt, 27-55 nt, 20-52 nt, 21-52 nt, 22-52 nt, 23-52 nt, 25 to 52 nt, 27 to 52 nt, 20 to 50 nt, 21 to 50 nt, 22 to 50 nt, 23 to 50 nt, 25 to 50 nt, 27 to 50 nt, 20 to 45 nt, 21 to 45 nt, 22 to 45 nt, 23 to 45 nt, 25 to 45 nt, 27 to 45 nt, 20 to 40 nt, 21 to 40 nt, 22 to 40 nt, 23 to 40 nt, 25 to 40 nt, 27 to 40 nt, 20-38 nt, 21-38 nt, 22-38 nt, 23-38 nt, 25-38 nt, 27-38 nt, 20-36 nt, 21-36 nt, 22-36 nt, 23-36 nt, 25-36 nt, 27-36 nt, 20-35 nt, 21-35 nt, 22-35 nt, 23-35 nt, 25-35 nt, 27-35 nt, 20-30 nt, 21-30 nt, 22-30 nt, 23-30 nt, 25-30 nt, 27-30 nt, 20-27 nt, 21-27 nt, 22-27 n single stranded polynucleotides of length t, 23-27 nt, 25-27 nt, 27 nt, 19-25 nt, 20-25 nt, 21-25 nt, 22-25 nt, 23-25 nt or 20-25 nt can be

In one example, the antisense region may include a sequence complementary to the sense region, for example, the antisense region may bind (hybridize) with the sense region, such that all or part of the nucleic acid sequence of the sense strand. and 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98 % or more, 98.5% or more, 99% or more, 99.5% or more, 99.8% or more, 99.9% or more, or 100% complementary nucleic acid sequence.

The stem-loop structure of the nucleic acid construct according to an example may include a loop structure, and the loop structure may include a single-stranded polynucleotide or consist of a single-stranded polynucleotide. The loop structure is 2 to 30nt, 2 to 27nt, 2 to 25nt, 2 to 23nt, 2 to 20nt, 2 to 16nt, 3 to 30nt, 3 to 27nt, 3 to 25nt, 3 to 23nt, 3 to 20nt, 3 to 16nt, 5-30nt, 5-27nt, 5-25nt, 5-23nt, 5-20nt, 5-16nt, 7-30nt, 7-27nt, 7-25nt, 7-23nt, 7-20nt, 7-16nt, 10 to 30 nt, 10 to 27 nt, 10 to 25 nt, 10 to 23 nt, 10 to 20 nt, or 10 to 16 nt, for example, a single-stranded polynucleotide having a length of 16 nt, or may consist of a single-stranded polynucleotide having a length in the above range.

In one example, the loop structure may exist in which the 3' end (or 5' end) of the section including the sense region and the 5' end (or 3' end) of the section including the antisense region are connected. .

The nucleic acid construct according to an example may include a polynucleotide sequence extending from the lower branch point of the stem structure. That is, the nucleic acid construct is a polynucleotide extending from the 5' end (or 3' end) of the segment containing the sense region in the stem structure, and/or the 3' end (or 5' end) of the segment containing the antisense region in the stem structure. ' at the end).

As used herein, an extended polynucleotide refers to a polynucleotide extending from the 5' end (or 3' end) of the segment containing the sense region in the stem structure, and the 3' end (or 5' end) of the segment containing the antisense region in the stem structure. ' may mean a polynucleotide extended at the end), or both.

In the nucleic acid construct, the extended polynucleotide may be present at the other end of the stem structure in which the loop structure is formed.

In one embodiment, the extended polynucleotide is 3 to 35 nt, 5 to 35 nt, 7 to 35 nt, 10 to 35 nt, 13 to 35 nt, 15 to 35 nt, 3 to 30 nt, 5 to 30 nt, 7 to 30 nt, 10 to 30 nt, 13 to 30 nt, 15 to 30 nt, 3 to 25 nt, 5 to 25 nt, 7 to 25 nt, 10 to 25 nt, 13 to 25 nt, 15 to 25 nt, 3 to 23 nt, 5 to 23 nt, 7 to 23 nt, 10 to 23 nt, 13 to 23 nt, 15 to 23 nt, 3 to 20 nt, 5 to 20 nt, 7 to 20 nt, 10 to 20 nt, 13 to 20 nt, 15 to 20 nt, 3 to 17 nt, 5 to 17 nt, 7 to 17 nt, 10 to 17 nt, 13 to 17 nt, 15 to 17 nt, 3 to 15 nt, 5 to 15 nt, 7 to 15 nt, 10 to 15 nt, or 13 to 15 nt in length.

The extended polynucleotide sequences may not be complementary to each other. In one example, the polynucleotide sequence extending from the 5' end of the section including the sense region in the stem structure and the polynucleotide sequence extending from the 3' end of the section including the antisense region in the stem structure are complementary to each other It may not be combined.

The extended polynucleotide sequence may include an antisense oligonucleotide (ASO) sequence and/or an anti-miRNA (anti-miRNA, anti-miR) sequence. In one example, the polynucleotide sequence extending from the 5' end of the segment containing the sense region in the stem structure, the polynucleotide sequence extending from the 3' end of the segment containing the antisense region in the stem structure, or both It may include an ASO sequence or an anti-miRNA sequence. The ASO sequence or the anti-miRNA sequence may include a sequence complementary to all or part of the sequence of the second target gene so as to regulate (increase and/or decrease) the expression of the second target gene.

By including the ASO sequence at the position in the nucleic acid construct according to an example, the nucleic acid construct (ASO sequence in the construct) according to an example complementarily binds to the mRNA sequence of the second target gene, thereby forming the second target gene. degrade mRNA and/or modulate (increase and/or decrease) selective splicing of the second target gene.

By including the anti-miRNA sequence at the position in the nucleic acid construct according to an embodiment, the nucleic acid construct (anti-miRNA sequence in the construct) according to an embodiment is a miRNA of a second target gene (the second target gene is combined with the second Inhibits the action of the miRNA of the second target gene (miRNA capable of inhibiting the expression of the second target gene by binding to the second target gene) by complementary binding with the miRNA sequence capable of inhibiting the expression of the target gene can do.

When the nucleic acid construct according to an embodiment is endogenously cleaved by DGCR8 and/or Droscha's complex in a cell, the polynucleotide strand including the ASO sequence and/or anti-miRNA sequence is separated separately to separate the ASO and/or Anti-miRNA activity may be exhibited, and such ASO and/or anti-miRNA activity may occur within the nuclear membrane of a cell.

The ASO sequence or anti-miRNA sequence included in the extended polynucleotide of the nucleic acid construct according to an example is from the 5' end of the section including the sense region in the stem structure, or the section including the antisense region in the stem structure. It can be introduced after 3 nt, after 4 nt, after 5 nt, after 6 nt, after 7 nt, after 8 nt, after 9 nt, or after 10 nt from the 3' end of In another example, the ASO sequence or anti-miRNA sequence contained within the extended polynucleotide of the nucleic acid construct is the 5' end of the segment containing the sense region in the stem structure or 3' of the segment containing the antisense region in the stem structure. It may be separated from the terminal by 3 nt or more, 4 nt or more, 5 nt or more, 6 nt or more, 7 nt or more, 8 nt or more, 9 nt or more, or 10 nt or more. When the ASO sequence or the anti-miRNA sequence is introduced according to the numerical range from the 5' end of the section including the sense region in the stem structure or the 3' end of the section including the antisense region in the stem structure, Compared to the case where it is not (e.g., if it is introduced after 1nt or 2nt after the 5' end of the segment containing the sense region in the stem structure or the 3' end of the segment containing the antisense region in the stem structure) It may not inhibit the action of Sha, or the gene suppression effect may be better.

The nucleic acid construct according to an embodiment may include chemically modified nucleotides. Specifically, the stem structure (section including the sense region, or section including the antisense region), the loop structure, and/or the extended polynucleotide sequence (the 5′ end of the section including the sense region in the stem structure) (or the polynucleotide extending at the 3' end), the polynucleotide extending at the 3' end (or the 5' end) of the segment containing the antisense region in the stem structure, or both) may include chemically modified nucleotides. can

In the nucleic acid construct according to an example, in the stem structure, loop structure, and/or extended polynucleotide sequence, C (cytidine, cytidine) and/or A (adenine, adenine) sequences may be chemically modified. have.

In the nucleic acid construct according to an example, in the loop structure, the extended polynucleotide sequence, or both, C (cytidine, cytidine) and/or A (adenine, adenine) sequences may be chemically modified.

In one example, the stem structure may include a chemically modified nucleotide at a “specific position”, and the “specific position” of the chemically modified nucleotide in the section including the sense region in the stem structure means the following position can be:

(1) 14, 17, from the 5' end of the stem structure (that is, the lower branching point where the stem ends in the section containing the sense region in the stem structure (the end of the stem structure leading to the extended polynucleotide that is not complementary)), one or more (eg, one or more, two or more, three or more, four or more, or all five) positions selected from the group consisting of 18, 20, and 27; or

(2) At least one selected from the group consisting of the 9th, 16th, 18th, 19th, and 22nd from the upper branching point (the end of the stem structure leading to the loop structure) where the stem ends in the section including the sense region among the stem structures (e.g. For example, one or more, two or more, three or more, four or more, or all five) positions.

In one example, the “specific position” of the chemically modified nucleotide of the antisense region included in the stem structure may refer to the following positions:

(1) 15, 16, from the 5' end of the stem structure (i.e., the lower branching point where the stem ends in the section containing the sense region in the stem structure (the end of the stem structure leading to the extended polynucleotide that is not complementary)), 19, 21, and one or more selected from the group consisting of 23 to 26 (eg, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or a position in the antisense region that complementarily binds to the nucleotide present at the position of 8); or

(2) At least one selected from the group consisting of the 10th to 13th, 15th, 17th, 20th, and 21st from the top branching point (the end of the stem structure leading to the loop structure) where the stem ends in the section including the antisense region among the stem structures (eg, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all 8).

In the present specification, the position counted from the 5' end of the stem structure (that is, the lower branch point where the stem ends in the section containing the sense region in the stem structure (the end of the stem structure leading to the extended polynucleotide that does not complement complementary)) The position in the section including the antisense region among the stem structures that complementarily binds with The positions and positions may correspond (usually) as shown in Table 1. For example, the 5' end of the stem structure (i.e., the lower branching point where the stem ends in the section containing the sense region in the stem structure (complementarily binding) The position in the section including the antisense region in the stem structure that is complementary to the 15th position from the end of the stem structure leading to the extended polynucleotide that does not It can correspond to the 21st position from the top bifurcation (the end of the stem structure leading to the loop structure) (interchangeably).

The 5' end of the stem structure (i.e., the lower branch point where the stem ends in the segment containing the sense region in the stem structure that complementarily binds to the segment containing the antisense region in the stem structure (the end of the stem structure extending toward the extended polynucleotide) )), the counted position Positions counted based on the top branching point where the stem ends (the end of the stem structure leading to the loop structure) in the section containing the antisense region among the stem structures One 35 2 34 3 33 4 32 5 31 6 30 7 29 8 28 9 27 10 26 11 25 12 24 13 23 14 22 15 21 16 20 17 19 18 18 19 17 20 16 21 15 22 14 23 13 24 12 25 11 26 10 27 9 28 8 29 7 30 6 31 5 32 4 33 3 34 2 35 One

The section including the sense region in the stem structure of the nucleic acid construct according to an example is at least one selected from the group consisting of 14, 17, 18, 20, and 27th from the 5' end of the stem structure (eg, at least one, 2 or more, 3 or more, 4 or more, or all 5) positions containing chemically modified nucleotides,

The section including the antisense region is at least one selected from the group consisting of 15, 16, 19, 21, and 23 to 26th from the 5' end of the stem structure (eg, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all) of the position {or, the upper branch point where the stem ends in the section including the antisense region in the stem structure (the end of the stem structure leading to the loop structure) ) at least one selected from the group consisting of 10 to 13, 15, 17, 20, and 21st (for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6) or more, 7 or more, or all 8) positions} and may include chemically modified nucleotides at positions complementary to binding with nucleotides present in the nucleotides.

The section including the sense region in the stem structure of the nucleic acid construct according to an embodiment may include a chemically modified nucleotide at the 14th position from the 5' end of the stem structure.

The section including the sense region in the stem structure of the nucleic acid construct according to an embodiment may include a chemically unmodified nucleotide at the 14th position from the 5' end of the stem structure.

The section including the sense region in the stem structure of the nucleic acid construct according to an example is at least one selected from the group consisting of 17, 18, 20, and 27th from the 5' end of the stem structure (eg, one or more, two types) It contains a chemically modified nucleotide at the position of the above, three or more, or all four),

The section including the antisense region is at least one selected from the group consisting of 19, 21, and 23 to 26th from the 5' end of the stem structure (eg, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all 6 types) comprising a chemically modified nucleotide at a position complementary to the nucleotide present at the position,

It may further include a chemically modified nucleotide at one or more positions selected from the group consisting of the following (1) to (3):

(1) a nucleotide present at the 14th position from the 5' end of the stem structure in the section including the sense region;

(2) in the section including the antisense region, a nucleotide present at a position complementary to a nucleotide present at the 15th position from the 5' end of the stem structure, and

(3) In a section including an antisense region, a nucleotide present at a position complementary to a nucleotide present at the 16th position from the 5' end of the stem structure.

The nucleic acid construct according to an embodiment may be any one nucleic acid construct selected from the group consisting of the following (1) to (4):

(1) the section including the sense region in the stem structure contains chemically modified nucleotides at positions 14, 17, 18, 20, and 27 from the 5' end of the stem structure,

The section including the antisense region includes chemically modified nucleotides at positions complementary to nucleotides present at positions 15, 16, 19, 21, and 23 to 26 from the 5' end of the stem structure, nucleic acid constructs;

(2) the section including the sense region in the stem structure contains chemically modified nucleotides at positions 17, 18, 20, and 27 from the 5' end of the stem structure,

The section including the antisense region includes chemically modified nucleotides at positions complementary to nucleotides present at positions 15, 16, 19, 21, and 23 to 26 from the 5' end of the stem structure, nucleic acid constructs;

(3) the section including the sense region in the stem structure contains chemically modified nucleotides at positions 17, 18, 20, and 27 from the 5' end of the stem structure,

The section including the antisense region contains chemically modified nucleotides at positions complementary to nucleotides present at positions 16, 19, 21, and 23 to 26 from the 5' end of the stem structure. ; and

(4) the section including the sense region in the stem structure contains chemically modified nucleotides at positions 17, 18, 20, and 27 from the 5' end of the stem structure,

The section including the antisense region comprises chemically modified nucleotides at positions complementary to nucleotides present at positions 19, 21, and 23 to 26 from the 5' end of the stem structure.

A nucleic acid construct according to an example is one selected from the group consisting of 1 to 13, 15, 16, 19, 21 to 26, and 28th or more (eg, 28 to 35th) from the 5' end of the stem structure. contain chemically unmodified nucleotides at the above positions, and/or,

The section including the antisense region is at least one position selected from the group consisting of 1 to 14, 17, 18, 20, 22, and 27 or more (eg, 28 to 35 th) from the 5' end of the stem structure { Alternatively, the ninth or less (eg, 1 to 9), 14, 16, 18, 19, from the top branching point (the end of the stem structure leading to the loop structure) where the stem ends in the section including the antisense region among the stem structures. and at least one position selected from the group consisting of 22 or more (eg, 22 to 35) nucleotides that are not chemically modified.

In addition to the chemical modification of the stem structure described above, the nucleic acid construct according to an embodiment has a loop structure, an extended polynucleotide sequence, or both, a C (cytidine, cytidine), and/or A (adenine, adenine) sequence This may be chemically modified.

In the nucleic acid construct according to an embodiment, at least one sequence selected from the group consisting of a polynucleotide sequence extended from the 5' end of the sense region, a polynucleotide sequence extended from the 3' end of the antisense region, and the loop structure is contains chemically modified nucleotides in the C (cytidine) and/or A (adenine) sequence;

In the stem structure, the section including the sense region includes chemically modified nucleotides at one or more positions selected from the group consisting of 14, 17, 18, 20, and 27th from the 5' end of the stem structure,

The section including the antisense region in the stem structure is complementary to a nucleotide present at one or more positions selected from the group consisting of 15, 16, 19, 21, and 23 to 26 from the 5' end of the stem structure. It may include a chemically modified nucleotide at the position.

That the nucleotide is not chemically modified may mean that it has the same composition as a nucleotide contained in a nucleic acid that is naturally or naturally present.

In the present specification, the chemical modification of a nucleotide (or nucleic acid) may mean that a sugar, a base, a binding site between nucleotides, or a combination thereof included in the nucleotide (or nucleic acid) is chemically modified. The chemical modification method is not particularly limited, and those skilled in the art can synthesize and modify the nucleotide (or nucleic acid) in a desired manner using methods known in the art. . Non-limiting examples of modified bases that can be introduced into nucleic acid molecules include hypoxanthine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene , 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (eg 5-methylcytidine), 5-alkyluridine (eg ribotimidine), 5-halo Uridine (eg 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (eg 6-methyluridine), propyne, and others (Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra). "Modified base" means a nucleotide base other than adenine, guanine, thymine, cytosine and uracil in the 1' position or the equivalent thereof.

In the chemical modification, the hydroxyl group at the 2' position of the ribose of at least one nucleotide included in the nucleic acid molecule is substituted with any one of a hydrogen atom, a fluorine atom, an -O-alkyl group, an -O-acyl group, and an amino group. It may be characterized in that, without being limited thereto, in order to increase the delivery capacity of a nucleic acid molecule, -Br, -Cl, -R, -R'OR, -SH, -SR, -N3 and -CN (R = alkyl, aryl, alkylene) may be substituted.

In one example, the chemical modification of the nucleotide means that the structure of the sugar contained in the nucleotide is modified to LNA (Locked Nucleic Acid) or the residue of the sugar is 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro Rho, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH ₂ -O-2'-bridge, 4'- (CH ₂ ) ₂ -O-2'-bridge, 2'-amino, and 2'-O-(N-methylcarbamate) may be modified with one or more selected from the group consisting of (methlycarbamate) .

The nucleic acid construct according to an example may exhibit one or more characteristics selected from the group consisting of the following (1) to (8), and specifically, a nucleic acid construct in which all nucleotides are not chemically modified, a previously known method ( For example, when compared to a nucleic acid construct chemically modified by an alternating modification method and/or a C/U sequence-based modification method, the following (1) to (8) One or more effects selected from the group consisting of, may be maintained, increased, or decreased:

(1) maintaining and/or increasing the interaction of said nucleic acids DGCR8, Drocha, and/or Dicer in vitro and/or in vivo (e.g., a nucleic acid construct by DGCR8, Drocha, and/or Dicer) maintaining and/or increasing the cutting rate of

(2) maintenance and/or increase in target gene silencing activity in vitro;

(3) increase in target gene silencing activity in vivo;

(4) reducing off-target effects;

(5) reduction of degradation by nucleases (eg, RNases) in vivo;

(6) increased intracellular uptake;

(7) increased stability in vitro and/or in vivo (eg, increased serum stability); and/or

(8) reduction in immune response (eg, immune response by TLR receptor activity in endosomes by the nucleic acid construct, or immune response by PKR activity of the nucleic acid construct exiting the cytoplasm, etc.).

The nucleic acid construct according to one embodiment can be usefully used as a therapeutic agent because the immune response is reduced in vivo without reducing the gene silencing inhibitory effect in vitro.

The alternating modification method is a method of chemically modifying adjacent nucleotides alternately. For example, according to the alternating modification method, the nucleotide located at an odd-numbered position from the 5' end of the sense strand is chemically modified, and the antisense strand complementary to a nucleotide located at an even-numbered position from the 5' end of the sense strand of nucleotides may be chemically modified. The C/U sequence-based modification method is a method of chemically modifying a nucleotide containing C and a nucleotide containing a base of U.

The "off-target effect" refers to an effect of unexpected degradation of off-target mRNA by the sense strand or suppression of expression of off-target genes by the sense strand and antisense strand when decomposition of off-target mRNA occurs by the sense strand By combining with this wrong target, it can include both the decomposition of off-target mRNA and the suppression of expression of off-target genes.

In one example, the nucleic acid construct is selected from the group consisting of (1) to (8) above when compared to siRNA (dsRNA having gene regulatory activity that is not processed by Dicer because of its short length) for the same target gene One or more effects may be maintained, increased, or decreased.

The nucleic acid construct according to an embodiment may be endogenously cleaved by DGCR8 and/or Drocha (eg, a complex of DGCR8 and Drocha). In addition, the nucleic acid construct according to an embodiment may be sequentially cleaved by DGCR8 and/or Droscha (eg, a complex of DGCR8 and Droscha) and Dicer. The cleavage by DGCR8 and/or Droscha may be made in the nuclear membrane of a cell, and cleavage by the Dicer may be made in the cytoplasm.

As used herein, “Drosha” refers to an RNase capable of producing a nucleic acid (Pre-miRNA) having a stem-loop structure by cutting hairpin structure RNA or Pri-miRNA (primary-microRNA) including a single-stranded-stem-loop structure. It may refer to endoribonuclease (endoribonuclease) in the III family.

As used herein, “DGCR8” refers to a protein that binds with Drocha to form a protein complex. It uses a double-stranded RNA binding domain to stabilize the binding of Droscha's pri-miRNA to aid in the RNA cleavage process of Droscha. It may mean a protein (dsRBD).

As used herein, “dicer” refers to a nucleic acid fragment of 19-25 nt (nucleotides) by cleaving dsRNA or dsRNA-containing molecules, for example, double-stranded RNA (dsRNA) or Pre-miRNA (precursor-microRNA). It may refer to an endoribonuclease in the RNase III family capable of producing a double-stranded nucleic acid of length (eg, miRNA or siRNA capable of exhibiting gene silencing activity).

When the nucleic acid construct according to an example is administered to a subject, the double-stranded region at the 4 bp position is cleaved in the 5' end (or 3' end) direction based on the GHG mismatching sequence of the sense region by the DGCR8 protein complex in the nucleus. It becomes a pre-miRNA analog and can be released into the cytoplasm (step 1: processing by Droscha and DGCR8 complex).

In one example, 1 to 5 nt, 2 to 5 nt, 2 to 3' end (or 5' end) of the antisense strand in the pre-miRNA analog (or cleaved product, cleaved product) cleaved by Drocha Overhangs of 4 nt, 2-3 nt, or 2 nt in length may occur.

The pre-miRNA analogue released into the cytoplasm includes a stem-loop structure, and the stem structure includes a sense region and an antisense region, and each sense region and/or antisense region has a predetermined length (eg, 19 nt, 20 nt, 21 nt, 22 nt, 23mt, 24 nt, or 25 nt) or longer, recognized as “long dsRNA (double strand RNA)” and cut by Dicer to about 19 to 25 nt in length can be adjusted (step 2; Dicer processing).

As used herein, "subject", "individual", or "patient" may refer to an organism to which the nucleic acid construct according to an embodiment can be administered. The subject is a mammal (eg, human) or a mammalian cell. (eg a human cell), an organism that is a donor or recipient of an explant cell, or the cell itself.

As used herein, the term “Drocha cleavage site” refers to a site at which the Droscha and DGCR8 protein complex cuts the nucleic acid construct according to an embodiment. In one example, Drocha contains two RNase III domains, which are typically capable of cleaving both the sense and antisense regions (strands) included in the stem structure.

As used herein, “Dicer cleavage site” refers to a site where Dicer cleaves the product cleaved by Droscha and DGCR8 protein complex. In one example, Dicer contains two RNase III domains, which are typically capable of cleaving both the sense and antisense regions (strands) included in the stem structure.

In the nucleic acid construct according to an example, the nucleotides present after the 16th (16th or more positions) from the 5' end of the sense region and the nucleotides of the antisense region complementary thereto are not chemically modified, so that by Dicer It may be easily recognized.

In one embodiment, 1 to 5 nt, 2 to 5 nt, at the 3' end (or 5' end) of the sense region (strand) in the double-stranded nucleic acid (or cleaved product, cleaved product) cleaved by Dicer; Overhangs of 2 to 4 nt, 2 to 3 nt, or 2 nt in length may occur.

The cleaved double-stranded nucleic acid (or cleaved product, cleaved product) is RNA-induced through RLC (RISC-loading complex) mediated by Dicer and the human immunodeficiency virus transactivating response RNA-binding protein (TRBP). silencing complex) (step 3). In this process, in vertebrates such as humans, asymmetric sensing occurs in which the thermodynamically weak strand of TRBP cleaved double-stranded nucleic acid is rearranged so that it can be recognized as a guide RNA by Ago2.

RISC is a ribonucleoprotein that recognizes and loads a double-stranded nucleic acid, and thermodynamically cuts the strong strand (passenger RNA) from the double-stranded nucleic acid loaded with Ago2 (Argonaute 2), a protein corresponding to the catalytic site in RISC, and thermodynamically to leave a weak strand (guide RNA) (step 4). Since the sequence of the antisense region (antisense sequence) included in the stem structure is designed to be a thermodynamically weak strand, the sense sequence is cleaved with high efficiency and the antisense sequence remains. The antisense sequence recognizes the mRNA of the target gene (step 5) and complementarily binds thereto to form a dsRNA and cleavage, whereby gene silencing can occur (step 6).

In one example, the product cleaved by the Drocha and DGCR8 protein complex may be cleaved to an appropriate length (eg, 19 to 30 nt, 20 to 30 nt, 22 to 27 nt) by Dicer, and the appropriate length is '20 to 25'+2 nt (eg, 19+2 nt, 20+2 nt, 21+2 nt, 22+2 nt, 23+2 nt, 24+2 nt, or 25+2 nt ( For example, it may be a nucleic acid having a 2 nt overhang structure at the 3' end of the sense strand).

According to one embodiment, a nucleic acid construct in which all nucleotides are not chemically modified (control) or a known method (eg, alternating modification) and/or a C/U sequence-based modification method (C/U) The nucleic acid construct according to an embodiment may have similar or increased Dicer reaction rate in vitro and/or in vivo compared to the nucleic acid construct chemically modified with sequence-based modification). The Dicer cleavage rate (%) or Dicer reaction rate analysis may be evaluated by a known method. In one embodiment, 10 pmole of the nucleic acid construct, 5 pmole of recombinant human Dicer, and 1 μl of Dicer reaction buffer (300 mM Tris-HCl (pH 6.8), 500 mM NaCl) in Dicer reaction rate analysis , 2 μl of DEPC-treated deionized water (DW), mixed with 2 μl of 25 mM MgCl ₂ , and incubated at 37° C. for 12 hours, each sample was collected and the thickness of the Dicer substrate band was measured through electrophoresis. By measuring, the dicer cleavage (%) degree can be calculated to analyze the dicer reaction rate. When calculating the Dicer cleavage rate (%) by the above method, 40 to 60% of the Droscha cleavage product of the nucleic acid construct according to an example is cleaved by reacting with Dicer for 1 hour, and reacting with Dicer for 2 hours to 90 to 100% can be cleaved.

The nucleic acid construct according to an embodiment may be siRNA for the same target gene, a nucleic acid construct in which all nucleotides are not chemically modified, or a known method (eg, alternating modification method and/or C/U sequence-based The gene silencing activity may be increased in vitro and/or in vivo compared to a nucleic acid construct chemically modified by a modification method (C/U sequence-based modification).

The "siRNA (small interfering RNA)" refers to a short (eg, 19 nt) double-stranded RNA (dsRNA) that mediates sequence-specifically efficient gene silencing. The nucleic acid construct according to an example is different from siRNA in that it can serve as a substrate for the Drocha and DGCR8 protein complex, and the nucleic acid construct according to an example is sequentially cleaved by the Drocha and DGCR8 protein complex and Dicer to produce The product can be understood as a kind of siRNA.

The nucleic acid construct according to an embodiment includes an antisense region capable of complementary binding to the first target gene in the stem structure, and thus is sequentially cleaved by the Drosha and DGCR8 protein complexes and Dicer. 1 Can regulate the expression of a target gene.

The first target gene or the aforementioned second target gene (gene whose expression is regulated by an ASO sequence and/or an anti-miRNA sequence) is a protein-coding gene, a proto-oncogene, an oncogene, a tumor suppressor gene, and a cell signal It may be one selected from the transgene.

Another aspect may provide a composition for inhibiting gene expression, including the nucleic acid construct. The nucleic acid construct is the same as described above. Another aspect provides a method for inhibiting the expression of a target gene comprising administering to a subject an effective amount of the nucleic acid construct and / or the composition for inhibiting gene expression. The method of suppressing the expression of the target gene may further include the step of identifying an individual in need of suppression of the expression of the gene before the step of administering. Another aspect provides the use of the nucleic acid construct for use in the preparation of a composition for inhibiting gene expression.

In one example, the method for suppressing the expression of the target gene may be to suppress the expression of the target gene in the target cell in vitro.

The gene may be selected from protein coding genes, proto-oncogenes, oncogenes, tumor suppressor genes, and cell signaling genes.

In one embodiment, the composition for inhibiting gene expression may further include a carrier. The carrier may be at least one selected from the group consisting of lipid molecules, liposomes, micelles, cationic lipids, cationic polymers, ligand conjugates, and cationic metals.

In one embodiment, the nucleic acid construct may be directly processed, complexed with cationic lipids, or packaged into liposomes for delivery, for example, encapsulation in liposomes, iontophoresis, or biodegradable polymers, hydrogels, cyclodextrins, poly to be administered to cells and/or subjects by a variety of methods known to those skilled in the art, including incorporation into other vehicles such as (lactic-co-glycolic) acid (PLGA) and PLCA microspheres, biodegradable nanocapsules and biocompatible microspheres. can

The term “liposome” refers to a vehicle composed of amphiphilic lipids arranged in one or more bilayers, eg, one bilayer or multiple bilayers. Liposomes include mono- and multi-membrane vehicles having a membrane formed from a lipophilic substance and an aqueous interior. The aqueous portion comprises a nucleic acid molecule. The lipophilic substance separates the aqueous interior from the aqueous exterior, which typically does not contain the nucleic acid molecule, but may include, in some instances. Liposomes are useful for transport and delivery of active ingredients to the site of action. Because the liposome membrane is structurally similar to a biological membrane, when the liposome is applied to a tissue, the bilayer of the liposome fuses with the bilayer of the cell membrane. As the liposome and cell integration progress, the aqueous content of the interior containing the nucleic acid molecule is delivered into the cell where the nucleic acid molecule can specifically bind to a target gene to mediate RNAi. In one example, the liposome is specifically targeted to direct the nucleic acid molecule to a particular type of cell.

The "micelle" is defined as a specific type of molecular assembly in which amphiphilic molecules are arranged in a globular structure, with the hydrophobic portions of the molecule all facing inward so that the hydrophilic portions remain in contact with the surrounding aqueous phase. When the environment is hydrophobic, the opposite arrangement exists.

Another aspect provides a pharmaceutical composition for preventing and / or treating a disease comprising the nucleic acid construct. Another aspect may be to provide a pharmaceutical composition for preventing or treating cancer or a proliferative disease comprising the nucleic acid construct. The nucleic acid construct is the same as described above. In addition to the nucleic acid construct, it may further include a pharmaceutically acceptable carrier. Another aspect may provide a method for preventing or treating a disease comprising administering the pharmaceutical composition to a subject in a pharmaceutically effective amount. The treatment method may further include the step of identifying an individual in need of prevention or treatment of a disease before the step of administering. Another aspect provides the use of the nucleic acid construct for use in the manufacture of a pharmaceutical composition for the prevention or treatment of cancer or proliferative disease.

The disease may include a genetic disease and/or a non-genetic disease.

The disease is cancer, proliferative disease, digestive disease, kidney disease, neurological disease, mental disease, blood and tumor disease, cardiovascular disease, respiratory disease, endocrine disease, infectious disease, musculoskeletal disease, gynecological disease, genitourinary disease, skin disease , and may be at least one selected from the group consisting of ophthalmic diseases, but is not limited thereto.

The cancer may be a solid cancer or a blood cancer, such as, but not limited to, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, skin or intraocular melanoma, Rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer It may be one or more selected from the group consisting of cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, osteosarcoma, etc. However, the present invention is not limited thereto.

The proliferative disease is myeloproliferative disease (MPD), primary myelofibrosis, chronic myeloproliferative disease (eg, polycythemia vera), thrombocythemia vera, idiopathic thrombocythemia (Essential Thrombocythemia), chronic myelogenous At least one selected from the group consisting of Chronic Myeloid Leukemia, and/or Idiopathic Myelofibrosis), Aplastic Anemia (eg, Severe Aplastic Anemia), etc. However, the present invention is not limited thereto.

The digestive diseases include peptic ulcer disease (PUD), gastroesophageal reflux disease (Gastroesophageal Re&ux Disease), constipation, diarrhea, irritable bowel syndrome (Constipation, Diarrhea and Irritable Bowel Syndrome), nausea and vomiting (Nausea and Vomiting), Select from the group consisting of In&ammatory Bowel Disease, Pancreatitis, Liver Cirrhosis and Complications, Viral Hepatitis, Drug-Induced Liver Injury, etc. It may be one or more, but is not limited thereto.

The kidney disease is fluid and electrolyte imbalance (Fluid and Electrolyte Disorders), acid-base disorders (Acid-base Disorders), drug-induced kidney disease (Drug-Induced Kidney Disease), renal dysfunction (Renal Impairment), acute kidney injury ( Acute Kidney Injury), Chronic Kidney Disease (Chronic Kidney Disease) may be at least one selected from the group consisting of, but is not limited thereto.

The neurological disease may be one or more selected from the group consisting of headache, epilepsy, Alzheimer's disease, Parkinson's disease, and the like, but is not limited thereto.

The mental disorders include Major Depressive Disorder, Schizophrenia, Generalized Anxiety Disorder, Panic Disorder, Bipolar Disorder, Attention De&cit Hyperactivity Disorder), alcohol, nicotine, caffeine addiction (Alcohol, Nicotine, and Caeine Addiction), sleep disorder (Sleep Disorder), may be one or more selected from the group consisting of eating disorders (Eating disorders), but is not limited thereto.

The blood and tumor diseases are Anemias, Lung Cancer, Gastric Cancer, Colorectal Cancer, Breast Cancer, Gynecologic Cancers, Prostate Cancer, It may be one or more selected from the group consisting of leukemias, lymphomas, etc., but is not limited thereto.

The cardiovascular disease is hypertension, heart failure, ischemic heart disease, acute coronary syndrome (ACS), arrhythmia (Arrhythmias), dyslipidemia (Dyslipidemia), stroke (Stroke) ), Venous Thromboembolism, Peripheral Arterial Disease, Hypovolemic Shock, etc. may be at least one selected from the group consisting of, but is not limited thereto.

The respiratory disease may be one or more selected from the group consisting of asthma, chronic obstructive pulmonary disease, allergic rhinitis, and the like, but is not limited thereto.

The endocrine disease may be one or more selected from the group consisting of diabetes (Diabetes Mellitus), thyroid disease (Thyroid Disorder), pituitary and adrenal gland disease (Pituitary & Adrenal Gland Disorders), and the like, but is not limited thereto.

The infectious disease is upper respiratory tract infection, pneumonia, urinary tract infection, tuberculosis, meningitis, gastrointestinal and intraperitoneal infections (Gastrointestinal/Intraabdominal Infections), skin At least one selected from the group consisting of Skin and Soft tissue Infection, Super&cial Fungal Infections / Deep mycoses, Sepsis, and Sexually Transmitted Infection (STI). may be, but is not limited thereto.

The musculoskeletal disease may be one or more selected from the group consisting of osteoarthritis, rheumatoid arthritis, osteoporosis, gout and hyperuricemia, and the like, but is not limited thereto.

The gynecological disease may be one or more selected from the group consisting of drug use in Pregnancy and Lactation, menopause, and urinary incontinence during pregnancy and lactation, but is not limited thereto.

The genitourinary disease may be one or more selected from the group consisting of Benign Prostatic Hyperplasia, Prostatitis, and the like, but is not limited thereto.

The skin disease may be at least one selected from the group consisting of atopic dermatitis, psoriasis, and the like, but is not limited thereto.

The ophthalmic disease may be glaucoma, but is not limited thereto.

As used herein, "administration" means introducing a predetermined substance to a patient (subject) by any suitable method, and the administration route of the pharmaceutical compositions is through any general route as long as the drug can reach the target tissue. may be administered. It may be intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc., but is not limited thereto. However, when administered orally, since the peptide is digestible, it is preferred that the oral composition be formulated to coat the active agent or to protect it from degradation in the stomach. Preferably, it may be administered in the form of an injection. In addition, long-acting formulations may be administered by any device capable of transporting the active agent to a target cell.

According to one embodiment, in the pharmaceutical composition or method, the pharmaceutical composition or the composition comprising the nucleic acid construct together with one or more additives selected from the group consisting of pharmaceutically acceptable carriers, diluents, and excipients. can be provided.

The pharmaceutically acceptable carrier, as commonly used in the formulation of pharmaceutical compositions, is lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, fine It may be at least one selected from the group consisting of crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. , but is not limited thereto. In addition to the above components, the pharmaceutical composition includes at least one selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. commonly used in the preparation of pharmaceutical compositions. can do.

The composition (a pharmaceutical composition or a composition for inhibiting gene expression) may be administered orally or parenterally. In the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc. can be administered. Since the protein or peptide is digested upon oral administration, oral compositions may be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the composition may be administered by any device capable of transporting the active agent to a target cell.

The appropriate dosage of the composition may be prescribed in various ways depending on factors such as formulation method, administration mode, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity.

The dosage of the composition may be in the range of 0.1 to 1000 mg/kg, or 0.1 to 200 mg/kg, based on an adult. For example, a composition comprising a dinucleic acid molecule at a concentration of 0.1 to 100 nmole may be administered at a dose of 0.1 to 1000 mg/kg, 1 to 500 mg/kg, or 1 to 100 mg/kg. Administration may be administered once a day or divided into several administrations. The daily dosage may be formulated as one preparation in a unit dosage form, formulated in an appropriate amount, or prepared by internalizing in a multi-dose container.

As used herein, "pharmaceutically effective amount" or "effective amount" means that the active ingredient (a nucleic acid construct according to an example) has a desired effect (eg, inhibiting the expression of a target gene or preventing cancer or proliferative disease and / or therapeutic effect), and it is dependent on factors such as formulation method, administration method, patient's age, weight, sex, morbidity, food, administration time, administration route, excretion rate, and response sensitivity. can be prescribed in a variety of ways.

The pharmaceutical composition may be prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method readily practiced by those skilled in the art, or may be prepared by internalizing in a multi-dose container. . In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

In addition, the pharmaceutical composition may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

The composition for suppressing gene expression, or the subject of the pharmaceutical composition, or the subject of the gene expression suppression method, prevention or treatment method, the patient (subject) includes humans, primates such as monkeys, or rodents such as rats and mice. It may be a mammal, or a cell or tissue isolated from the mammal, or a culture thereof, but is not limited thereto.

The construct according to an example may not only effectively reduce the expression of the first target gene, but also inhibit selective splicing, degrade the mRNA of the second target gene, and/or inhibit the action of miRNA .

1A and 1B show the sequence and structure of a nucleic acid construct (pri-miRNA construct) according to an example. Specifically, Figure 1a shows a schematic diagram for designing a nucleic acid construct (pri-miRNA construct) according to an example. In Figure 1a, the underlined sequence represents the Drocha recognition motif, the shaded sequence represents the antisense sequence, the framed sequence represents the sense sequence, and the rest of the sequences represent the rest of the pri-miRNA. It represents a randomly constructed ribonucleotide sequence that can be applied. 1B shows a nucleic acid construct (pri-miRNA construct) according to an example of a GFP target (upper construct) and an HPRT target (lower construct). Figure 1c is a sample in which the single strand of each group of GFP target and HPRT target pri-miRNA separated into two strands and the two strands of each group are ligated using T4 RNA ligase2 (NEB), and then the unreacted single strand is removed. The result of separation by PAGE gel (left gel picture) and its schematic diagram (right picture) are shown.

2A to 2C show the results of whether a nucleic acid construct (siRNA precursor) according to an example is successfully cleaved by Drosha and Dicer. Specifically, FIG. 2a shows pri-miR Let 7B, a naturally occurring miRNA precursor by the activity of Droschawa Dicer enzymes isolated from human cells, and a 25 bp double-stranded 2 nt well known substrate for Dicer enzymes. The result of separating the cut result of Dicer substrate siGFP (25+2 Linear) with an overhang structure by PAGE gel (left gel photo) and its schematic diagram (right figure) are shown, and FIG. The site and the Dicer cleavage site of Dicer substrate linear ribonucleic acid, which is a Dicer substrate, are shown. Fig. 2c shows the PAGE gel separation of pri-miGFP or pri-miHPRT sequentially cleaved by the activity of Droscha and Dicer enzymes. The result (left gel photo) and its schematic diagram (right figure) are shown.

3 and 4 are results showing the intracellular gene suppression efficiency of siRNA and pri-miRNA.

3 shows the results of comparing the HPRT1 mRNA expression level of the control group (PBS group) as 100% after measuring the reduction in the expression of HPRT1 mRNA by pri-miHPRT in the HCT116 cell line using RT-PCR.

Figure 4 shows the GFP mean fluorescence intensity (MFI) of the decrease in the expression of GFP by pri-miGFP in the KB-GFP cell line (a stable cell line that is genetically engineered in KB cells to continuously express GFP fluorescent protein). The results are shown by comparing the GFP average fluorescence intensity of the control group (PBS group) as 100% after measurement using FACS.

5 shows the results of comparing the degree of reduction in HPRT1 mRNA expression by pri-miHPRT in HCT116 wild-type (WT) and HCT116 Drosha knockout (KO) cell lines.

6 shows the structures of the Non-Mod, SS-Mod, ST-Mod, Seq-Mod, PP-Mod, PP(-1)-Mod, PP(-2)-Mod, and PP(-3)-Mod groups. , is a schematic diagram showing the sequence, and whether or not chemical modification. The sequence indicated by a border in FIG. 6 indicates a nucleotide position in which a 2'-OH residue is modified with a 2'-O-Methyl group.

7a and 7b show that each single strand of each group of FIG. 6 separated into two strands and the two strands of each group of FIG. 6 are linked using T4 RNA ligase2 (NEB), and the unreacted single strand The result of separating the removed sample by PAGE gel is shown.

FIG. 8 shows the result of measuring the decrease in GFP expression level by ribonucleic acid in each group of FIG. 6 in the KB-GFP cell line using FACS. Specifically, the upper graph of FIG. 8 is a graph comparing the target gene silencing effect of Non-Mod, SS-Mod, ST-Mod, Seq-Mod, and PP-Mod groups, and the lower graph of FIG. 8 is SS-Mod , is a graph comparing the target gene silencing effect of Seq-Mod, PP-Mod, PP(-1)-Mod, PP(-2)-Mod, and PP(-3)-Mod groups.

Figure 9a shows the result of confirming that the ribonucleic acid of each group of Figure 6 is cleaved by the activity of the Droscha enzyme over time to form a product, separated using PAGE gel, and a schematic diagram thereof, Figure 9b is for each group Shows the result of confirming that the Droscha cleavage product of ribonucleic acid is cleaved by the activity of the Dicer enzyme over time to form a product by separation using PAGE gel and a schematic diagram thereof. Figure 9c is the result of confirming that the ribonucleic acid of each group of Figure 6 is degraded by nuclease present in C57bl/6NCrSlc mouse serum over time (top) and the RNA structure remaining without degradation for each time It is a graph (bottom) showing the band intensity of 0 min as 1 as the standard. Figure 9d is a comparison result of the immune-induced reduction of the nucleic acid construct according to the chemical modification by measuring the expression level of TNF-α in human immune cells (Primary Peripheral Blood Mononuclear Cells, PBMC) by ribonucleic acid of each group.

Figure 10a shows pri-miHPRT ASO1 (top) and pri-miHPRT ASO1 (top) and pri- in which ASO1 or ASO2 is introduced into a single-stranded region extended from the 3' end of a section including an antisense region to impart a second target gene regulatory function to pri-miHPRT. miHPRT ASO2 (bottom) shows the structures and sequences of two nucleic acid constructs. The sequence indicated by the frame in FIG. 10A indicates a sequence region composed of DNA that functions as an antisense oligonucleotide for the survival motor neuron 2 gene (SMN2-ASO), and the shaded sequence among the sequences within the frame indicates that the sugar structure is LNA. The substituted sequence is indicated, and the sequence marked with * indicates the sequence in which the phosphorodiester bond is substituted with the phosphorothioate bond, and the rest of the sequences within the frame indicate the DNA sequence.

Figure 10b shows two strands of the 5' end fragment of pri-miHPRT and the HPRT 3' end fragment to which ASO1 is applied or the HPRT 3' end fragment to which ASO2 is applied using T4 RNA ligase2 (NEB) to connect pri-miHPRT ASO1 and pri- miHPRT ASO2 The result of separating the non-reacted single-stranded sample by PAGE gel after making two nucleic acid constructs (left gel photo) and a schematic diagram (right figure) are shown.

11 shows the results of confirming that the ribonucleic acids of each group of pri-miHPRT ASO1 and pri-miHPRT ASO2 are cleaved by the activity of the Drocha enzyme to form a product by separation using PAGE gel.

12 shows the degree of reduction in the expression of SMN2 Δ7 mRNA (SMN2 mRNA with Exon 7 removed by splicing) and HPRT1 mRNA by pri-miHPRT ASO1 and pri-miHPRT ASO2 in HCT116 cell line. Specifically, the result of comparing the expression level of HPRT1 mRNA (left orange graph) and the expression level of SMN2 Δ7 mRNA when HCT116 cells were treated with ribonucleic acid of each group (right sky blue graph) are shown. The SMN2 Δ7 mRNA expression level was corrected using the SMN2 full mRNA expression level measured using RT-PCR.

Figure 13 is in the HCT116 cell line in order to confirm the degree of change in the expression level of SMN2 Δ7 mRNA by pri-miHPRT, pri-miHPRT ASO2 and 3' ASO in the cell line using PCR amplification and PAGE gel of Exon 6 to 8 sites of SMN2 mRNA. This is the confirmed result.

14a shows the structure and sequence of pri-miHPRT Anti-miR21 in which Anti-miR21 is introduced into a single-stranded region extended from the 3' end of the section including the antisense region to impart a second target gene regulatory function to pri-miHPRT. indicates In Fig. 14a, the sequence indicated by the frame indicates the sequence region composed of DNA functioning as Anti-miR21, and the shaded sequence among the sequences within the frame indicates the sequence in which the sugar structure is substituted with LNA.

Figure 14b is a single strand that did not react after making pri-miHPRT Anti-miR21 by linking the 5' end fragment of pri-miHPRT and the HPRT 3' end fragment to which Anti-miR21 was applied using T4 RNA ligase2 (NEB). The result of separating the sample from which was removed by PAGE gel (left gel picture) and its schematic diagram (right picture) are shown.

15 shows the results of confirming that the ribonucleic acids of each group of pri-miHPRT and pri-miHPRT Anti-miR21 are cleaved by the activity of the Droscha enzyme over time to form a product, separated using PAGE gel.

16 shows the results of comparing the expression levels of miR21 and HPRT1 mRNA when HCT116 cells were treated with ribonucleic acids of each group of pri-miHPRT, pri-miHPRT Anti-miR21, and 3' Anti-miR21 in HCT116 cell line. . The miR21 expression level was corrected using the U6 snRNA expression level measured using RT-PCR.

17 is a result of confirming using Western Blot to confirm the degree of change in the expression level of PDCD4 by pri-miHPRT, pri-miHPRT Anti-miR21 and Anti-miR21 and the expression level of Vinculin as an internal control in the HCT116 cell line.

18 shows a schematic diagram of a process in which a construct according to an example can be introduced into a cell to silence a target gene and additionally exhibit ASO activity and Anti-miRNA activity.

The present invention will be described in more detail with reference to the following examples, but the scope of the rights is not limited to the following examples.

Example 1. Preparation of siRNA precursors

The RNA strands listed in Table 2 below were ordered from IDT or Bioneer, and chemically synthesized ones were purchased and used.

siGFP and siHPRT described in Table 2 below were prepared to compare the gene suppression effect of pri-miRNA with that of an existing short-length siRNA. Dicer substrate siGFP described in Table 2 was prepared to confirm the activity of the Dicer enzyme isolated from the cell as a Dicer substrate nucleic acid.

Sample Name Sequence (5'->3') SEQ ID NO: siGFP Sense GCAAGCUGACCCUGAAGUUdTdT SEQ ID NO: 1 Antisense AACUUCAGGGUCAGCUUGCdTdT SEQ ID NO: 2 siHPRT Sense CAGACUUUGUUGGAUUUGAdTdT SEQ ID NO: 3 Antisense UCAAAUCCAACAAAGUCUGdTdT SEQ ID NO: 4 Dicer substrate siGFP Sense GCAAGCUGACCCUGAAGUUAUCACC SEQ ID NO: 5 Antisense GGUGAUAACUUCAGGGUCAGCUUGCCA SEQ ID NO: 6 pri-miLet7B 5' fragment CAAGGCCGGGCCUGGCGGGGUGAGGUAGUAGGUUGUGUGGUUUCAGGGCAGUGAUGUUGCCCCUCGGAAGAUAACUAUAC SEQ ID NO: 7 3' fragment AACCUACUGCCUUCCCUGAGGAGCCCAGUGACA SEQ ID NO: 8 pri-miGFP 5' fragment CCAUUCCCAGCCCUUGCGCUACUCCACGGCAAGCUGACCCUGAAGUUCAUGUGGACAGGUAAGGAGAUGAACUUCAGG SEQ ID NO: 9 3' fragment GUCAGCUUGCCGUGGCGUAGCGCUCAAUCCCUAUAGUG SEQ ID NO: 10 pri-miHPRT 5' fragment CCAUUCCCAGCCCUUGCGCUACUCCAUGCCAGACUUUGUUGGAUUUGAAUGUGGACAGGUAAGGAGAUUCAAAUCCAAC SEQ ID NO: 11 3' fragment AAAGUCUGGCAUGGCGUAGCGCUCAAUCCCUAUAGUG SEQ ID NO: 12

In Table 2, dT means deoxythymidine.

In the case of siGFP, siHPRT, and Dicer substrate siGFP, the Antisense and Sense strands were mixed in 1X PBS buffer to a concentration of 10 μM, respectively, and then heated at 95 ° C for 3 minutes using a thermal cycler (Bio-Rad T100TM) at -1.0 ° C/s. The temperature was decreased from 95 °C to 4 °C at a rate to hybridize.

The strands corresponding to the 3' fragment of each gene target were reacted at 37 °C for 2 hours using T4 Polynucleotide Kinase (NEB) to phosphorylate the 5' end of the RNA. The 5' fragment strand and the phosphorylated 3' fragment strand were mixed in 1X PBS aqueous solution to a concentration of 10 µM, respectively, and after 3 minutes at 95 °C, the temperature was decreased from 95 °C to 4 °C at a rate of -1.0 °C/s to hybridize. did it The hybridized RNA mixture was reacted for 12 hours at 25 °C using T4 RNA ligase (NEB) to connect the double-stranded Nick structure to prepare a pri-miRNA structure.

The linked RNA product was mixed with 10 μl of 2X RNA loading dye (NEB) per 10 μl and denatured at 70° C. for 20 minutes to prepare a sample for PAGE gel electrophoresis. The RNA strands were separated from unreacted RNA strands by ligase by gel running on 15% polyacrylamide gel with 8% urea added at 200V for 40 minutes. After staining the gel with Gel red (Biotium), the RNA band portion of the product linked with Gel Doc ^TM EZ (Bio-Rad) was confirmed. The siRNA precursor was separated by cutting the PAGE gel part of the band corresponding to the RNA of the 5' fragment and 3' fragment strands and shaking in 1X Tris-Borate-EDTA buffer (TBE buffer) for 24 hours.

1A and 1B show the sequence and structure of a nucleic acid construct (pri-miRNA construct) according to an example. Specifically, Figure 1a shows a schematic diagram for designing a nucleic acid construct (pri-miRNA construct) according to an example. In Figure 1a, the underlined sequence represents the Drocha recognition motif, the shaded sequence represents the antisense sequence, the framed sequence represents the sense sequence, and the rest of the sequences represent the rest of the pri-miRNA. It represents a randomly constructed ribonucleotide sequence that can be applied. 1B shows a nucleic acid construct (pri-miRNA construct) according to an example of a GFP target (upper construct) and an HPRT target (lower construct).

After ligase treatment, the separated pri-miGFP and each strand of 5' fragment and 3' fragment of each group that did not react with pri-miHPRT were mixed with 5 μl of 2X RNA loading dye (NEB) at 0.5 pmol at 70 ° C for 20 minutes. Samples were prepared for PAGE gel electrophoresis by denaturing. After gel running on 15% Polyacrylamide gel with 8% Urea added at 200V for 40 minutes, the gel was stained with Gel red to obtain a gel image with Gel Doc ^TM EZ, and the results are shown in Fig. 1c.

As shown in FIG. 1c , it was confirmed that, in the sample separated after ligase treatment, the unreacted single strand was removed and the length was extended so that a band appeared at an upper position than the unreacted strand.

Example 2. Dicer and Droscha Enzyme Isolation for In-vitro Cleavage Experiments

For Drosha enzyme isolation, 10 μg of each of the plasmids pXO-Drosha-flag and pXO-DGCR8-HA (provided by Professor Bitnaeri Kim, Seoul National University) were diluted in Opti-MEM (Gibco) and 15 μL of lipofectamine 2000 (Invitrogen) was added. After mixing so that the volume of the entire mixture becomes 1 ml, it was placed at room temperature for 5 minutes. HEK293T cells (human kidney-derived cells, GE dharmacon) cultured in DMEM medium (10% Fetal bovine serum, 1% Penicillin/Streptomycin) at a density of 2.0x10 ⁶ cells/plate in a mixed solution of plasmid and lipofectamine 2000 on a 100 mm round plate. was subjected to plasmid transfection.

48 hours after plasmid transfection, the cells expressing the flag-Drosha and HA-DGCR protein complexes were washed twice with 5 ml of DPBS (Hyclone, without calcium magnesium) after removing the DMEM medium to remove all the medium on the cell surface. After scraping using a cell scraper (SPL) on a round plate covered with 500 μL of 1X IP buffer (20 mM Tris pH 7.5, 100 mM NaCl), all cultured HEK293T cells were removed and transferred to an Eppendorf tube (Axygen). . All cells were disrupted by sonication (10% amplitute, 5 sec running, 5 sec cooling, total 30 sec, on Ice) using an ultrasonic disruptor (Branson S-450D). The disrupted cell lysate was centrifuged (SmartR17, 13000xg, 15 min, 4 ℃) and the supernatant from which all the settled debris was removed was used.

After moving 10 μL of well shaken ANTI-FLAG M2 Affinity Gel (invitrogen) to the ep tube, 1 ml of 1X IP buffer was added and inverted 3 times. After centrifuging at 3000xg for 30 seconds, the affinity gel was washed by removing the supernatant except for the affinity gel that had subsided. After repeating this step three times, the HEK293T lysate supernatant was added to the affinity gel and mixed, followed by shaking incubation at 4 °C. During shaking incubation, flag-Drosha and HA-DGCR8 protein complexes are attached to affinity gel through antigen-antibody reaction selective to flag peptide. After 2 hours, centrifuge at 3000xg for 30 seconds to sink the affinity gel, remove the upper lysate, and repeat the washing step 8 times to remove proteins not attached to the affinity gel. 50 μL of Elution buffer (1X IP buffer diluted to a concentration of 0.3 g/mL of Flag-peptide (invitroge)) was mixed with 10 μL of the thus-made affinity gel, followed by shaking incubation at 4 ° C to separate the proteins attached to the affinity gel. After 2 hours, the mixture was centrifuged to obtain a solution containing flag-Drosha and HA-DGCR8 protein complex.

For Dicer enzyme separation, 10 μg of pCAGGS-hsDicer (Addgene) was diluted in Opti-MEM (Gibco), 15 μL of lipofectamine 2000 was added, mixed so that the total volume of the mixture was 1 ml, and then placed at room temperature for 5 minutes. The mixed solution of plasmid and lipofectamine 2000 was treated with HEK293T cells cultured in DMEM medium at a density of 2.0x10 ⁶ cells/plate on a 100 mm round plate. Afterwards, the process was carried out in the same manner as in the process of separation of the Droscha enzyme.

Example 3. In-vitro Cleavage Experiment Using Drocha Enzyme

6 μL of Droscha enzyme isolated from HEK293T cells isolated in Example 2, 1 μl 50 mM MgCl ₂ , 0.2 μl of Ribolock RNase inhibitor (Thermofisher), and 1X IP buffer per 1 pmol of the RNA sample prepared in Example 1 The mixture was added to make a total of 10 μl and incubated for 2 hours at 37 °C in a thermal cycler so that Drocha cut pri-miRNA. 10 μl of the reaction-completed sample was mixed with 2 μl of 6X DNA loading dye (NEB) to prepare a sample for PAGE gel electrophoresis. 12 μl was loaded into each well of a 15-well 15% polyacrylamide gel electrophoresis (PAGE). After gel running at 200V for 50 minutes, the gel was stained with Gel red to obtain a gel image with Gel Doc ^TM EZ, and the results are shown in FIGS. 2A and 2C. The right figure of FIG. 2a and the right figure of FIG. 2b, and FIG. 2c show a schematic diagram in which the prepared pri-miRNA (pri-miR-Let7B, pri-miGFP, pri-miHPRT) is cleaved by Drosha. As shown in Fig. 2a, pri-miR-Let7B mimicking the naturally occurring primary miRNA structure prepared above was successfully cleaved by Drocha, and a fragment of about 76 nt was identified. It was confirmed that it had normal activity. As shown in FIG. 2C , the pri-miRNA prepared above was successfully cleaved by Drocha, and a fragment of about 62 nt was identified. It was confirmed that the pri-miRNA prepared above can act as a substrate for Droscha.

Example 4. In-vitro Cleavage Experiment Using Dicer Enzyme

0.5 μl of proteinase K (Sigma) was mixed with 10 μl (1 pmole) of Dicer substrate siGFP prepared in Example 1 or the reaction product containing the pri-miRNA cut by the Droscha enzyme in Example 3, followed by 30 at 37 ° C. left a minute After 3 minutes at 95 °C, the temperature was decreased from 95 °C to 4 °C at a rate of -1.0 °C/s to remove the activity of all proteins in the mixture, resulting in the product of Drocha cleavage of pri-miRNA (pri-miRNA). Drosha Cleaved) was prepared. Mix 2 μl of Dicer enzyme isolated from HEK293T cells in Example 2 with 10 pmole or 10.5 μl of a reaction containing pri-miRNA Drosha Cleaved diluted in 1X IP buffer (5 mM MgCl ₂ added) to make a total of 12.5 μl. The mixed mixture was incubated at 37 °C for 2 hours in a thermal cycler to cause a cleavage reaction by Dicer. After the reaction was completed, a sample to be subjected to PAGE gel electrophoresis was prepared by mixing 2.5 μl of 6X DNA loading dye (NEB). 15 μl of the sample was loaded into each well of a 15-well 15% PAGE Gel. After gel running at 200V for 50 minutes, the gel was stained with Gel red to obtain a gel image with Gel Doc ^TM EZ, and the results are shown in FIGS. 2A and 2C. As shown in FIG. 2a, the Dicer substrate siGFP prepared above was successfully cleaved by Dicer digestion, and an RNA double-stranded fragment of about 20 bp was confirmed, confirming that the Dicer enzyme isolated from the cell had normal activity. . As shown in FIG. 2C , the product produced by Drocha cleavage of pri-miRNA was successfully cleaved by Dicer to confirm an RNA double-stranded fragment of about 20 bp. From the above results, it was confirmed that the pri-miRNA prepared according to one example could act as a substrate for Dicer by the product produced by Droscha cleavage.

Example 5. Confirmation of gene silencing effect using Real Time (RT)-PCR (HPRT target)

To confirm the gene silencing effect, HCT116 cells (human colorectal cancer-derived cell line) were seeded at a density of 0.1x10 ⁶ cells/well in a 96-well transparent culture plate with RPMI medium (10% Fetal bovine serum, 1% Penicillin/Streptomycin). . The two RNA samples prepared in Example 1 were siHPRT and pri-miHPRT samples at 0.05 pmole (final 0.1 nM concentration), 0.25 pmole (final 0.5 nM concentration), 0.5 pmole (final 1 nM concentration), 2.5 pmole (final 5). nM concentration) and 5 pmoles (final 10 nM concentration) were diluted in DPBS (without calcium magnesium) to make a total volume of 48.5 μl. After mixing with 1.5 μl Lipofectamine® RNAiMAX (Invitrogen) at room temperature for 5 minutes, 450 μl of After adding RPMI medium to 500 μl, the two samples were treated at 100 μl per well (N=4).

After 48 hours of treatment, RNA was extracted from the cells using CellAmp ^TM Direct RNA Prep Kit for RT-PCR (TAKARA). The extracted RNA was mixed with One Step TB Green® PrimeScript ^TM RT-PCR Kit II (Takara) and Forward Primer and Reverse primer to prepare a final volume of 20 μl. At this time, the RT-PCR primer is a forward and reverse primer for the HPRT gene to check the silencing effect of the two RNA samples, and a total of 4 forward and reverse primers for the GAPDH gene to be used as an internal control for result correction. prepared. Each primer sequence is shown in Table 3 below, ordered from Bioneer, and chemically synthesized was purchased and used.

PCR amplification was performed using the RT-CFX96 Touch Real-Time PCR Detection System (Biorad). Amplification was repeated 40 times in a cycle of 5 minutes at 42 °C, 10 seconds at 95 °C, 5 seconds at 95 °C, 30 seconds at 60 °C, and 30 seconds at 72 °C. Ct refers to the threshold cycle, and the average Ct value of the internal control GAPDH mRNA is subtracted from the average Ct value of HPRT mRNA. -delta Ct). The change in HPRT mRNA expression level was calculated using the ΔΔCt calculation method, and the results are shown in FIG. 3 .

Sample Name Sequence (5'->3') SEQ ID NO: HPRT1 FW GACTTTGCTTTCCTTGGTCAG SEQ ID NO: 13 HPRT1 RV GGCTTATATCCAACACTTCGTGGG SEQ ID NO: 14 GAPDH FW GGATTTGGTCGTATTGGG SEQ ID NO: 15 GAPDH RV GGAAGATGGTGATGGGATT SEQ ID NO: 16

As shown in FIG. 3 , when cells were treated for different concentrations and expressed as a bar graph based on the PBS group, the HPRT1 mRNA expression level of the pri-miHPRT group decreased in a similar manner to that of siHPRT, which was pri-miRNA. This means that the target gene expression level was reduced by the gene silencing activity of

Example 6. Confirmation of gene silencing effect using FACS (GFP target)

To confirm the gene silencing effect, GFP-KB cells, which are modified KB cells stably expressing GFP fluorescent protein, were seeded at a density of 0.5x10 ⁶ cells/well in a 24-well culture plate with RPMI medium. Two RNA samples, siGFP and pri-miGFP, were mixed with 0.05 pmole (final 0.1 nM concentration), 0.25 pmole (final 0.5 nM concentration), 0.5 pmole (final 1 nM concentration), 2.5 pmol (final 5 nM concentration) and 5 pmole (final 10 nM concentration). ) was diluted in DPBS (without calcium magnesium) to make a total volume of 145.5 μl, mixed with 4.5 μl Lipofectamine® RNAiMAX (Invitrogen) at room temperature for 5 minutes, and then treated with 450 μl of RPMI in each well. After 48 hours of incubation, the GFP expression level of GFP-KB cells in each well was measured with a Novocyte 2060R flow cytometer (ACEA Biosciences) by removing the cells from each well with Tryple Express (Gibco). The GFP gene silencing effect was analyzed using ACEA NovoExpress software, and the results are shown in FIG. 4 .

As shown in FIG. 4 , when cells were treated for different concentrations and the amount of GFP expression based on the PBS group was expressed as a bar graph, the same tendency as in Example 5 was shown. In addition, it can be seen that in order for the pri-miRNA structure to induce an appropriate silencing effect, additional modifications are required to the pri-RNA structure to cause sequential cleavage by delivering most of it into the nucleus. As shown in FIG. 4 , when cells were treated for different concentrations and the GFP expression level was expressed as a bar graph based on the PBS group, the GFP expression level of the pri-miGFP group decreased in a similar trend to that of siGFP, which was the pri-miGFP gene. It means that the amount of GFP expression decreased by the silencing activity. This means that even if the target sequence of pri-miRNA is changed, gene silencing activity with a similar tendency is shown.

Example 7. Confirmation of gene silencing effect in Drosha Knock-Out HCT116 cells and wild-type HCT116 cells using Real Time (RT)-PCR (HPRT target)

In order to confirm the gene silencing effect of siRNA precursors according to the presence or absence of Drosha protein in cells, wild-type HCT116 cells (WT) and Drosha Knock-Out HCT116 cells (Drosha KO) were each treated with RPMI medium (10% Fetal bovine serum, 1 % Penicillin/Streptomycin) and seeded at a density of 0.1x10 ⁶ cells/well in a 96-well transparent culture plate. The pri-miHPRT sample prepared in Example 1 was diluted with 0.5 pmol (final 0.5 nM concentration), 1 pmol (final 1 nM concentration), and 5 pmole (final 5 nM concentration) in DPBS (without calcium magnesium) to increase the volume. After mixing with 3 μl Lipofectamine® RNAiMAX (Invitrogen) at room temperature for 5 minutes, 900 μl of RPMI medium was added to make 1000 μl, and the sample was adjusted to 1000 μl per well incubating Drosha KO cells and WT cells. 100 μl each was treated 4 times (N=4).

After 48 hours of treatment, RNA was extracted from the cells using CellAmp ^TM Direct RNA Prep Kit for RT-PCR (TAKARA). The extracted RNA was mixed with One Step TB Green® PrimeScript ^TM RT-PCR Kit II (Takara) and Forward Primer and Reverse primer to prepare a final volume of 20 μl. At this time, the RT-PCR primer is a forward and reverse primer for the HPRT gene to check the silencing effect of one RNA sample, and a total of 4 forward and reverse primers for the GAPDH gene to be used as an internal control for result correction. prepared.

PCR amplification was performed using the RT-CFX96 Touch Real-Time PCR Detection System (Biorad). Amplification was repeated 40 times in a cycle of 5 minutes at 42 °C, 10 seconds at 95 °C, 5 seconds at 95 °C, 30 seconds at 60 °C, and 30 seconds at 72 °C. Ct refers to the threshold cycle, and the average Ct value of the internal control GAPDH mRNA is subtracted from the average Ct value of HPRT mRNA. -delta Ct). Changes in HPRT mRNA expression levels were obtained using the ΔΔCt calculation method, and the results are shown in FIG. 5 .

As shown in FIG. 5 , when cells were treated for different concentrations and expressed in a bar graph based on the PBS group of each cell group, the same tendency as in Example 5 ( FIG. 3 ) was shown. The expression level of HPRT1 mRNA in the Drosha KO group was significantly higher than that of wild-type HCT116 cells (WT group), indicating that the gene silencing activity of pri-miHPRT was decreased in the Drosha KO group compared to the WT group. Therefore, it can be seen that the action of Drocha is necessary for the pri-miRNA construct to cause effective gene repression in cells.

Example 8. Preparation of chemically modified siRNA precursor (GFP target)

Each of the RNA strands listed in Table 4 below was chemically synthesized by IDT. In Table 4 below, nucleotides indicated in bold and underlined in the table mean that the 2'-OH group of the ribose sugar is chemically modified with a 2'-O-methyl group.

Sample Name Sequence (5'->3') SEQ ID NO: Non-Mod 5' fragment CCAUUCCCAGCCCUUGCGCUACUCCACGGCAAGCUGACCCUGAAGUUCAUGUGGACAGGUAAGGAGAUGAACUUCAGG SEQ ID NO: 17 3' fragment GUCAGCUUGCCGUGGCGUAGCGCUCAAUCCCUAUAGUG SEQ ID NO: 18 SS-Mod 5' fragment CCA UU CCCA G CCC UUGCGCUACUCCACGGCAAGCUGACCCUGAAGUUCAUGUGG ACA GGU AA GG A GAUGAACUUCAGG SEQ ID NO: 19 3' fragment GUCAGCUUGCCGUGGCGUAGCGCU CAA U CCC U A U A GUG SEQ ID NO: 20 ST-Mod 5' fragment CCAUUCCCAGCCCUUG C G C U AC U CCAC GG CAA G C UG ACCC UG AA GUU CA UGUGGACAGGUAAGGAG A UG AAC UU CA GG SEQ ID NO: 21 3' fragment GU CA G C UUG CC GUGG C GU A G C G C UCAAUCCCUAUAGUG SEQ ID NO: 22 Seq-Mod 5' fragment CCA UU CCCA G CCC UUG C G C U AC U CCAC GG CAA G C UG ACCC UG AA GUU CA UGUGG ACA GGU AA GG A G A UG AAC UU CA GG SEQ ID NO: 23 3' fragment GU CA G C UUG CC GUGG C GU A G C G C U CAA U CCC U A U A GUG SEQ ID NO: 24 PP-Mod 5' fragment CCA UU CCCA G CCC UUGCGCUACUCCACG G CA AG C U GACCCU G AAGUUCAUGUGG ACA GGU AA GG A GAUGAACUUC AGG SEQ ID NO: 25 3' fragment G U C A G CU UG CCGUGGCGUAGCGCU CAA U CCC U A U A GUG SEQ ID NO: 26 PP(-1)-Mod 5' fragment CCA UU CCCA G CCC UUGCGCUACUCCACG G CA AG C U GACCCU G AAGUUCAUGUGG ACA GGU AA GG A GAUGAACUUC AGG SEQ ID NO: 27 3' fragment G U C A G CU UG CCGUGGCGUAGCGCU CAA U CCC U A U A GUG SEQ ID NO: 28 PP(-2)-Mod 5' fragment CCA UU CCCA G CCC UUGCGCUACUCCACG G CA AG C U GACCCU G AAGUUCAUGUGG ACA GGU AA GG A GAUGAACUUC AGG SEQ ID NO: 29 3' fragment G U C A G CU U G CCGUGGCGUAGCGCU CAA U CCC U A U A GUG SEQ ID NO: 30 PP(-3)-Mod 5' fragment CCA UU CCCA G CCC UUGCGCUACUCCACG G CA AG C U GACCCU G AAGUUCAUGUGG ACA GGU AA GG A GAUGAACUUC AGG SEQ ID NO: 31 3' fragment G U C A G CU UG CCGUGGCGUAGCGCU CAA U CCC U A U A GUG SEQ ID NO: 32

In the case of Non-Mod, it is the same sample as pri-miGFP prepared in Example 1, and contains nucleotides that are not chemically modified, and SS-Mod is a region that does not form a stem (ie, an extended polynucleotide sequence of a nucleic acid construct). and nucleotides modified in the C and A sequences in the loop structure), ST-Mod includes modified nucleotides in the C and A sequences at the site forming the stem (stem structure of the nucleic acid construct), and Seq-Mod is It contains modified nucleotides in the C, A sequence of the entire strand of the nucleic acid construct (extended polynucleotide sequence, stem structure, and loop structure of the nucleic acid construct).

Specifically, the SS-Mod includes modified nucleotides in C and A sequences at sites that do not form a stem (extended polynucleotide sequence and loop structure) so that the 5' fragment strand is 1 from the 5' end of SEQ ID NO: 19, 2, 3, 6, 7, 8, 9, 11, 12, 13, 55, 56, 57, 61, 62, and 65 th sequence contains modified nucleotides, and the 3' fragment strand is 5' of SEQ ID NO: 20 modified nucleotides at sequences 25, 26, 27, 29, 30, 31, 33, and 35 from the terminus.

ST-Mod includes nucleotides modified in C and A sequences at the site (stem structure) forming the stem, and the 5' fragment strand is 17, 19, 21, 22, 24, 25, from the 5' end of SEQ ID NO: 21, 26, 27, 30, 31, 32, 34, 37, 38, 39, 40, 43, 44, 48, 49, 67, 70, 71, 72, 75, and 76 nucleotides comprising modified nucleotides 3 ' The fragment strand includes modified nucleotides in the 3, 4, 6, 10, 11, 16, 19, 21, and 23rd sequences from the 5' end of SEQ ID NO: 22.

Seq-Mod includes modified nucleotides in the C and A sequences of the entire strand of the nucleic acid construct, and the 5' fragment strand is 1, 2, 3, 6, 7, 8, 9, 11, 12, 13, 17, 19, 21, 22, 24, 25, 26, 27, 30, 31, 32, 34, 37, 38, 39, 40, 43, 44, 48, 49, 55, 56, 57, 61, 62, 65, 67, 70, 71, 72, 75, and 76 nucleotides containing modified nucleotides, and the 3' fragment strand is 3, 4, 6, 10, 11, 16, 19, 21, 23, 25, 26, 27, 29, 30, 31, 33, and 35 nucleotides.

PP-Mod contains modified nucleotides in the C, A sequence in the non-stem-forming region (ie, the extended polynucleotide sequence and loop structure of the nucleic acid construct),

14, 17 from the 5' end of the stem structure (or the lower branching point where the stem ends in the section containing the sense region at the site forming the stem (the end of the stem structure leading to the extended polynucleotide that is not complementary)) , 18, 20, and 27 positions, and

In the section including the antisense region, a position complementary to nucleotides present at positions 15, 16, 19, 21, and 23 to 26 from the 5' end of the stem structure {or, the antisense region in the stem structure contains modified nucleotides at positions 10 to 13, 15, 17, 20, and 21} from the top branching point where the stem ends (the end of the stem structure leading to the loop structure) in the containing section.

The 5' fragment strand of the PP-Mod is 1, 2, 3, 6, 7, 8, 9, 11, 12, 13, 29, 32, 33, 35, 42, 55, 56, 57, 61, 62, 65, 76, 77, and 78 nucleotides comprising modified nucleotides and the 3' fragment strand is 1, 3, 5, 8, 9, 25, from the 5' end of SEQ ID NO: 26; and modified nucleotides in sequences 26, 27, 29, 30, 31, 33, and 35.

PP(-1)-Mod contains modified nucleotides in the C, A sequence at a site that does not form a stem,

17, 18 from the 5' end of the stem structure (or the lower branching point where the stem ends in the section containing the sense region at the site forming the stem (the end of the stem structure leading to the extended polynucleotide that is not complementary)) , 20, and 27 positions, and

In the section including the antisense region, modified nucleotides are included at positions complementary to nucleotides present at positions 15, 16, 19, 21, and 23 to 26 from the 5' end of the stem structure.

The 5' fragment strand of the PP(-1)-Mod is 1, 2, 3, 6, 7, 8, 9, 11, 12, 13, 32, 33, 35, 42, 55, 56, 57, 61, 62, 65, 76, 77, and 78 nucleotides containing modified nucleotides and the 3' fragment strand is 1, 3, 5, 8, 9, from the 5' end of SEQ ID NO: 28; and modified nucleotides in sequences 25, 26, 27, 29, 30, 31, 33, and 35.

PP(-2)-Mod contains modified nucleotides in the C, A sequence at a site that does not form a stem,

17, 18 from the 5' end of the stem structure (or the lower branching point where the stem ends in the section containing the sense region at the site forming the stem (the end of the stem structure leading to the extended polynucleotide that is not complementary)) , 20, and 27 positions, and

In the section including the antisense region, modified nucleotides are included at positions complementary to nucleotides present at positions 16, 19, 21, and 23 to 26 from the 5' end of the stem structure.

The 5' fragment strand of PP(-2)-Mod) is 1, 2, 3, 6, 7, 8, 9, 11, 12, 13, 32, 33, 35, 42 from the 5' end of SEQ ID NO: 29 , 55, 56, 57, 61, 62, 65, 76, 77, and 78 nucleotides, and the 3' fragment strand is 1, 3, 5, 8, 25 from the 5' end of SEQ ID NO: 30 , 26, 27, 29, 30, 31, 33, and 35 nucleotides.

PP(-3)-Mod contains modified nucleotides in the C, A sequence at a site that does not form a stem,

17, 18 from the 5' end of the stem structure (or the lower branching point where the stem ends in the section containing the sense region at the site forming the stem (the end of the stem structure leading to the extended polynucleotide that is not complementary)) , 20, and 27 positions, and

In the section including the antisense region, modified nucleotides are included at positions complementary to nucleotides present at positions 16, 19, 21, and 23 to 26 from the 5' end of the stem structure.

The 5' fragment strand of the PP(-3)-Mod is 1, 2, 3, 6, 7, 8, 9, 11, 12, 13, 32, 33, 35, 42, 55, 56, 57, 61, 62, 65, 76, 77, and 78 nucleotides containing modified nucleotides, and the 3' fragment strand is 1, 3, 5, 25, 26, from the 5' end of SEQ ID NO: 32; and modified nucleotides in sequences 27, 29, 30, 31, 33, and 35.

Structures, sequences of the Non-Mod, SS-Mod, ST-Mod, Seq-Mod, PP-Mod, PP(-1)-Mod, PP(-2)-Mod, and PP(-3)-Mod groups, and chemical modification is shown in FIG. 6 .

Each 3' fragment strand was reacted at 37 °C for 2 hours using T4 Polynucleotide Kinase (NEB) to phosphorylate the 5' end of the RNA. The 5' fragment strand and the phosphorylated 3' fragment strand corresponding to each modification pattern were mixed in 1X PBS aqueous solution to a concentration of 10 μM, respectively, and after 3 minutes at 95 °C using a thermal cycler (Bio-Rad T100TM) - Hybridization was carried out by decreasing the temperature from 95 °C to 4 °C at a rate of 1.0 °C/s. The hybridized RNA mixture was reacted at 25 °C for 12 hours using T4 RNA ligase (NEB) to connect the double-stranded Nick structure. The linked RNA product was mixed with 10 μl of 2X RNA loading dye (NEB) per 10 μl and denatured at 70° C. for 20 minutes to prepare a sample for PAGE gel electrophoresis. The RNA strands were separated from unreacted RNA strands by ligase by gel running on 15% polyacrylamide gel with 8% urea added at 200V for 40 minutes. After staining the gel with Gel red (Biotium), the RNA band portion of the product linked with Gel Doc ^TM EZ (Bio-Rad) was confirmed. The siRNA precursor (nucleic acid construct according to an example, pri-miRNA) is separated by cutting the PAGE gel part of the band corresponding to the RNA of the part where the 5' fragment and 3' fragment strands are connected and shaking in 1X TBE buffer for 24 hours. did.

2X RNA loading of 0.5 pmol of each strand of 5' fragment and 3' fragment of each group that did not react with Non-Mod, SS-Mod, ST-Mod, Seq-Mod, and PP-Mod separated after ligase treatment Samples were prepared for PAGE gel electrophoresis by mixing with 5 μl of dye (NEB) and denaturing at 70 °C for 20 minutes. After running the gel on 15% Polyacrylamide gel with 8% Urea added at 200V for 40 minutes, the gel was stained with Gel red to obtain a gel image with Gel Doc ^TM EZ, and the results are shown in FIGS. 7a and 7b.

As shown in FIGS. 7A and 7B , in the sample separated after ligase treatment, the unreacted single strand was removed, and it was confirmed that the band appeared at an upper position than the unreacted strand as the length was extended.

Example 9. In-vitro Drosha Cleavage reaction amount confirmation experiment by time according to chemical transformation pattern

Each sample (Non-mod, SS-Mod, Non-mod, SS-Mod, ST-Mod, Seq-Mod, and PP-Mod) Add 30 μL of Droscha enzyme isolated from HEK293T cells, 5 μl 50 mM MgCl ₂ , 1 μl of Ribolock RNase inhibitor (Thermofisher), and 1X IP buffer to 5 pmole for a total of 50 The resulting mixture was incubated at 37 °C in a thermal cycler to allow Droscha to cut pri-miRNA. After 0, 30, 60, and 120 minutes after the reaction, 10 μl of each sample was collected. 10 μl of the sample collected by time was mixed with 2 μl of 6X DNA loading dye (NEB) to prepare a sample for PAGE gel electrophoresis. 12 μl was loaded into each well of a 15-well 15% polyacrylamide gel electrophoresis (PAGE). After gel running at 200V for 50 minutes, the gel was stained with Gel red to obtain a gel image with Gel Doc ^TM EZ, and the results are shown in FIG. 9a.

As shown in Fig. 9a, in the non-mod group that did not contain chemically modified nucleotides, all siRNA precursors were immediately cleaved by Drocha enzyme and converted to products, but the other group containing chemically modified nucleotides (SS- mod, ST-mod, Seq-Mod, and PP-Mod groups) were cleaved by the Droscha enzyme and it was confirmed that it took more than 2 hours to convert all of them into products. Through this, it was confirmed that when chemically modified nucleotides (2'-O-Methyl modification) were applied to impart stability to pri-miRNA, the action of Drocha was inhibited.

Example 10. Confirmation of gene silencing effect using FACS (GFP target)

In order to confirm the gene silencing effect, GFP-KB cells (cells expressing GFP fluorescent protein), which are modified KB cells stably expressing GFP fluorescent protein, were mixed with RPMI medium in a 12 well culture plate at a density of 1.0x10 ⁶ cells/well. seeded. Non-Mod, SS-Mod, ST-Mod, Seq-Mod, PP-Mod, PP(-1)-Mod, PP(-2)-Mod, and PP(-3)-Mod prepared in Example 8 Eight chemically modified pri-miRNA samples of ® After mixing with RNAiMAX (Invitrogen) at room temperature for 5 minutes, 50 μl of each well incubated with 450 μl of RPMI was treated. After 48 hours of incubation, the GFP expression level of GFP-KB cells in each well was measured with a Novocyte 2060R flow cytometer (ACEA Biosciences) by removing the cells from each well with Tryple Express (Gibco). The GFP gene silencing effect was analyzed with ACEA NovoExpress software, and the results are shown in FIG. 8 . For statistical analysis, two-way ANOVA analysis (Graphpad Prism 7) was used to compare gene silencing activity for target genes between chemically modified pri-miGFPs.

As shown in the upper graph of FIG. 8 , it was confirmed that the ST-Mod group or the Seq-Mod group had significantly lower gene silencing activity for the target gene than the Non-mod group. It was confirmed that the group treated with PP-Mod and SS-Mod showed an excellent gene silencing effect by reducing the expression of GFP.

As shown in the graph at the bottom of Figure 8, in the stem structure of PP-Mod and PP-Mod, from the single-stranded region extending from the 5' end of the section including the sense region to the 3' end from the lower branch point where the single-stranded region starts PP(-1)-Mod, PP(-2)-Mod, or PP(-3)-Mod except 1 (14nt), 2 (14 and 15nt), or 3 (14, 15 and 16nt) respectively was confirmed to show a similar level of gene silencing activity to that of PP-Mod.

Through this, if chemical modification is included in the stem region, the gene suppression effect of pri-miRNA may be inhibited, but the PP-Mod group, which is an siRNA precursor with site-specific chemical modification, did not inhibit the gene suppression effect of pri-miRNA. confirmed that it is not.

Example 11. In-vitro Dicer Cleavage reaction amount confirmation experiment by time according to the pattern of chemical transformation

To compare the degree of cleavage of Droscha Cleavage reactants of pri-miRNA applied with chemical modification by Dicer, 40 μL of Droscha enzyme, 5 μl 50 mM MgCl2, Ribolock RNase inhibitor (Thermofisher) 1 in 5 pmole of each pri-miRNA sample μl, and 1X IP buffer, mixed to make a total of 50 μl, and reacted at 37°C for 2 hours, followed by inactivation using proteinase K. A schematic diagram of a process in which a product cleaved by Drosha is cleaved by Dicer is shown in FIG. 8A . 8 μl of Dicer enzyme was added to the reaction mixture and reacted at 37 ° C. After 0, 30, 60, and 120 minutes, 12.5 μl of each sample was collected. Samples collected by time were mixed with 2.5 μl of 6X DNA loading dye (NEB) to prepare samples for PAGE gel electrophoresis. 15 μl was loaded into each well of 15-well 15% PAGE Gel. After gel running at 200V for 50 minutes, the gel was stained with Gel red to obtain a gel image with Gel Doc ^TM EZ, and the results are shown in FIG. 9b. As shown in FIG. 9b , the production of mature siRNA was significantly reduced in the ST-mod group or the Seq-Mod group containing chemically modified nucleotides in the stem region. However, in the PP-Mod group containing site-specific chemically modified nucleotides in the stem region, similar to the non-modified Non-Mod group or the SS-Mod group modified in the non-stem-forming site, all Drocha cleavage The product was cleaved by Dicer to produce mature siRNA.

According to the results of FIGS. 9A and 9B, when A and C sequence-specific chemically modified nucleotides were included in the stem region, the gene silencing activity was reduced due to inhibition of the cleavage process by Dicer, but In the PP-Mod group, which is an siRNA precursor with a site-specific chemical modification that does not inhibit the action of the siRNA, all Droscha cleavage products are cleaved by Dicer and matured similarly to the Non-Mod group or SS-Mod group. production was confirmed.

Example 12. Serum stability confirmation experiment by time according to the pattern of chemical transformation.

In order to check the degree of degradation by nuclease in the serum of pri-miRNA to which chemical modification of nucleotides has been applied, blood from untreated C57bl/6NCrSlc (female, 18-22g) mouse was collected and then 4 ℃, 2000 g, 15 Serum is separated by centrifugation for min. 5.5 pmole of the oligo prepared in Example 8 was mixed with MgCl2, C57bl/6NCrSlc mouse serum, 4.4 μL and PBS, and adjusted to a final volume of 44 μL. At this time, the final concentration of each sample is 0.125 μl, and the final concentration of MgCl2 is 5 mM. The purpose of this study was to compare the stability of each oligo in serum while incubating at 37 °C for 2 hours. At 0, 10, 30, 60, and 120 minutes after the reaction, 8 μl of each sample was collected and mixed with 2 μl of 1X TBE buffer and 2 μl of 6X gel loading dye (NEB) to stop the reaction at each time. 15 μl was loaded into each well of 15-well 15% PAGE Gel. After gel running at 200V for 50 minutes, the gel was stained with Gel red to obtain a gel image with Gel Doc ^TM EZ, and the results are shown on the left side of FIG. 9C. To compare the degree of time-dependent degradation of oligo by serum nuclease in each group, the band intensity values of the bands indicated by arrows for each modification pattern were measured for each modification pattern using Image Lab software (biorad). By setting the band intensity value to 1, the band intensity value of the remaining time period is shown in the graph on the right side of FIG. 9c. As shown in FIG. 9c , in the case of the Non-Mod group or the ST-Mod group, it was confirmed that most of the ribonucleic acid constructs were decomposed within 10 minutes of treating the serum, so that the stability in the serum was not improved. In the Seq-Mod group or PP-Mod group, including chemically modified nucleotides in the single-stranded region and the stem region, it was confirmed that the undecomposed ribonucleic acid structure remains even after 2 hours. confirmed that there is. In the case of the SS-Mod group, even though chemical modification of nucleotides was limitedly applied to the single-stranded region, it was confirmed that the ribonucleic acid construct that was not decomposed after 2 hours remained, thereby improving the stability in serum.

8 and 9c, it was confirmed that the modification pattern according to SS-Mod and PP-Mod is a chemical modification pattern that can improve the stability of the pri-miRNA precursor in serum.

Example 13. Immunogenicity assay according to the pattern of chemical modification (Immune assay)

In order to examine the effect of reducing the immune induction of pri-miRNA by chemical modification of the sequence (2'-O-Methyl modification), PBMCs (human peripheral blood mononuclear cells, ATCC) were treated with RPMI medium (10% Fetal bovine serum, 1% Penicillin). /Streptomycin) was dispensed in a 24-well plate at a concentration of 5 × 10 ^ 5 cells/well, 450 μl each. After stabilizing the cells by culturing at 37° C. and 5% CO 2 conditions for 24 hours, the four chemically modified pri-miRNA samples (Non-mod, Seq-Mod, and PP prepared in Example 8 above) were placed in each well. -Mod) 250 pmole (final concentration of 500 nM) was diluted in DPBS (without calcium magnesium) to make a total volume of 144 μl. After mixing with 6 μl Lipofectamine® RNAiMAX (Invitrogen) at room temperature for 5 minutes, 450 μl of RPMI was added. PBMC were transfected by processing 50 μl of each cultured well. Cells from wells treated with only 50 μl of PBS (NC in Fig. 9d) and wells treated only with Lipofectamine® RNAiMAX (Invitrogen) without nucleic acid (Lipo-only in Fig. 9d) were used as controls. At this time, the number of wells treated per each pri-miRNA sample was n=3, and statistical significance was obtained after cytokine quantitative analysis.

After additional incubation at 37° C. and 5% CO 2 conditions for 24 hours, the medium was collected and centrifuged at 400 g 4° C. for 5 min to separate the supernatant. The amounts of human TNF-α and IFN-α in the supernatant were quantified using an enzyme immunoassay, and the results are shown in FIG. 9D . For cytokine analysis of the supernatant, the Human TNF-alpha Quantikine ELISA Kit (R&D system) was used, and the absorbance was measured at 450 nm with Infinite M200 Pro (Tecan) after proceeding according to the manufacturer's manual. One-way ANOVA analysis (Graphpad Prism 7) was used to compare the degree of immune response induction between Non-Mod pri-miGFP and chemically modified pri-miGFP.

Through the results of FIG. 9d, when the PBMC cell line was treated with the Non-Mod group (Non-Mod in FIG. 9d), which is a chemically unmodified pri-miRNA, no treatment group (NC) or control group (in FIG. 9d ) Lipo-only), it was confirmed that an increase in the expression of TNF-α, a type of immune cytokine, appears by the immune response. The Seq-Mod group, in which chemical modifications specifically for A and C sequences were introduced into pri-miRNA, or the PP-Mod group, in which site-specific chemical modifications that do not inhibit the action of Dicer were introduced into the stem region of pri-miRNA. In this case, it was confirmed that the increase in the expression of TNF-α did not appear due to the decrease in the immune response. These results showed consistent results with previous papers (Journal of Controlled Release 343 (2022) 57-65) that when a chemical modification was introduced into the double-stranded region of a nucleic acid construct, recognition as an in vivo immunogenicity was inhibited.

In the results of FIG. 8, the SS-Mod group and the PP-Mod group had superior gene suppression activity than the Seq-Mod group in which A and C sequence-specific chemical modifications were introduced into the stem region, and FIGS. 9c and FIG. Through the result of 9d, it was confirmed that it was suitable for application as an in vivo therapeutic agent because it showed excellent stability in serum and low immunogenicity compared to pri-miRNA (Non-Mod) without chemical modification.

Example 14. Preparation of siRNA precursors containing chemically modified ASO sequences

Each of the RNA strands listed in Table 5 below was chemically synthesized by IDT. In Table 5 below, nucleotides indicated in bold and underlined indicate a sequence region consisting of DNA that functions as SMN2-ASO (antisense oligonucleotide for the survival motor neuron 2 gene). Acid), and it means that the partial phosphodiester bond marked with * between the nucleotide sequences is modified with a phosphorothioate bond.

Sample Name Sequence (5'->3') SEQ ID NO: 3' SMN2-ASO1 AAAGUCUGGCAUGGCGUAGCGCUC T*[+C]*[+A]*C*[+T]*[+T]*T*[+C]*[+A]*T*[+A]*[+A]*T *[+G]*[+C]*T*[+G]*[+G] SEQ ID NO: 33 3' SMN2-ASO2 AAAGUCUGGCAUGGCGUAGCGCUCAAUCC T*[+C]*[+A]*C*[+T]*[+T]*T*[+C]*[+A]*T*[+A]*[+A]*T *[+G]*[+C]*T*[+G]*[+G] SEQ ID NO: 34

pri-miHPRT, an siRNA precursor into which ASO was introduced by hybridizing 3' SMN2-ASO1 or 3' SMN2-ASO2 with the pri-miHPRT 5' fragment strand [SEQ ID NO: 11] constituting the pri-miHPRT of Example 1 and Table 2 ASO1 or pri-miHPRT ASO2 was prepared. At this time, in the stem structure, the group introducing SMN2 ASO 2 nucleotides later toward the 3' end from the lower branch point where the single-stranded site starts in the single-stranded site extended from the 3' end of the section including the antisense region in the stem structure was pri- miHPRT ASO1, a group in which SMN2 ASO was introduced 7 nucleotides after the 3' end from the bottom branching point where the single-stranded region starts in the single-stranded region extending from the 3' end of the section containing the antisense region in the stem structure was pri- It was named miHPRT ASO2. The structures of the pri-miHPRT ASO1 and pri-miHPRT ASO2 are shown in FIG. 10A.

The pri-miHPRT ASO1 contains modified sugars at positions 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, and 42 from the 5' end of SEQ ID NO: 33. In the pri-miHPRT ASO1, a phosphodiester bond between nucleic acids 25 to 42 from the 5' end of SEQ ID NO: 33 was modified into a phosphorothioate bond.

The pri-miHPRT ASO2 contains modified sugars at positions 31, 32, 34, 35, 38, 39, 40, 41, 43, 44, 46, and 47 from the 5' end of SEQ ID NO: 34. In the pri-miHPRT ASO2, a phosphodiester bond between nucleic acids 30 to 47 from the 5' end of SEQ ID NO: 34 was modified into a phosphorothioate bond.

Each strand was reacted at 37 °C for 2 hours using T4 Polynucleotide Kinase (NEB) to phosphorylate the 5' end of RNA. Each of the phosphorylated strands and the pri-miHPRT 5' fragment strand [SEQ ID NO: 11] constituting the pri-miHPRT of Table 2 of Example 1 were mixed in 1X PBS aqueous solution to a concentration of 10 μM, respectively, and a thermal cycler (Bio- Rad T100TM) was used at 95 °C for 3 minutes, and then the temperature was decreased from 95 °C to 4 °C at a rate of -1.0 °C/s for hybridization. The hybridized RNA mixture was reacted at 25 °C for 12 hours using T4 RNA ligase (NEB) to connect the double-stranded Nick structure. The linked RNA product was mixed with 10 μl of 2X RNA loading dye (NEB) per 10 μl and denatured at 70° C. for 20 minutes to prepare a sample for PAGE gel electrophoresis. The RNA strands were separated from unreacted RNA strands by ligase by gel running on 15% polyacrylamide gel with 8% urea added at 200V for 40 minutes. After staining the gel with Gel red (Biotium), the RNA band portion of the product linked with Gel Doc ^TM EZ (Bio-Rad) was confirmed. For pri-miHPRT ASO1 and pri-miHPRT ASO2, the 5' fragment and 3' fragment strands were separated by cutting the PAGE gel part of the band corresponding to the RNA and shaking in 1X TBE buffer for 24 hours.

0.5 pmol of each strand of pri-miHPRT ASO1, pri-miHPRT ASO2 and pri-miHPRT 5' fragment, 3' SMN2 ASO1, and 3' SMN2 ASO2 that was not reacted with pri-miHPRT ASO1 separated after ligase treatment with 2X RNA loading dye (NEB) 5 Mixed with μl and denatured at 70 °C for 20 minutes to prepare a sample for PAGE gel electrophoresis. After gel running for 40 minutes at 200V on 15% Polyacrylamide gel with 8% Urea added, the gel was stained with Gel red to obtain a gel image with Gel Doc ^TM EZ, and the results are shown in FIG. 10b.

As shown in FIG. 10b , in the pri-miHPRT ASO1 and pri-miHPRT ASO2 samples separated after ligase treatment, the unreacted single strand was removed, and it was confirmed that the band appeared at the upper position than the unreacted strand as the length was extended.

Example 15. Time-dependent in-vitro Drosha Cleavage reaction amount confirmation experiment of siRNA precursor containing chemically modified ASO sequence

Prepared in Example 8 above to compare the cleavage degree of pri-miHPRT to which nucleotides are not chemically modified or pri-miHPRT (pri-miHPRT ASO1 and pri-miHPRT ASO2) to which chemical modifications are applied To 2 pmole of each sample, 30 μL of Droscha enzyme isolated from HEK293T cells, 5 μl 50 mM MgCl ₂ , Ribolock RNase inhibitor (Thermofisher) 1 μl, 1X IP buffer, and the mixture to make a total of 50 μl in the thermal cycler. incubated at 37 °C to allow Droscha to cut pri-miRNA. 30 minutes after the reaction, 12.5 μl was collected from each sample. In 0.5 pmole of each sample prepared above, 7.5 μL of Drocha enzyme isolated from HEK293T cells and 6X DNA loading dye (NEB) were mixed in a 1:5 ratio to remove activity, 7.5 μL, 1.25 μl 50mM MgCl ₂ , Ribolock RNase inhibitor (Thermofisher) 0.25 μl, 1X IP buffer was added, and the mixture was mixed so that a total of 12.5 μl was used as a control of the reaction. 12.5 μl of the collected sample or control sample was mixed with 2.5 μl of 6X DNA loading dye (NEB) to prepare a sample for PAGE gel electrophoresis. 15 μl was loaded into each well of a 15-well 15% polyacrylamide gel electrophoresis (PAGE). After gel running at 200V for 50 minutes, the gel was stained with Gel red to obtain a gel image with Gel Doc ^TM EZ, and the results are shown in FIG. 11 .

As shown in FIG. 11, the pri-miHPRT ASO1 group in which SMN2 ASO was introduced 2 nucleotides after the lower branch point where the single-stranded polynucleotide site extended from the 3' end of the section including the antisense region in the stem structure starts was It was confirmed that no Droscha cleavage product was formed. In addition, the pri-miHPRT ASO2 group, in which SMN2 ASO was introduced after 7 nucleotides of the single-stranded region from the branching point where the single-stranded region extended from the 3′ end of the antisense region, was similar to the pri-miHPRT group, the siRNA precursor was It was confirmed that the product was cleaved by Through this, when a chemically modified sequence is introduced into the single-stranded polynucleotide site extending from the 3' end of the section including the antisense region in the stem structure of pri-miRNA to impart a second target gene regulatory function, single-stranded It was confirmed that the introduction of ASO after 7 chemically unmodified sequences from the lower bifurcation of the site to the 3' end did not inhibit the action of Drocha.

Example 16. Confirmation of gene inhibitory effect and selective splicing inhibitory effect of siRNA precursor containing chemically modified ASO sequence

To confirm the simultaneous expression of the gene silencing effect and splicing inhibitory effect of pri-miHPRT ASO1 or pri-miHPRT ASO2, HCT116 cells were treated with RPMI medium (10% Fetal bovine serum, 1% Penicillin/Streptomycin) in 96-well transparent culture plate was seeded at a density of 0.1x10 ⁶ cells/well. Three samples of pri-miHPRT, pri-miHPRT ASO2, and single-stranded SMN2-ASO2 were diluted in 0.25 pmole (final concentration of 0.5 nM) in DPBS (without calcium magnesium) to a total volume of 48.5 μl and 1.5 μl Lipofectamine® RNAiMAX After mixing with (Invitrogen) at room temperature for 5 minutes, 450 μl of RPMI medium was added, adjusted to 500 μl, and 100 μl of each of the three samples was treated (N=4).

After 48 hours of treatment, RNA was extracted from the cells using CellAmp ^TM Direct RNA Prep Kit for RT-PCR (TAKARA). The extracted RNA was mixed with One Step TB Green® PrimeScript ^TM RT-PCR Kit II (Takara) and Forward Primer and Reverse primer to prepare a final volume of 20 μl.

At this time, the primers for RT-PCR are forward and reverse primers for the HPRT gene to check the gene silencing effect by the three RNA samples, and a total of 4 forward and reverse primers for the GAPDH gene to be used as an internal control to correct the results. In addition, forward and reverse primers for SMN2 mRNA (SMN2 Δ7) with exon 7 removed and SMN2 mRNA without exon 7 removed (SMN2 full) A total of four forward and reverse primers were prepared. Primer sequences for SMN2 Δ7 and SMN2 full are shown in Table 6 below, ordered from Bioneer, and chemically synthesized were purchased and used. PCR amplification was performed using the RT-CFX96 Touch Real-Time PCR Detection System (Biorad). Amplification was repeated 40 times in a cycle of 5 minutes at 42 °C, 10 seconds at 95 °C, 5 seconds at 95 °C, 30 seconds at 60 °C, and 30 seconds at 72 °C. With the measured Ct value, the expression level of HPRT mRNA and the change in the expression level of SMN2 Δ7 compared to the expression level of SMN2 full were calculated using the ΔΔCt calculation method, and the results are shown in the graph of FIG. 12 .

Sample Name Sequence (5'->3') SEQ ID NO: SMN2 Δ7 FW TCTGGACCACCAATAATTCCCC SEQ ID NO: 35 SMN2 Δ7 RV ATGCCAGCATTTCCATATAATAGCC SEQ ID NO: 36 SMN2 full FW GCTATCATACTGGCTATTATATGGGTTTT SEQ ID NO: 37 SMN2 full RV CTCTATGCCAGCATTTCTCCTTAAT SEQ ID NO: 38

As shown in the lower graph of FIG. 12 , in the stem structure of pri-miHPRT, at the single-stranded polynucleotide site extending from the 3' end of the section including the antisense region, 2 nucleotides from the lower branch point where the single-stranded site starts The pri-miHPRT ASO1 group in which SMN2 ASO was introduced showed less reduction in HPRT1 mRNA expression than the pri-miHPRT group in which the chemically modified nucleotide was not introduced, confirming that the gene suppression function was lost. In addition, in the stem structure of pri-miHPRT, pri-miHPRT ASO2 introduced SMN2 ASO 7 nucleotides after the lower branch point where the single-stranded region starts at the single-stranded polynucleotide site extending from the 3' end of the section including the antisense region Similar to the pri-miHPRT group, it was confirmed that the gene suppression activity was maintained even though the chemically modified nucleotide was introduced by reducing the HPRT1 mRNA expression level in the group.

As shown in the upper graph of FIG. 12, pri-miHPRT ASO1 and pri-miHPRT ASO2 are 3' SMN2-ASO1 (SMN2-ASO1) and It was confirmed that 3' SMN2-ASO2 (SMN2-ASO2) exhibits the same level of splicing inhibitory effect as the same level.

11 and 12, when ASO is introduced into the single-stranded polynucleotide site extended from the 3' end of the section including the antisense region in the stem structure of pri-miRNA, from the lower branch point where the single-stranded site starts Since introduction after the seven chemically unmodified sequences does not inhibit the action of Drocha, it was confirmed that the function of the siRNA precursor and the second target gene regulatory function could be effectively expressed without inhibition of the gene suppression effect.

Example 17. Confirmation of Exon 7 expression of SMN2 mRNA by siRNA precursor containing chemically modified ASO sequence

To confirm the preservation of exon 7 of SMN2 mRNA due to the splicing inhibitory effect of pri-miHPRT (pri-miHPRT ASO2) introduced with SMN2-ASO, HCT116 cells were treated with RPMI medium (10% Fetal bovine serum, 1% Penicillin/Streptomycin). ) and seeded at a density of 0.1x10 ⁶ cells/well in a 96-well transparent c μlture plate. Three samples of pri-miHPRT, pri-miHPRT ASO2 and single-stranded SMN2-ASO2 were diluted at 0.5 pmol (final concentration of 5 nM) in DPBS (without calcium magnesium) to a total volume of 8.5 μl and 1.5 μl Lipofectamine® RNAiMAX After mixing with (Invitrogen) at room temperature for 5 minutes, 90 μl of RPMI medium was added to adjust the volume to 100 μl, and 100 μl of the three samples were processed per well.

After 48 hours of treatment, RNA was extracted from the cells using CellAmp ^TM Direct RNA Prep Kit for RT-PCR (TAKARA). The extracted RNA was mixed with One Step TB Green® PrimeScript ^TM RT-PCR Kit II (Takara) and Forward Primer and Reverse primer to prepare a final volume of 20 μl.

At this time, primers for PCR are forward and reverse primers for exon 6, 7, and 8 sections of SMN2 mRNA to check SMN2 Exon 7 splicing by 3 RNA samples, and internal control for result correction (loading control) A total of four forward and reverse primers were prepared for the GAPDH gene to be used as The primer sequences for SMN2 exon 6 to 8 sections (SMN2 E5F, SMN2 E8R) were used in Table 7 below, and the primer sequences for GAPDH were used in Table 3. The primers used below were ordered from Bioneer, and chemically synthesized ones were purchased and used. PCR amplification was performed using the RT-CFX96 Touch Real-Time PCR Detection System (Biorad). Amplification was repeated for 30 cycles at 42 °C for 5 minutes, 95 °C for 10 seconds, 95 °C for 5 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds.

Sample Name Sequence (5'->3') SEQ ID NO: SMN2 E5F CTGCCTCCATTTCCTTCTG SEQ ID NO: 39 SMN2 E8R TGGTGTCATTTAGTGCTGCTC SEQ ID NO: 40

To check the length of the PCR amplification product for exon 6-8 section of SMN2 mRNA or the amplification product for GAPDH, take 5 μl from each sample and mix with 5 μl of 1X TBE buffer and 2 μl of 6X loading dye (NEB). A total of 12 μl was prepared to prepare a sample for PAGE gel electrophoresis. After loading each well of 15% PAGE gel and electrophoresing at 200V for 50 minutes, the gel was stained with Gel red to obtain a gel image with Gel Doc ^TM EZ. The same experiment as above was performed three times to obtain three images, one of which is shown in FIG. 13 . The thickness of the band obtained from each image was measured using Image Lab software (biorad), and in order to compare the degree of exon 7 splicing in each group, it represents the PCR amplification product for exon 6-8 section of SMN2 mRNA. [Top band intensity/bottom band intensity] was calculated on PAGE gel. The value measured in the control group was set to 1, and the average value and standard error value of the values in each group are shown in the upper graph.

As shown in FIG. 13 , the upper band corresponds to SMN2 Δ7 mRNA from which Exon 7 has been removed by selective splicing, and the lower band corresponds to SMN2 full mRNA containing Exon 7 because splicing does not proceed. will do In the pri-miHPRT group in which SMN2-ASO was not introduced, it was confirmed that the thickness of the lower band was similar to that of the control group (NC), and in the 3' SMN2 ASO2 group, the thickness of the lower band became thinner and the thickness of the upper band became thicker, so that the thickness of the SMN2 mRNA was reduced. It was confirmed that SMN2 full mRNA expression including Exon 7 was increased through splicing inhibition. It was confirmed that pri-miHPRT ASO2 showed a similar tendency to single-stranded 3' SMN2 ASO and increased the expression level of SMN2 full mRNA containing Exon 7 due to the splicing inhibitory effect.

Example 18. Preparation of siRNA precursor containing chemically modified anti-miR21 sequence

Each of the RNA strands listed in Table 8 below was chemically synthesized by IDT. In Table 8 below, nucleotides indicated in bold and underlined indicate a sequence region consisting of DNA functioning as Anti-miR21, and [+] indicates that a ribose sugar is modified with LNA.

Sample Name Sequence (5'->3') SEQ ID NO: 3' Anti-miR21 AAAGUCUGGCAUGGCGUAGCGCUCAAUCC [+G][+A][+T][+A][+A][+G][+C][+T] SEQ ID NO: 41

An siRNA precursor was prepared by hybridizing the pri-miHPRT 5' fragment strand constituting the pri-miHPRT of Example 1 and Table 2 with Anti-miR21. At this time, the group containing Anti-miR21 after 7 nucleotides toward the 3' end from the lower branch point where the single-stranded region starts in the single-stranded region extended from the 3' end of the section containing the antisense region in the stem structure is "pri" -miHPRT Anti-miR21". The structure of the pri-miHPRT Anti-miR21 is shown in Figure 14a.

In the pri-miHPRT Anti-miR21, the sugars of sequences 30 to 37 of SEQ ID NO: 41 were modified.

For the strands in Table 8, phosphorylation of the 5' end of RNA was used by reacting at 37°C for 2 hours using T4 Polynucleotide Kinase (NEB). The phosphorylated 3' anti-miR21 strand and the pri-miHPRT 5' fragment strand [SEQ ID NO: 11] constituting the pri-miHPRT in Table 2 were mixed in 1X PBS aqueous solution at a concentration of 10 μM, respectively, and a thermal cycler (Bio-Rad T100TM) ) was used at 95 °C for 3 minutes and then the temperature was decreased from 95 °C to 4 °C at a rate of -1.0 °C/s for hybridization. The hybridized RNA mixture was reacted at 25 °C for 12 hours using T4 RNA ligase (NEB) to connect the double-stranded Nick structure. The linked RNA product was mixed with 10 μl of 2X RNA loading dye (NEB) per 10 μl and denatured at 70° C. for 20 minutes to prepare a sample for PAGE gel electrophoresis. The RNA strands were separated from unreacted RNA strands by ligase by gel running on 15% polyacrylamide gel with 8% urea added at 200V for 40 minutes. After staining the gel with Gel red (Biotium), the RNA band portion of the product linked with Gel Doc ^TM EZ (Bio-Rad) was confirmed. For pri-miHPRT Anti-miR21, the PAGE gel part of the band corresponding to the RNA of the 5' fragment and 3' fragment strands was cut out and separated by shaking in 1X TBE buffer for 24 hours.

0.5 pmol of each strand of pri-miHPRT 5' fragment and 3' Anti-miR21 that was not reacted with pri-miHPRT Anti-miR21 separated after ligase treatment was mixed with 5 μl of 2X RNA loading dye (NEB) and heated at 70 °C for 20 minutes. Samples were prepared for PAGE gel electrophoresis by denaturing for a minute. After gel running for 40 minutes at 200V on 15% Polyacrylamide gel with 8% Urea added, the gel was stained with Gel red to obtain a gel image with Gel Doc ^TM EZ, and the results are shown in FIG. 14B .

As shown in FIG. 14b , in the pri-miHPRT Anti-miR21 sample isolated after ligase treatment, the unreacted single strand was removed, and it was confirmed that the band appeared at an upper position than the unreacted strand as the length was extended.

Example 19. Time-dependent in-vitro Drosha Cleavage reaction amount confirmation experiment of siRNA precursor containing chemically modified anti-miR21 sequence

Prepared in Example 8 above to compare the degree of cleavage of pri-miHPRT (pri-miHPRT Anti-miR21) to which chemically modified Anti-miR21 was introduced and pri-miHPRT to which chemically modified Anti-miR21 was not introduced by drossary over time. To 2 pmole of each sample, 30 μL of Droscha enzyme isolated from HEK293T cells, 5 μl 50 mM MgCl ₂ , Ribolock RNase inhibitor (Thermofisher) 1 μl, 1X IP buffer, and the mixture to make a total of 50 μl in the thermal cycler. incubated at 37 °C to allow Droscha to cut pri-miRNA. 30 minutes after the reaction, 12.5 μl was collected from each sample. In 0.5 pmole of each sample prepared above, 7.5 μL of Drocha enzyme isolated from HEK293T cells and 6X DNA loading dye (NEB) were mixed with 6X DNA loading dye (NEB) in a 1:5 ratio to remove activity, 7.5 μL, 1.25 μl 50 mM MgCl ₂ , Ribolock RNase Inhibitor (Thermofisher) 0.25 μl, 1X IP buffer was added, and the mixture was mixed to make a total of 12.5 μl and used as a control of the reaction. 12.5 μl of the collected sample or control sample was mixed with 2.5 μl of 6X DNA loading dye (NEB) to prepare a sample for PAGE gel electrophoresis. 15 μl was loaded into each well of a 15-well 15% polyacrylamide gel electrophoresis (PAGE). After gel running at 200V for 50 minutes, the gel was stained with Gel red to obtain a gel image with Gel Doc ^TM EZ, and the results are shown in FIG. 15 .

As shown in FIG. 15 , it was confirmed that all of the siRNA precursors reacted with pri-miHPRT to which pri-miHPRT Anti-miR21 was not introduced with chemically modified nucleotides were cut by Drosha to form products. 11 and 15, in order to impart a second target gene regulatory function, in the stem structure of pri-miRNA, a single-stranded polynucleotide site chemically modified at the 3' end of the section including the antisense region When introducing the miR21 sequence, it was confirmed that the introduction after 7 chemically unmodified sequences from the lower branch point of the single-stranded region to the 3' end did not inhibit the action of Drocha.

Example 20. Confirmation of the gene inhibitory effect and miR21 inhibitory effect of the siRNA precursor containing the chemically modified Anti-miR21 sequence

To confirm the simultaneous expression of the pri-miHPRT anti-miR21 gene silencing effect and miR21 inhibitory effect, HCT116 cells were incubated with RPMI medium (10% Fetal bovine serum, 1% Penicillin/Streptomycin) in a 96-well transparent culture plate with 0.1x10 ⁶ cells. It was seeded at a density of /well. Three samples of pri-miHPRT, pri-miHPRT antimiR21, and single-stranded 3' anti-miR21 were diluted at 0.25 pmol (final concentration of 0.5 nM) in DPBS (without calcium magnesium) to a total volume of 48.5 μl and 1.5 μl Lipofectamine ® After mixing with RNAiMAX (Invitrogen) at room temperature for 5 minutes, 450 μl of RPMI medium was added to make 500 μl, and 100 μl of each of the three samples was processed per well (N=4).

After 48 hours of treatment, RNA was extracted from the cells using CellAmp ^TM Direct RNA Prep Kit for RT-PCR (TAKARA). Extracted RNA and M-MLV reverse transcriptase kit (Biorad) and Stem-Loop primer for miR21 or U6 snRNA were used to prepare a final volume of 20 μl. cDNA was synthesized by treatment at 42 °C for 5 minutes and at 37 °C for 1 hour, and the enzyme was inactivated by treatment at 95 °C for 5 minutes. The extracted RNA and miR21 cDNA were mixed with One Step TB Green® PrimeScript ^TM RT-PCR Kit II (Takara) and Forward Primer and Reverse primer to prepare a final volume of 20 μl.

At this time, the primers for RT-PCR are forward and reverse primers for the HPRT gene to check the gene silencing effect by the three RNA samples, and a total of 4 forward and reverse primers for the GAPDH gene to be used as an internal control to correct the results. Stem-Loop primer for miR21 (miR21 Stem-Loop), forward and reverse primer (miR21 FW, RV) and Stem-Loop primer (U6 Stem-Loop) for U6 snRNA to be used as an internal control for result correction, forward, reverse primer (U6 FW, RV) A total of 6 types were prepared. The primer sequence is described in Table 9 below, and the chemically synthesized one was purchased and used by ordering from Bioneer. miR21 was subjected to PCR amplification using the RT-CFX96 Touch Real-Time PCR Detection System (Biorad). Amplification was repeated 40 times in a cycle of 5 minutes at 42 °C, 10 seconds at 95 °C, 5 seconds at 95 °C, 30 seconds at 60 °C, and 30 seconds at 72 °C. Changes in the expression level of HPRT mRNA and the expression level of miR21 were calculated using the ΔΔCt calculation method using the measured Ct values, and the results are shown in the graph of FIG. 16 .

Sample Name Sequence (5'->3') SEQ ID NO: miR21 Stem-Loop GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACA SEQ ID NO: 42 miR21 FW CGGCGGTAGCTTATCAGACTGATGT SEQ ID NO: 43 miR21 RV GTGCAGGGTCCGAGGT SEQ ID NO: 44 U6 Stem-Loop CGCTTCACGAATTTGCGTGTCAT SEQ ID NO: 45 miR21 FW CTCGCTTCGGCAGCACA SEQ ID NO: 46 miR21 RV AACGCTTCACGAATTTGCGT SEQ ID NO: 47

As shown in FIG. 16 , pri-miHPRT (pri-miHPRT Anti-miR21) into which Anti-miR21 was introduced had the same level of gene suppression effect as pri-miHPRT without chemical modification, and at the same time, single-stranded 3' It was confirmed that the miR21 inhibitory effect at the same level as that of anti-miR21 (anti-miR21).

Example 21. Confirmation of expression of miR21 downstream gene expression by siRNA precursor containing chemically modified Anti-miR21 sequence

To confirm the expression of miR21 regulatory genes by the miR21 inhibitory effect of pri-miHPRT anti-miR21, HCT116 cells were treated with RPMI medium (10% Fetal bovine serum, 1% Penicillin/Streptomycin) in a 12-well transparent culture plate with 1.0x10 ⁶ cells. It was seeded at a density of /well. Three samples of pri-miHPRT, pri-miHPRT anti-miR21 and single-stranded 3' anti-miR21 were diluted in DPBS (without calcium magnesium) at 5 pmole (final concentration of 5 nM) to make a total volume of 97 μl. After mixing with μl Lipofectamine® RNAiMAX (Invitrogen) at room temperature for 5 minutes, 100 μl of each well cultured in 900 μl RPMI was treated. To compare the PDCD4 expression level in each well 48 hours after treatment, cells were lysed using CelLytic ^TM M (Sigma) and then centrifuged at 4 °C 12000 rcf for 15 minutes to extract the protein by taking only the supernatant. The extracted protein was prepared by measuring the concentration using Bradford reagent (Thermo), diluting the protein to the same concentration, mixing it with 5x protein sample buffer (Elpis biotech) to make a total of 50 μl, and then denaturing at 95 ° C. for 5 minutes. 15 μl was loaded into each well of a 15-well 12% SDS PAGE gel. After gel running at 80V for 30 minutes and then at 120V for 2 hours, the protein electrophoresed on SDS-PAGE gel was transferred to a nitrocellulose membrane at 200V for 1 hour. The membrane to which the protein was transferred was blocked for 40 minutes using Skim milk diluted to 5% in TTBS buffer. The rabbit host's primary antibody against PDCD4 (anti-PDCD4, Cell Signaling Technology) and the mouse host's primary antibody against Vinculin (anti-Vinculin, Abcam) to be used as an internal control for result correction were respectively 1:2000 and 1 : After dilution in 5% BSA TTBS buffer at a volume ratio of 5000, it was treated by shaking with a membrane at 4 °C for 12 hours. After that, two antibodies anti-Rabbit IgG (Abcam) and anti-Mouse IgG (Abcam) conjugated with Horseradish peroxidase (HRP) were diluted in 5% Skim milk TTBS buffer at a volume ratio of 1:5000, and the membrane treated with anti-PDCD4 and Each membrane treated with anti-Vinculin was treated by shaking at room temperature for 2 hours. After treating each membrane with 1 ml of ECL solution (Abfrontier), Chemiluminecense was detected with Chemi-Doc MP ^TM (Bio-Rad) to confirm the protein band, and the results are shown in FIG. 17 . The same experiment as above was performed three times to obtain three images, one of which is shown in FIG. 13 . The intensity of the band obtained from each image was measured using Image Lab software (biorad), and [PDCD4 band intensity/Vinculin band intensity] was calculated to compare the PDCD4 expression levels in each group. The value measured in the control group was set to 1, and the average value and standard error value of the values in each group are shown in the upper graph.

As shown in FIG. 17 , a change in the expression level of PDCD4 protein, which is one of the genes downregulated by miR21 in each group, was confirmed. In the pri-miHPRT group in which Anti-miR21 was not introduced, it was confirmed that the PDCD4 expression level was not significantly different from that of the Control group. It was confirmed that through the PDCD4 expression increases.

Claims (23)

a stem-loop structure; The stem-loop structure includes a stem structure having a double-stranded polynucleotide of 33 to 38 bp in length and a loop structure having a single-stranded polynucleotide of 3 to 25 nt in length, The stem structure includes a section including an antisense region of 20 to 25 nt in length including a sequence complementary to the first target gene, and a sense region having a length of 20 to 25 nt capable of complementary binding to a section including the antisense region. Including a section including wherein the stem structure comprises the sense region 13 nt after the 5' end of the stem structure, A polynucleotide of 10 to 30 nt in length extended from the 5' end of the section including the sense region in the stem structure and a polynucleotide of 10 to 30 nt in length extended from the 3' end of the section including the antisense region in the stem structure. including, A nucleic acid construct comprising one or more features selected from the group consisting of the following (1) to (4): (1) a polynucleotide sequence extending from the 5' end of the segment containing the sense region in the stem structure, the polynucleotide sequence extending from the 3' end of the segment containing the antisense region in the stem structure, or both are ASO (antisense oligonucleotide) sequence or anti-miRNA sequence; (2) the polynucleotide sequence extending at the 5' terminus, the polynucleotide sequence extending at the 3' terminus, or both comprise chemically modified nucleotides; (3) the stem structure comprises chemically modified nucleotides; and (4) the loop structure comprises chemically modified nucleotides. The polynucleotide sequence of claim 1, wherein the polynucleotide sequence extending from the 5' end of the section including the sense region in the stem structure and the polynucleotide sequence extending from the 3' end of the section including the antisense region in the stem structure are complementary to each other. A nucleic acid construct that does not bind aggressively. The method of claim 1, wherein the section including the sense region in the stem structure is chemically modified at one or more positions selected from the group consisting of 14, 17, 18, 20, and 27th from the 5' end of the stem structure. modified) containing nucleotides, The section including the antisense region in the stem structure is complementary to a nucleotide present at one or more positions selected from the group consisting of 15, 16, 19, 21, and 23 to 26 from the 5' end of the stem structure. A nucleic acid construct comprising a chemically modified nucleotide at a position. The nucleic acid construct according to claim 3, wherein the section including the sense region in the stem structure comprises a chemically modified nucleotide at the 14th position from the 5' end of the stem structure. The nucleic acid construct according to claim 3, wherein the section including the sense region in the stem structure comprises a chemically unmodified nucleotide at the 14th position from the 5' end of the stem structure. The nucleic acid construct according to claim 3, further comprising a chemically modified nucleotide at one or more positions selected from the group consisting of the following (1) to (3): (1) a nucleotide present at the 14th position from the 5' end of the stem structure in the section including the sense region; (2) a position complementary to a nucleotide present at the 15th position from the 5' end of the stem structure in the section including the antisense region, and (3) In a section including an antisense region, a position complementary to a nucleotide present at the 16th position from the 5' end of the stem structure. The method according to claim 3, wherein the section including the sense region in the stem structure is one selected from the group consisting of 1 to 13, 15, 16, 19, 21 to 26, and 28 to 35 from the 5' end of the stem structure. A nucleic acid construct comprising a chemically unmodified nucleotide at the above position. According to claim 3, wherein the section including the sense region in the stem structure is at least one position selected from the group consisting of 1 to 14, 17, 18, 20, 22, and 27 to 35 from the 5' end of the stem structure. A nucleic acid construct comprising a chemically unmodified nucleotide at a position complementary to a nucleotide present in the nucleotide. The method of claim 1, wherein at least one sequence selected from the group consisting of a polynucleotide sequence extended from the 5' end of the sense region, a polynucleotide sequence extended from the 3' end of the antisense region, and the loop structure is C A nucleic acid construct comprising chemically modified nucleotides in the (cytidine) and A (adenine) sequences. The polynucleotide sequence extending from the 5' end of the sense region, the polynucleotide sequence extending from the 3' end of the antisense region, and the loop structure comprising a C (cytidine) and A (adenine) sequence A nucleic acid construct comprising a chemically modified nucleotide to. The method of claim 1, wherein at least one sequence selected from the group consisting of a polynucleotide sequence extended from the 5' end of the sense region, a polynucleotide sequence extended from the 3' end of the antisense region, and the loop structure is C contains chemically modified nucleotides in the (cytidine) and A (adenine) sequences; The section including the sense region in the stem structure includes a chemically modified nucleotide at one or more positions selected from the group consisting of 14, 17, 18, 20, and 27 from the 5' end of the stem structure. do, The section including the antisense region in the stem structure is complementary to a nucleotide present at one or more positions selected from the group consisting of 15, 16, 19, 21, and 23 to 26 from the 5' end of the stem structure. comprising a chemically modified nucleotide at the position, nucleic acid construct. According to claim 1, wherein the chemically modified nucleotide, the structure of the sugar contained in the nucleotide is modified with LNA (Locked Nucleic Acid), or, The residue of the sugar contained in the nucleotide is 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl ], 4′-thio, 4′-CH ₂ —O-2′-bridge, 4′-(CH ₂ ) ₂ —O-2′-bridge, 2′-amino, and 2′-O- (N-methylcarbamate) (methlycarbamate) will be modified with one or more selected from the group consisting of, a nucleic acid construct. The nucleic acid construct according to claim 1, which can be endogenously cleaved by the complex of DGCR8 and Drocha. The nucleic acid construct according to claim 13, which can be sequentially cleaved by the complex and Dicer. The nucleic acid construct according to claim 1, which modulates the expression of the first target gene. The nucleic acid construct according to claim 1, wherein the first target gene is selected from a protein coding gene, a proto-oncogene, an oncogene, a tumor suppressor gene, and a cell signaling gene. The nucleic acid construct according to claim 1, wherein the ASO sequence complementarily binds to the mRNA sequence of the second target gene, thereby degrading the mRNA of the second target gene. The nucleic acid construct of claim 1 , which induces selective splicing of the second target gene. The nucleic acid construct according to claim 1, wherein the anti-miRNA sequence is complementary to the miRNA sequence of the second target gene and inhibits the action of the miRNA of the second target gene. According to claim 1, wherein the ASO sequence or the anti-miRNA sequence, In the stem structure, the nucleic acid construct is introduced after 7 nt from the 5' end of the section including the sense region, or 7 nt after the 3' end of the section including the antisense region in the stem structure. A composition for inhibiting gene expression, comprising the nucleic acid construct according to any one of claims 1 to 20. The composition for inhibiting gene expression according to claim 21, wherein the gene is selected from a protein coding gene, a proto-oncogene, an oncogene, a tumor suppressor gene, and a cell signaling gene. 21. A cancer, a proliferative disease, a digestive disease, a kidney disease, a neurological disease, a psychiatric disease, a blood and tumor disease, a cardiovascular disease, a respiratory disease, an endocrine disease comprising the nucleic acid construct according to any one of claims 1 to 20 , Infectious diseases, musculoskeletal diseases, obstetrics and gynecological diseases, genitourinary diseases, skin diseases, and a pharmaceutical composition for the prevention or treatment of one or more diseases selected from the group consisting of ophthalmic diseases.

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