KR102134954B1 - siRNA for inhibiting CEACAM6 expression and therapeutic agent for adenocarcinoma comprising thereof - Google Patents

Abstract

The present invention relates to an siRNA delivery complex comprising siRNA and a pHLIP (low pH-induced transmembrane structure) for inhibiting CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6) expression, the acidic microparticles using the siRNA delivery complex of the present invention Since it effectively suppresses CEACAM6 in tumor cells in the environment, it can be useful as a therapeutic agent for adenocarcinoma.

Description

SiRNA for inhibiting CEACAM6 expression and adenocarcinoma therapeutic agent comprising the same {siRNA for inhibiting CEACAM6 expression and therapeutic agent for adenocarcinoma comprising thereof}

The present invention relates to siRNA for suppressing the expression of CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6) and a therapeutic agent for adenocarcinoma comprising the same.

Cancer is a major cause of mortality in developed countries. For the purpose of cancer treatment, many chemotherapy agents have been developed in the past 50 years. Most chemotherapeutic agents can be classified into alkylating agents, metabolic antagonists, anthracyclines, plant alkaloids, topoisomerase inhibitors, and anti-tumor agents. All of these agents affect cell division or the synthesis of DNA, resulting in a therapeutic effect through a mechanism that functions in some form.

Adenocarcinoma is a cancer that occurs especially in the cells that make up the gland, including the mucous membranes of the stomach, intestines, bronchi, uterus (body), and gallbladder, as well as the glandular tissue or excretion of the prostate, ovaries, thyroid, and interest. Occurs in the coffin. Most of the most common gastric cancers represent adenocarcinoma, but most of cervical cancers are squamous cell carcinomas and adenocarcinomas are rare. It is similar to a benign adenoma and has a malignancy, which is especially called a malignant adenoma.

As the adenocarcinoma progresses, the stage of the cancer can be confirmed and treatment is performed using surgery, chemotherapy, and radiation.

In recent years, instead of conventional chemotherapeutic agents, the development of molecular targeting drugs targeting molecules specifically expressed in cancer cells has been developed. With the advent of these molecular target drugs, side effects inherent in conventional chemotherapeutic agents are avoided, and cancer treatments contributing to the quality of life (QOL) of cancer patients are possible. In addition to the small molecule drug, such a molecular target drug includes a drug such as an antibody, and a therapeutic antibody is a molecule inherently present in the living body, and besides the advantage of low toxicity to the living body, the effector function is mediated. Since it also has the advantage of exerting a therapeutic effect by specifically injuring a target cell by an action mechanism, many therapeutic antibodies are commercially available, but solid cancer targeted therapeutics have not been tried depending on the type of cancer.

(0001) Republic of Korea Patent KR10-2017-0128567 (0002) Republic of Korea Patent KR10-2009-0111299

In order to solve the above object, the present invention suppresses the expression of CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6) gene, and includes a base sequence represented by SEQ ID NO: 1 and a low pH-induced transmembrane structure (pHIP) Phosphorus siRNA delivery complex and to provide a method for producing the same.

In order to achieve the above object, the present invention inhibits the expression of CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6) gene, and includes a base sequence represented by SEQ ID NO: 1 and a low pH-induced transmembrane structure (pHIP) A siRNA delivery complex is provided.

In addition, the present invention comprises the steps of cutting a PNA oligomer comprising a base sequence represented by SEQ ID NO: 1 and cysteine (Cys) from a resin using an m-cresol solution;

Reverse-phase high-speed liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) by binding TAMRA (5-carboxytetramethylrhodamine) to the amino acid (N) terminal of the PNA represented by SEQ ID NO: 1 Purifying the PNA oligomer synthesized by; And

It provides a method for producing a siRNA delivery complex that inhibits the expression of the CEACAM6 gene, comprising the step of forming a disulfide bond with the C-terminus of the PNA oligomer at the PHIP sequence represented by SEQ ID NO: 3 at the C-terminus of the PNA oligomer.

In addition, the present invention provides a pharmaceutical composition for preventing and treating adenocarcinoma containing the siRNA delivery complex.

The siRNA delivery complex that inhibits the expression of the CEACAM6 gene of the present invention is a targeted therapeutic agent that precisely injects siRNA regulating CEACAM6 into pHLIP using an acidic microenvironment, and is usefully used to prevent and treat adenocarcinoma while minimizing side effects occurring in normal tissue Can.

1 is a graph showing the results of Kaplan-Meier survival analysis of CEACAM6 expression in lung adenocarcinoma (log-rank test, P = 0.0262).
2 is a graph showing the results of RT-PCR measurement of CEACAM6 mRNA expression levels in A549 cell lines transfected with non-target siRNA, GAPDH siRNA and four siCEACAM6 sequences.
3 is a graph showing the results of purifying the reaction mixture (pHLIP-PNA) of PNA (siCEACAM6) and pHLIP using HPLC.
4 is a graph showing the results of Western blot analysis of A549 cells cultured for 48 hours with pHLIP-siCEACAM6 (100, 250, 500 nM) and pHLIP-scr at pH 6.2.
5 is a graph showing the results of flow cytometry analysis of A549 cells cultured with pHLIP-siCEACAM6 and pHLIP-scr (500nM) at pH 7.4 (blue) or 6.2 (red) for 48 hours.
6 is a graph showing the effect of CEACAM6 inhibition on A549 cell viability at neutral and acidic pH values: statistical analysis and data performed with ANOVA mean ± SD; n=3; *** P <0.001.
FIG. 7 is a graph showing the results of intravenous injection of pHLIP-siCEACAM6 and cisplatin and measurement of tumor volume in nude mice with (left) to 44.8 mm ³ A549 tumors: statistical analysis and data performed with Student's t-test Is expressed as mean±SD when n=5; *** P <0.001.
(Right) Photo showing lung tumors of mice 3 weeks after injection of pHLIP-scr, pHLIP-siCEACAM6 and cisplatin.
8 is a graph quantifying the cell number of Ki-67 in lung tumors treated with pHLIP-scr, pHLIP-siCEACAM6 and cisplatin: Error bars are SD (n = 5 tumors per group).
9 is a graph quantifying the cell number of apoptotic cells (CC3) in lung tumors treated with pHLIP-scr, pHLIP-siCEACAM6 and cisplatin: Error bars are SD (n = 5 tumors per group).
10 is a photograph confirming histological and immunohistochemical staining of lung tumors of mice treated with vehicle, pHLIP-scr, pHLIP-siCEACAM6 and cisplatin.
11 is a photograph of lung tumors of mice treated with pHLIP-siCEACAM6 (4 mg/kg) under a confocal microscope: PNA-TAMRA (red), nucleus (blue).
12A is a schematic diagram showing the experimental design of a combination treatment of siCEACAM6 and cisplatin combination therapy.
12B is a graph showing the measurement of tumor volume by intravenous injection of pHLIP-siCEACAM6 or cisplatin at the indicated time (arrow) in mice with ˜44.8 mm ³ A549 tumors: statistics performed with Student's t-test Analysis and data are presented as mean ± SD. when n=5; ** P <0.01, *** P <0.001.
13 is a graph quantifying the number of cells of Ki-67 in lung tumors treated with vehicle, pHLIP-siCEACAM6, cisplatin and pHLIP-siCEACAM6 with cisplatin: Error bars are SD (n = 5 tumors per group).
14 is a graph quantifying the number of cells of apoptotic cells (CC3) in lung tumors treated with vehicle, pHLIP-siCEACAM6, cisplatin and pHLIP-siCEACAM6 and cisplatin: Error bars are SD (n = 5 tumors per group ).
FIG. 15 is a confocal microscope photograph of lung tumors of mice treated with vehicle, pHLIP-siCEACAM6, cisplatin and pHLIP-siCEACAM6 with cisplatin: PNA-TAMRA (red), nucleus (blue).
16 is a view showing the structure of the pHLIP-scr and pHLIP-siCEACAM6 prepared in the present invention.

Hereinafter, the present invention will be described in detail.

Accordingly, the present inventors linked the production of a new siRNA delivery complex by connecting a peptide vector (pHLIP) that works only in an acidic environment and siRNA (siCEACAM6) for CEACAM6 in the form of peptide nucleic acid (PNA) to suppress CEACAM6 overexpressing in adenocarcinoma. The present invention was completed because a siRNA (siCEACAM6) linked to a peptide targeting a tumor-specific acidic microenvironment was passed through a cancer cell membrane.

The present invention is a siRNA (small-interfereing RNA) delivery complex, has the following structural formula, inhibits the expression of CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6) gene, the following structural formula is the sequencing sequence represented by SEQ ID NO: 1 It provides a siRNA delivery complex comprising a pHLIP (low pH-induced transmembrane structure).

[Structural Formula 1]

In one embodiment of the present invention, the nucleotide sequence represented by SEQ ID NO: 1 is a siRNA of the CEACAM6 gene with SEQ ID NO: 2 produced in the form of peptide nucleic acid (PNA) (see FIG. 16).

In one embodiment of the present invention, the pHLIP (low pH-induced transmembrane structure) is a peptide vector that selectively functions as a nanosyringe in the acidic environment of cancer represented by SEQ ID NO: 3.

"PHLIP" of the present invention is a peptide vector using an acidic microenvironment of cancer. Cancer cells consume more glucose than normal cells to survive, and most of them metabolize to lactic acid regardless of oxygen concentration, and the lactic acid and hydrogen ions thus formed are released outside the cancer cells, and the extracellular environment of the tumor becomes acidified. A peptide vector (low pH-induced transmembrane structure, pHLIP) that selectively functions as a nanosyringe in a cancerous acidic environment was developed by Reshetnyak, and can be used to inject a substance with a small molecular weight into the cell. It passes through the cell membrane only in an acidic environment and forms a stable spiral structure, acting as a syringe. When a carrier is connected to the C-terminal of the peptide vector, disulfide bonds are released and function in the cytoplasm, and thus, in the present invention, the C-terminal of the PNA oligomer comprising the nucleotide sequence of SEQ ID NO: 1 was linked.

"CEACAM6" of the present invention is a glycoprotein belonging to the CEA family, which is overexpressed in most adenocarcinomas and expressed very low in normal cells.

"PNA (peptide nucleic acid)" of the present invention is a neutral gene control molecule capable of binding to a DNA or RNA having a structure in which a nucleic acid base is bonded to a backbone side chain that is a peptide-like, PNA/DNA double chain PNA molecules with a base complementary to a portion of either chain nucleotide sequence for the DNA double chain have a greater thermal stability than the DNA/DNA bond, and forcibly unwind the double chain to the DNA double chain. And DNA-PNA double chain with strong binding ability. In order to produce PNA, it can be prepared to have a desired nucleotide sequence by using coupling-capping-deprotecting using a solid phase synthesis method or the like. The length of the base sequence of the PNA is not greatly limited, but it is preferred to have about 5 to 30 bases, preferably about 10 to 20 base sequences.

In addition, since it is made of PNA, it is stable because it does not have polarity, and is not easily degraded in vivo, and is suitable for injecting into cells due to its very strong binding to target DNA or RNA. In the present invention, PNA siRNA is injected as a material to be injected through pHLIP. It was produced in the form.

"AEEEA" of the structural formula 1 was added before and after the PNA sequence for the role of a hydrophilic linker.

In addition, the PNA oligomer comprising the base sequence and cysteine (Cys) represented by SEQ ID NO: 1 using m-cresol solution to cut from the resin (resin);

Reverse-phase high-speed liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) by binding TAMRA (5-carboxytetramethylrhodamine) to the amino acid (N) terminus of the PNA represented by SEQ ID NO: 1 Purifying the PNA oligomer synthesized by; And

It provides a method for producing a siRNA delivery complex that inhibits the expression of the CEACAM6 gene, comprising the step of forming a disulfide bond with the C-terminus of the PNA oligomer at the PHIP sequence represented by SEQ ID NO: 3 at the C-terminus of the PNA oligomer.

In addition, the present invention provides a pharmaceutical composition for preventing and treating adenocarcinoma comprising the following siRNA delivery complex.

[Structural Formula 1]

In one embodiment of the present invention, the adenocarcinoma may prevent and treat one or more adenocarcinomas of the group consisting of lung adenocarcinoma, gastric adenocarcinoma, colorectal cancer, cervical adenocarcinoma, and pancreatic cancer, and more preferably lung adenocarcinoma. It can be prevented and treated.

In addition, a substance such as cisplatin in the siRNA delivery complex of the present invention can have a synergistic effect such as combination therapy.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-mentioned active ingredient. The pharmaceutically acceptable carrier should be compatible with the active ingredient of the present invention, and may include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these ingredients. It can be used in combination, and if necessary, other conventional additives such as antioxidants, buffers, bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, and emulsions. In particular, it is preferable to provide a formulation in a lyophilized form. For the preparation of a lyophilized formulation, a method commonly known in the art to which the present invention pertains may be used, and a stabilizer for lyophilization may be added. Furthermore, it can be preferably formulated according to each disease or ingredient using methods disclosed in Remington's pharmaceutical Science (Mack Publishing company, Easton PA) by appropriate methods in the art.

The content and method of administration of the active ingredients contained in the composition of the present invention can be determined by a person skilled in the art based on the symptoms and severity of the disease in a typical patient. It can also be formulated in various forms such as powders, tablets, capsules, liquids, injections, ointments, syrups, and can also be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles.

The composition of the present invention can be administered orally or parenterally. The route of administration of the composition according to the present invention is not limited to these, for example, oral cavity, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, sublingual Or topical administration is possible. The dosage amount of the composition according to the present invention varies in range depending on the patient's weight, age, sex, health status, diet, administration time, method, excretion rate, or disease severity, and is easy for a person skilled in the art. Can decide. In addition, the compositions of the present invention can be formulated into suitable formulations using known techniques for clinical administration.

In addition, the present invention provides a method for preventing and treating lung adenocarcinoma, comprising administering a siRNA delivery complex according to the present invention to a patient in need thereof.

Hereinafter, the present invention will be described in more detail through the following examples. However, the following implementations are provided for illustrative purposes only to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

< Example 1> Experimental materials and methods

<1-1> PNA- pHLIP synthesis

All PNA oligomers (siCEACAM6, TAMRA-ooo-GATCACAGTCTCTGGAAGT-ooo-Cys; scrambled siRNA (scr), TAMRA-ooo-TGGTTTACATGTCGACTAA-ooo-Cys) were purchased from PANAGENE (Daejeon, Korea). The PNA oligomer sequence comprising -ooo- (11-amino-3,6,9-trioxaundecanoic acid, hydrophilic linker) and cysteine was synthesized through solid phase synthesis using an automatic synthesizer with BAG PNA monomer (PNAGENE's PNA oligomer) Using synthetic techniques). The oligomer was cleaved from the resin using m-cresol: TFA (1: 4) cocktail solution. The single isomer, 5-carboxytetramethylrhodamine (TAMRA), was only bound to the amino acid (N) terminus of PNA. PNA oligomers were purified by reverse phase high speed liquid chromatography (RP-HPLC) and MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight). The two PNA oligomers have a melting temperature (Tm) of 79°C and 75°C, respectively. To generate the pHLIP-PNA construct, the following PHIP sequence was synthesized: AAEQNPIYWARYADWLFTTPLLLLDLDLVDADEGTCG (SEQ ID NO: 3). The C-terminus of the PNA oligomer was conjugated to pHLIP by disulfide bonds. To synthesize the pHLIP-PNA structure, pHLIP and PNA (peptide: PNA 1.5:1) were reacted overnight in a mixture of DMF/Tris (pH 7.5) in the dark. After binding, pH-LIP-PNA was purified by RP-HPLC and MALDI-TOF was characterized. ^{13,700 M -1 cm -1 (A)} , 6,600 M -1 cm -1 (C), 11,700 M -1 cm -1 (G), 8,800 M -1 cm -1 (T), 200 M -1 cm - ^It was dried in the order of ¹ (F, Phenylalanine) and 32,300 M ^-1 cm ^-1 (TAMRA) and then lyophilized (FIG. 16).

<1-2> Cell culture

The human lung cancer cell line (A549) was purchased from American Type. RPMI containing L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 g/ml) and 10% FBS was added to the culture and maintained at 37°C at 5% CO ₂ . In all pH controlled cell culture experiments, cells were cultured with HEPES or 10% FBS in RPMI buffered to pH 7.4 with pH 6.2 and MES.

<1-3> CEACAM6 siRNA Transformation

siRNA double strands were purchased from Dharmacon Research, Inc (Lafayette, CO). In order to confirm the specificity of the inhibition, a non-target siRNA as a control was used as a negative control.

The siRNA sequences used in the CEACAM6 siRNA experiment are as follows:

Control (Negative control): UGGUUUACAUGUCGACUAA (SEQ ID NO: 7)

Seq #1: GAUCACAGUCUCUGGAAGU (SEQ ID NO: 2),

Seq #2: GAACAUGGCUAAAUACAAU (SEQ ID NO: 4),

Seq #3: GAGGGUAACUUAACAGAGU (SEQ ID NO: 5),

Seq #4: CUACAUACUCCAACUGAAA (SEQ ID NO: 6).

The human lung cancer cell line (A549) was transfected with 10 nM siRNA double strands using Lipofectamine RNAiMax (Thermo Fisher Scientific) according to the manufacturer's instructions. After 48 hours, it was confirmed that the CEACAM6 gene was inhibited by real-time RT-PCR.

<1-4> Quantitative real-time RT- PCR

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) and cDNA was synthesized according to the manufacturer's protocol using the iScript ^™ cDNA synthesis kit (Bio-Rad, Hercules, CA). Quantitative real-time RT-PCR (qRT-PCR) was performed using iQ SYBR Green supermix (Bio-Rad). Primer sequences are as follows: GAPDH (forward, 5'-CCTGCACCACCAACTGCTTA-3' (SEQ ID NO: 8), reverse, 5'-GTCTTCTGGGTGGCAGTGAT (SEQ ID NO: 9)); CEACAM6 (forward, 5'-GACAGTTCCATGTATACCCG-3' (SEQ ID NO: 10), reverse, 5'-ACAGCATCCTTGTCCTCC-3' (SEQ ID NO: 11)). Thermal cycling and fluorescence detection were performed using the S1000 Thermal cycler real-time PCR system (Bio-Rad). The reaction was performed in a CFX96 Real-Time PCR System (Bio-Rad) using the following thermal conditions: initial denaturation step at 95°C for 3 minutes and denaturation at 40 cycles for 10 seconds at 95°C and annealing/extension 55 30 seconds at ℃. Relative gene expression levels were evaluated as a ratio to GAPDH expression.

<1-4> Western Blot

The human lung cancer cell line (A549) was incubated with HEPES with MES or 1% FBS in RPMI buffered to pH 7.4 with pH 6.2, and treated with pHLIP-PNA suspended for 48 hours in reaction buffer. Proteins were separated by electrophoresis on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) and transferred to a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, Rockford, IL). The membrane was blocked with 5% skim milk and then mouse anti-CEACAM6 monoclonal antibody (9A6, Santa Cruz Biotechnology Inc.) or mouse anti-GAPDH monoclonal antibody (6C5, Santa Cruz Biotechnology Inc.) was incubated with loading control. . Protein complexes were detected with enhanced chemiluminescent reagents (Thermo Fisher Scientific).

<1-5> Flow cell analysis

For flow cytometry, the human lung cancer cell line (A549) was incubated with HEPES or pH 6.2 and MES with 10% FBS in RPMI buffered at pH 7.4 and treated with pHLIP-PNA suspended in reaction buffer for 48 hours. After washing the cells to remove the free pHLIP-PNA, it was suspended again in 500 μl PBS (pH 7.4) and analyzed by flow cytometry (Becton Dickinson, San Jose, CA).

<1-6> Cell viability analysis

The Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan) was used to measure the inhibitory effect of CEACAM6 on human lung cancer cell line (A549). The human lung cancer cell line (A549) (5 x 10 ⁴ cells/ml) was treated with pHLIP-siCEACAM6 for 48 hours at a dose of 0, 100, 250, 500 or 1,000 nM in a 96-well plate, and the control of pHLIP-scr It was used as. All treatments were performed at the indicated pH. After 48 hours, the degree of cell growth was evaluated using CCK-8 analysis (Dojindo, Kumamoto, Japan). CCK-8 solution (10 μl) was added to each well and incubated at 37° C. for 2 hours. Absorbance at 450 nm was measured with a multi-line reader (Lambda Bio-20, Beckman). Cell viability was expressed as a percentage of control cells.

<1-7> Tumor xenograft experiment in vivo

All animal experiments were conducted according to a protocol approved by the Korea Institute of Biotechnology, Institutional Animal Care and Use Committee. A human lung cancer cell line (A549) was injected into the right side of 5-week-old BALB/c athymic nude mice (5 mice 1.2 x 10 ⁷ cells/mouse). Tumor growth was measured using a caliper to measure length (L), width (W) and height (H) and using the formula V = (L × W × H) × 0.5. Upon tumor formation (~44.8 mm ³ ), mice were injected intramuscularly (iv) with pHLIP-PNA and intravenously (ip) with cisplatin,

<1-7> Confocal Laser microscopy

To prepare for confocal preparation of the obtained tumor tissue, the tumor is flash frozen in the OCT and then cut into 2 μm sections. Tissue sections were fixed with cold acetone, washed with PBS, reacted with hydrogen peroxide to inactivate endogenous peroxidase, and washed with PBS. Cell nuclei were stained by adding DAPI. All washes were performed using PBS at pH 7.4 to wash the surface bound pHLIP. Images were collected using a confocalmicroscope (Zeiss LSM 710 META, Jena, Germany) and processed using Zensoftware (version 8.0, Zeiss, Jena, Germany).

<1-8> Histology and other analytical techniques

Mice were killed by choking carbon dioxide. Staining was performed on formalin-fixed, paraffin-fixed tumor sections (4 μm thick). For immunostaining, BenchMark XT automatic staining machine (Ventana Medical Systems Inc., Tucson, AZ, USA) was used. anti-cleaved caspase 3 (1:100, BioCare Medical) and anti-cleaved caspase 3 antibody (9A6, 1:4000, Santa Cruz Biotechnology Inc., Dallas, TX, USA) Ki67 (1: 100, VP-K452, vector laboratory ) Was used. The number of positive cells per tumor site was quantified.

<1-9> Cancer genome map ( TCGA ) analysis

A TCGA data set (http://tcga-data.nci.nih.gov/) containing multidimensional genomic data combined with a clinical pathological report of 515 patients with lung adenocarcinoma was cBioPortal tool (http://www.cbioportal. org). RNA sequence data of TCGA lung adenocarcinoma patients was used for mRNA expression analysis of CEACAM6. For univariate survival analysis, Kaplan-Meier plots using alog-rank test were presented using the overall survival data of lung cancer adenocarcinoma patients.

<1-10> Statistical Analysis

Two-way ANOVA was performed to compare the three groups in cell viability analysis. For animal experiments, mouse and tumor weights for each group were compared using the Student t-test. Immunostaining results of each group were compared using Student t-test. Survival curves were estimated using the Kaplan-Meier method and compared using the log rank test. P value ≤ 0.05 was judged to be statistically significant. All statistical tests were analyzed on both sides and performed using the SPSS statistical package, version 18.0 (IBM, NY, USA).

< Example 2> Experimental results

<2-1> TCGA Data analysis results

According to the TCGA data set of 515 lung adenocarcinoma patients analyzed with the cBioPortal tool (http://www.cbioportal.org), 50 of the 515 patients showed overexpression of CEACAM6 mRNA. In Kaplan-Meier survival analysis, high level of CEACAM6 mRNA expression was low in overall survival (P = 0.0262) (FIG. 1).

<2-2> pHLIP -PNA siCEACAM6 Active assay results

Efficient four siRNA sequences of CEACMA6 expression were screened to construct pHLIP-PNA siCEACAM6. Human lung cancer cell line (A549) was transformed with non-target siRNA, GAPDH siRNA and four siCEACAM6 sequences as described in Examples 1-3. All four sequences were found to inhibit CEACAM6 mRNA expression at similar levels 48 hours after transfection at 10 nM concentration compared to non-target siRNA (FIG. 2 ), to tether siCEACAM6 sequence #1 to pHLIP Selected.

The pHLIP-PNA siCEACAM6 structure was synthesized by combining the carboxy(C)-terminus of the PNA oligomer with pHLIP, and pHLIP-scr was used as a control. PHLIP-PNA was modified with the TAMRA label attached to the PNA. The preparation of pHLIP-PNA was verified by RP-HPLC and mass spectrometry (Fig. 3).

In addition, to investigate whether the pHLIP-PNA conjugate can deliver siCEACAM6 to the human lung cancer cell line (A549) and reduce the CEACAM6 protein, the human lung cancer cell line (A549) was pHLIP-siCEACAM6 (100, 250, 500 nM) at pH 6.2. ) And pHLIP-scr for 48 hours. Western blot analysis showed that the human lung cancer cell line (A549) overexpressing pHLIP-siCEACAM6 at low pH significantly lowered the protein expression level of endogenous CEACAM6 according to the dose of pHCE-siCEACAM6, thus intracellular delivery and activity of siCEACAM6. Inhibited (Figure 4).

In addition, the human lung cancer cell line (A549) association was measured by flow cytometry at neutral and acidic pH values, and the human lung cancer cell line (A549) was mixed with pHLIP-siCEACAM6 and pHLIP-scr (500 nM) for 48 hours at pH 7.4 or 6.2. Cultured together. The pHLIP-siCEACAM6 human lung cancer cell line (A549) was observed at low pH. The attached PNA sequence had little effect on the binding of the pHLIP-PNA construct (Figure 5).

In addition, as a result of performing cell viability analysis at neutral and acidic pH values, inhibition of CEACAM6 by pHLIP-siCEACAM6 reduced the viability of the human lung cancer cell line (A549) at pH 6.2 depending on the dose (FIG. 6). Through it, it was found that the connection of the pHLIP peptide to PNA targets CEACAM6 mRNA and inhibits the expression of CEACAM6 protein by the delivered siRNA.

<2-3> CEACAM6 Analysis of lung tumor inhibition in expression-suppressing animal models

It was analyzed whether siCEACAM6 can inhibit CEACAM6 expression in vivo. For in vivo analysis, BALB/c nude mice were injected subcutaneously with 1.2×10 ⁷ A549 cells. Two weeks after tumor initiation, physiological saline as a negative control with pHLIP-siCEACAM6 was injected through the tail vein of the mouse. To determine the optimal amount of pHLIP-siCEACAM6 administration, two groups of mice (n = 5 mice/group) were dosed twice daily (2 mg/kg and 4 mg/kg) over a three-week treatment period. In addition, since cisplatin is the backbone of lung cancer treatment, cisplatin-treated mice were used as a positive control. Cisplatin 2 mg/kg was administered intraperitoneally twice or three times a week.

In addition, to evaluate the effect of pHLIP-siCEACAM6 delivery on lung tumor development, tumor growth was examined every 2 or 3 days. Treatment with pHLIP-siCEACAM6 significantly inhibited tumor growth compared to control mice treated with pHLIP-scr (pHLIP-siCEACAM6 2 mg/kg: 23.0%, pHLIP-siCEACAM6 4 mg/kg: 35.5%, all P <0.001). .

Significant reduction in tumor growth was also observed in the cisplatin treated group compared to vehicle (2 cisplatin/week: 35.0%, cisplatin 3/week: 46.7%, all P <0.001) (FIG. 7 ).

In addition, to further study the mechanism of tumor regression, lung tumors were harvested and stained with proliferation and apoptosis markers. Compared to the pHLIP-scr group, it was confirmed that the proliferation of tumors treated with pHLIP-siCEACAM6 was reduced (pHLIP-scr: 21%, pHLIP-siCEACAM6: 15%, P=0.004) (FIG. 8 ).

Cisplatin treatment also resulted in a reduction in proliferation (vehicle 27%, cisplatin 11%, P <0.001) (Figure 9)

These data indicate that in vivo silencing of CEACAM6 in tumors leads to inhibition of proliferation. Cleaved caspase 3 (CC3) staining, a marker of apoptosis, was only increased in the pHLIP-siCEACAM6 group, thus confirming that siCEACAM6 inhibited lung tumor growth (P=0.025) (FIGS. 8, 9 and 10 ).

In addition, as a result of examining the ability of pHLIP-siCEACAM6 to deliver siCEACAM6 in lung adenocarcinoma in vivo, tumor tissues were isolated for confocal imaging analysis.

In pHLIP-siCEACAM6 labeled with TAMRA, a strong fluorescence signal was observed on the tumor cell surface of pHLIP-siCEACAM6 treated animals, indicating efficient intracellular delivery of siCEACAM6 in vivo (FIG. 11 ).

<2-4> s iCEACAM 6 Cisplatin Analysis results of lung tumor inhibition according to simultaneous treatment

To analyze whether chemotherapy and CEACAM6 targeting could enhance the anti-tumor effect, mice were randomly selected 2 weeks after subcutaneous injection of 1.2 x 10 ⁷ A549 cells (n = 5 mice/group): (i) vehicle, (ii) pHLIP-siCEACAM6, (iii) cisplatin and (iv) pHLIP-siCEACAM6 + cisplatin.

The optimal dose of pHLIP-siCEACAM6 was determined to be 2 mg/kg each for treatment with iCEACAM6 and cisplatin. Two weeks after the injection of the cell line, pHLIP-siCEACAM6 was injected twice into the mouse through the tail vein, and cisplatin was injected twice a week for a three-week treatment period (FIG. 12A).

Tumor volume was measured every 2 or 3 days, and after 3 weeks of systemic treatment, mice in the pHLIP-siCEACAM6 treated group had a 21.8% volume reduction compared to the vehicle (P = 0.004). Cisplatin treatment showed 29.9% of tumor reduction (P <0.001). The group treated with pHLIP-siCEACAM6 and cisplatin at the same time confirmed a 47% growth reduction in tumor volume (P<0.001) (FIG. 12B ). siCEACAM6s showed remarkable therapeutic activity in primary tumors and enhanced effectiveness with cytotoxic chemotherapy.

To further evaluate the biological effects of siCEACAM6 treatment, tumor samples for proliferation and apoptosis were stained, resulting in siCEACAM6 (P=0.121) or cisplatin alone (P=0.271) (P=0.271) in tumors administered with siCEACAM6 and cisplatin in combination (siCEACAM6: 19.8%, cisplatin: 18.4, pHLIP-siCEACAM6: 16.6%). However, the combination treated tumors increased CC3 positive cell numbers compared to siCEACAM6 (P=0.022) or cisplatin alone (P=0.016) (FIGS. 13, 14 and 15).

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> siRNA for inhibiting CEACAM6 expression and therapeutic agent for adenocarcinoma comprising thereof <130> PN1711-425 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Converting CEACAM6 siRNA to PNA <400> 1 gatcacagtc tctggaagt 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> CEACAM6 siRNA <400> 2 gaucacaguc ucuggaagu 19 <210> 3 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> pHLIP (low pH-induced transmembrane structure) <400> 3 Ala Ala Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu 1 5 10 15 Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Asp Leu Val Asp Ala Asp 20 25 30 Glu Gly Thr Cys Gly 35 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> CEACAM6 siRNA #2 <400> 4 gaacauggcu aaauacaau 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> CEACAM6 siRNA #3 <400> 5 gaggguaacu uaacagagu 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> CEACAM6 siRNA #4 <400> 6 cuacauacuc caacugaaa 19 <210> 7 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> CEACAM6 siRNA Negative control <400> 7 ugguuuacau gucgacuaa 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 8 cctgcaccac caactgctta 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 9 gtcttctggg tggcagtgat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CEACAM6 forward primer <400> 10 gacagttcca tgtatacccg 20 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CEACAM6 reverse primer <400> 11 acagcatcct tgtcctcc 18

Claims (7)

As a pharmaceutical composition for the prevention and treatment of lung adenocarcinoma comprising a small-interfereing RNA (siRNA) delivery complex of the following structural formula to suppress the expression of CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6) gene,
The siRNA delivery complex of the following structural formula includes a peptide nucleic acid (PNA) consisting of the nucleotide sequence of SEQ ID NO: 1 and a low pH-induced transmembrane structure (pHIP) consisting of the amino acid sequence of SEQ ID NO: 3,
The pHLIP is a peptide vector that selectively functions as a nanosyringe in an acidic environment,
The siRNA delivery complex is a siRNA delivery complex in which a peptide vector (pHLIP) that acts only in an acidic environment and a siRNA for CEACAM6 (siCEACAM6) in the form of peptide nucleic acid (PNA) are used to inhibit CEACAM6 overexpressing in adenocarcinoma. , SiRNA (siCEACAM6) linked to the peptide targeting the tumor-specific acidic microenvironment passes through the cancer cell membrane,
The nucleotide sequence represented by SEQ ID NO: 1 is a siRNA of the CEACAM6 gene represented by SEQ ID NO: 2 produced in the form of peptide nucleic acid (PNA),
In the siRNA delivery complex, a PNA oligomer containing a base sequence represented by SEQ ID NO: 1 and cysteine (Cys) is cut from a resin using an m-cresol solution, and TAMRA (5-carboxytetramethylrhodamine) is represented by SEQ ID NO: 1. The PNA oligomer synthesized by reverse phase high-speed liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) is purified by binding only to the amino acid (N) terminal of the PNA to be obtained, and the PNA oligomer is purified. It was prepared by reacting the pHLIP sequence represented by SEQ ID NO: 3 on the C-terminus of the PNA oligomer to form a disulfide bond with the C-terminus,
The siRNA delivery complex inhibits CEACAM6 mRNA expression, which siRNA overexpresses in lung adenocarcinoma 48 hours after transfection at a concentration of 10 nM, lowers cell proliferation capacity (Ki-67 index) and increases cell death (cleaved caspase 3) in lung adenocarcinoma cells. Characterized in that, to prevent and treat lung adenocarcinoma pharmaceutical composition:

. Pharmaceutical composition for the prevention and treatment of lung adenocarcinoma comprising s iRNA (small-interfereing RNA) delivery complex of the following structural formula that suppresses the expression of CEACAM6 (Carcinoembryonic antigen-related cel l adhesion molecule 6) gene, and cisplatin as an active ingredient as,
The siRNA delivery complex of the structural formula below is an example of lung adenocarcinoma characterized in that it contains a peptide nucleic acid (PNA) consisting of the nucleotide sequence of SEQ ID NO: 1 and a low pH-induced transmembrane structure (pHIP) consisting of the amino acid sequence of SEQ ID NO: 3. Pharmaceutical compositions for room and treatment:

According to claim 2,
The pHLIP is a composition characterized in that it is a peptide vector that functions as a nanosyringe (nanosyringe) selectively in an acidic environment.
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