Selective delivery of tumor suppressor miRNA (miR126) to therapy-resistant metastatic melanoma cells.

Abstract

The present invention discloses a carrier having a tumor suppressor that is selectively directed to a tumor target, for the treatment of metastatic melanoma resistant to targeted therapy.

Description

[Technical Field]

[0001] explanation Melanoma is a cancer that arises from the malignant transformation of melanocytes, the pigment cells of the epidermis (Situm M et al, 2014). According to the World Health Organization (WHO), an estimated 300,000 people worldwide are newly diagnosed with melanoma each year, and 60,000 die from it. Melanoma is a rapidly increasing, highly malignant cancer, having increased by 75% in the last 30 years (Saginala K et al, 2021). Incidence and mortality rates vary by country, but are high in Australia, New Zealand, Europe, and North America (Bolick NL et al., 2020). Recent research has focused on improving therapeutic strategies for advanced melanoma, with the use of immune checkpoint inhibitors being promising (Guo W et al., 2021). Effective treatment of metastatic melanoma has also been achieved by targeting specific molecular pathways (Ostrowski SM et al, 2020). However, new biomarkers and drug targets are needed to improve diagnosis and treatment (Davis LE et al, 2019). [Background technology]

[0002] Molecular analysis of the melanoma development process has revealed that activating mutations in the BRAF gene are found in more than 50% of melanomas, with the most common mutation being a substitution of glutamate to valine at codon 600 (V600E). Drugs such as vemurafenib and dabrafenib can selectively inhibit the mutated BRAF protein (Wellbrock C et al, 2016). While the clinical use of these drugs has shown promising results, early recurrence and treatment resistance remain challenges. To overcome this, novel therapies are being developed that focus on combining multiple compounds to inhibit various pathways crucial for cancer cell survival (Long GV et al, 2014). Based on this evidence, simultaneous inhibition of the mitogen-activated protein kinase (MAPK / ERK) pathway and the phosphatidylinositol 3 kinase-protein / AKT (PI3K / AKT) pathway is an effective therapeutic strategy. PIK-75, a PI3K / AKT pathway inhibitor, has been identified as a potential drug to overcome resistance by inhibiting both pathways in combination therapy with vemurafenib or dabrafenib (Pedini F et al, 2019).

[0003] Non-coding RNAs, such as microRNAs (miRs), play important roles in various cellular processes. While miR126 has been shown to act as a tumor suppressor in several types of cancer, it has also been demonstrated to inhibit the growth and metastasis of metastatic melanoma by regulating the expression of tumorigenic molecules (Felli N et al, 2013), particularly the PI3K regulatory subunit p85β (Guo C et al, 2008).

[0004] miR126 has been shown to suppress tumor growth in metastatic melanoma, and its potential to enhance the efficacy of other treatments has been evaluated. This includes combination therapy with PIK-75 and vemurafenib or dabrafenib. Overexpression of miR126 suppresses tumor growth and enhances the efficacy of PIK-75 alone or in combination with vemurafenib or dabrafenib. It has also shown efficacy against early-stage cell lines derived from patient biopsies and against melanoma cell lines resistant to either vemurafenib or dabrafenib, suggesting the possibility of overcoming drug resistance (Pedini F et al, 2019).

[0005] The use of nucleic acids (including miRs) in therapy requires overcoming several challenges, particularly those related to their rapid degradation or disappearance. One effective strategy to address these critical challenges is the development of cancer nanomedicine, which involves the practical application of nanotechnology in cancer therapy. Nanoparticles (NPs) are attracting attention as promising candidates for cancer therapy due to their unique properties, such as sustained and controlled drug release, excellent availability and biocompatibility, and the ability to maintain physicochemical properties in the human body over long periods (Ojha A et al, 2022; Arasi MB et al. 2020). More generally, the conventional application of nanotechnology in cancer therapy is to improve pharmacokinetics and reduce the systemic toxicity of chemotherapy by selectively targeting and delivering anticancer drugs to tumor tissue.

[0006] Chitosan-based nanoparticles (CS) have been identified as promising candidates for drug nucleic acid delivery due to their positive charge in acidic environments, biocompatibility, and stability (Dahlman JE et al, 2014; Sargazi S et al, 2022). [Overview of the project]

[0007] The inventors of this application have developed a novel therapeutic agent for the treatment of tumors, particularly metastatic melanoma exhibiting resistance to therapy. In particular, the inventors have remarkably found a method for directly delivering mature miR-126-3p to tumor cells. The system is based on the use of functionalized particles having a targeting moiety for specific tumor markers. These nanoparticles have been shown to be highly effective in treating metastatic melanoma resistant to targeted therapy in preclinical models. [Brief explanation of the drawing]

[0008] [Figure 1] Chemical modification of OMe-miR126 enhances its stability in human plasma without altering its functionality. A: Chemical modification of the standard miR126-3p sequence: OMe-miR126. B (left panel) Immunoblotting analysis: Comparison of downmodulation of the target protein (p85β) by OMe-miR126 and the commercially available miR126-3p sequence (Dharm-miR126). B (right panel) Immunoblotting analysis: Time-course evaluation of p85β targeting by OMe-miR126. C: Stability of OMe-miR126 and Dharm-miR126 sequences in PBS containing 50% human plasma at 37°C. D: Inhibition of cell proliferation after OMe-miR-126 overexpression. The experiments reported in B and D were performed using the A375M-VR cell line. [Figure 2] Synergistic effects of the OMe-miR126 sequence and BRAF inhibitors or PI3K inhibitors. A. Comparison of proliferation inhibition of A375M via the OMe-miR126 or Dharm-miR126-3 sequence with combination therapy of OMe-CTRL and vemurafenib (top left), dabrafenib (top middle), PIK-75 (top right), vemurafenib + PIK-75 (bottom left), and dabrafenib + PIK-75 (bottom right). B. Comparison of proliferation inhibition of A375M-DR via the OMe-miR126 or Dharm-miR126-3 sequence with combination therapy of OMe-CTRL and PIK-75. IC50 values ​​for each treatment are reported. [Figure 3]Synergistic effects of the OMe-miR126 sequence with BRAF inhibitors or PI3K inhibitors. Excess Over Bliss (EOB) score. The obtained values ​​suggest that OMe-miR126 shows synergistic effects with single agents (vemurafenib, dabrafenib, or PIK-75) or combinations thereof (vemurafenib + PIK-75, or dabrafenib + PIK-75) in A375M WT cell lines and A375M-DR cell lines. [Figure 4] Synthesis and analysis of single-chain antibody 9.2.27 (scFv-9.2.27). A: Schematic diagram and Western blot of purified scFv-9.2.27. B: Flow cytometry analysis of A375M-DR showed that scFv-9.2.27 can selectively interact with the surface of melanoma cells. C: Treatment of A375M-DR with scFv-9.2.27 showed no toxic effects up to 1 μg / ml. [Figure 5] Synthesis and characterization of nanoparticles. A: Schematic diagram of scFv-CS126 nanoparticle synthesis (left). Various scFv:CS weight ratios were investigated, and a scFv:CS weight ratio of 1:10 was selected for Ab-CS126 synthesis (right). B: The average zeta potential of CS126 was +37.8 ± 4.3 mV, and for Ab-CS126 it was +33.3 ± 1.2 mV. C: NanoSight evaluation of nanoparticle size distribution and concentration. [Figure 6] Characterization of CS126 and Ab-CS126 nanoparticles. A: Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of CS126 and Ab-CS126. B: WES analysis of bound scFv-9.2.27 allowed for the estimation of the amount of 250 pg of scFv / 6.8 x 10^7 Ab-CS126. C: The integrity of the OMe-miR126 sequence captured by CS126 in 50% human plasma was demonstrated up to 48 hours of incubation. [Figure 7]Evaluation of CS and Ab-CS internalization in A375M-DR cells. A: Visualization by fluorescence microscopy (left) and FACS analysis showed that approximately 100% of cells were FITC-positive after incubation with 1.6 × 10⁸ particles for 20 minutes. B: qRT-PCR results revealed that the amounts of OMe-miR126 captured by CS126 and Ab-CS126 were 1.2 × 10⁻⁸ and 1.7 × 10⁻¹⁰ pMole / nanoparticles, respectively. C: qRT-PCR analysis of OMe-miR126 introduction in A375M-DR cell lines after exposure to the same number of different nanoparticles. D: The delivery efficiency (DE) of Ab-CS126 was approximately 9.5 times higher than that of CS126. [Figure 8] Stability of CS-FITC and Ab-CS-FITC: In vitro and in vivo experiments. A: In vivo plasma analysis of mice injected with fluorescent nanoparticles showed subdetectable fluorescence after 60 minutes (top panel). In in vitro experiments, the fluorescence of CSFITC and Ab-CSFITC was retained for up to 6 days (bottom panel). B: Distribution analysis of nanoparticles showed that the majority of the FITC signal was present in the lungs, liver, kidneys, and spleen of injected mice. [Figure 9] Evaluation of the tumor-suppressing effect of OMe-126-containing nanoparticles in in vivo experiments. A: Schematic diagram of the animal model. B: Statistical analysis of tumor growth. C: Fluorescence IVIS images at different time points of one representative mouse selected from six treatment groups. In the right panel, the liver and lungs of each mouse are individually imaged with IVIS spectra. [Figure 10]Detection of apoptosis in cryopreserved tissue sections of mouse liver and lung. A. The Tune assay for detecting apoptosis was performed on cryopreserved tissue sections of liver and lung from NSG mice. As shown in the figure, mice injected with the A357M-DR cell line and administered dabrafenib, PIK-75, and Ab-CS126 showed a much stronger fluorescence signal compared to the control group, which is due to the progression of apoptosis specifically induced at tumor invasion sites by the combined treatment. B. Immunostaining with anti-scFv-9.2.27 and a relative negative control was performed on kidney sections of mice treated with CS126 or Ab-CS126 to confirm the distribution of Ab-CS126 in animal organs. [Figure 11A] CSPG4 expression in ovarian cancer cell lines (SK-OV3 and A2780) and lung cancer cell lines (H1975 and HCC827). [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 12] Synergistic effect of OMe-miR126 sequence and PI3K inhibitor in ovarian cancer cell line SK-OV3.

[0009] Objective of the present invention In its first purpose, the present invention describes a double-stranded RNA containing a mature tumor-suppressive miR-126-3p sequence.

[0010] In a second object, the present invention discloses a nanocarrier system having double-stranded RNA containing a mature tumor-suppressive miR-126-3p sequence.

[0011] In certain embodiments, the nanocarrier system is linked to the targeting portion.

[0012] In a third object, the double-stranded RNA of the present invention, the mature tumor-suppressive miR-126-3p sequence of the present invention, and the nanocarrier system of the present invention are disclosed for medical use.

[0013] In a preferred embodiment, the medical use of the present invention is for the treatment of tumors that express the tumor marker CSPG4.

[0014] In a more preferred embodiment, the medical use of the present invention is disclosed in the treatment of tumors including melanoma, ovarian cancer, and lung cancer.

[0015] In an even more preferred embodiment, the medical use of the present invention is disclosed in the treatment of melanoma.

[0016] In an even more preferred embodiment, the nanocarrier system of the present invention is disclosed for medical use in the treatment of metastatic melanoma resistant to targeted therapy.

[0017] The double-stranded RNA of the present invention, the mature tumor-suppressive miR-126-3p sequence of the present invention, and the nanocarrier system of the present invention are disclosed for medical use in treating tumors in combination with a PI3K / AKT inhibitor.

[0018] Specifically, the double-stranded RNA of the present invention, the mature tumor-suppressive miR-126-3p sequence of the present invention, and the nanocarrier system of the present invention are disclosed for medical use in treating metastatic melanoma resistant to targeted chemotherapy in combination with a PI3K / AKT inhibitor.

[0019] Detailed Description of the Present Invention According to a first object, the present invention describes a double-stranded RNA comprising a mature tumor-suppressive miRNA sequence.

[0020] For the purposes of the present invention, the double-stranded miRNA comprises the mature tumor-suppressive miR-126-3p sequence.

[0021] More specifically, the double-stranded RNA is represented by a double-stranded oligonucleotide characterized by a sense strand sequence corresponding to SEQ ID NO: 6 described below and an antisense strand sequence corresponding to SEQ ID NO: 7. [Table 1]

[0022] According to a preferred embodiment, the double-stranded RNA containing the mature tumor-suppressing miR-126-3p sequence of the present invention is chemically modified.

[0023] In particular, all pyrimidine residues in the sense strand and one uridine residue in the antisense strand are modified by O-methylation (2'-OMe) at the 2'-O-pentose (see Figure 1A).

[0024] More specifically, in the antisense chain, O-methylation occurs at the fourth uridine residue (corresponding to the 14th nuclear residue).

[0025] Such modifications have the effect of improving thermal stability.

[0026] Furthermore, both the sense strand and the antisense strand are further modified by adding two 2'-deoxythymidine residues to their respective 3' ends.

[0027] Such modifications have the effect of protecting the molecule from cleavage by nucleases.

[0028] The active tumor suppressor molecule is ultimately represented by the standard miR-126-3p antisense chain having the following structure. [Table 2]

[0029] According to a second object, the present invention discloses a nanocarrier system having double-stranded RNA containing a mature tumor-suppressive miR-126-3p sequence.

[0030] According to one embodiment of the present invention, the nanocarrier system of the present invention targets tumor cells.

[0031] For the aforementioned purpose, the nanocarrier system is connected to the target portion.

[0032] According to one embodiment, the nanocarrier system may be represented by nanoparticles carrying double-stranded RNA containing a mature tumor-suppressive miR-126-3p sequence.

[0033] In certain embodiments, the nanoparticles are represented by chitosan nanoparticles.

[0034] For the purposes described above, chitosan nanoparticles can be prepared by mixing a chitosan solution with a solution containing mature tumor-suppressive miR-126-3p sequence double-stranded RNA.

[0035] Preferably, the concentration of the chitosan solution is 1 mg / ml.

[0036] According to one embodiment of the present invention, the molecular weight of chitosan may be 30 to 40 kDa.

[0037] Preferably, the double-stranded RNA is diluted with a distilled aqueous solution.

[0038] The two solutions are preferably mixed in a 1:1 volume ratio.

[0039] After mixing, the mixture is stirred at a constant speed for about 15 minutes, and then left at room temperature to allow for the spontaneous formation of nanoparticles (NPs).

[0040] The formed chitosan nanoparticles are preferably stored at approximately -25°C to approximately -15°C.

[0041] For the purposes of this invention, the chitosan nanoparticles are characterized by having a size in the range of 130 nm to 160 nm.

[0042] As mentioned above, chitosan nanoparticles contain double-stranded RNA, including mature tumor-suppressing miRNAs.

[0043] According to a preferred embodiment, the double-stranded RNA comprises the mature tumor-suppressive miR-126-3p sequence disclosed above.

[0044] According to another embodiment of the present invention, the nanocarrier system is represented by lipid-based vesicles represented by liposomes (LPNs).

[0045] Lipid-based vesicles can be incorporated into multiple lipid compositions that can either contain the mature tumor-suppressing miR-126-3p sequence disclosed above, or complex with nucleic acids such as the mature tumor-suppressing miR-126-3p sequence disclosed above.

[0046] According to one embodiment of the present invention, a disclosed nanocarrier system having double-stranded RNA containing mature tumor-suppressing miR-126-3p targets tumor cells.

[0047] In one embodiment, the tumor cells are selected from melanoma, ovarian cancer, and lung cancer cells.

[0048] In a preferred embodiment, the tumor cells are metastatic melanoma cells.

[0049] In particular, the nanocarrier system of the present invention can be appropriately targeted by conjugation with the targeting portion.

[0050] More specifically, the targeted portion is covalently bonded to the nanocarrier system.

[0051] In the object of the present invention, the targeted portion targets the tumor marker CSPG4 (Ab-CS126).

[0052] In a preferred embodiment, the targeted portion is represented by an antibody or an antibody fragment.

[0053] For the purposes of the present invention, the antibody may be selected from the group including human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, and multispecific antibodies.

[0054] For the purposes of the present invention, antibody fragments may be selected from the group comprising Fv, Fab, F(ab')2, Fab', dsFv, scFv, or sc(Fv)2.

[0055] In a preferred embodiment, the targeted portion is represented by scFv.

[0056] In a more preferred embodiment, the targeted portion is represented by scFv-9.2.27.

[0057] In one embodiment of the present invention, the disclosed scFv-9.2.27 comprises the amino acid VH sequence of SEQ ID NO: 3 (see table below).

[0058] In one embodiment of the present invention, the disclosed scFv-9.2.27 comprises a VH sequence encoded by the nucleotide sequence of SEQ ID NO: 2 (see table below).

[0059] In one embodiment of the present invention, the disclosed scFv-9.2.27 contains the amino acid VL sequence of SEQ ID NO: 5 (see table below).

[0060] In one embodiment of the present invention, the disclosed scFv-9.2.27 includes a VL sequence encoded by the nucleotide sequence of SEQ ID NO: 4 (see the table below). [Table 3]

[0061] The conjugation of the nanocarrier system can be carried out according to methods known in the art.

[0062] For example, the conjugation of chitosan nanoparticles can be carried out according to methods known in the art, such as using EDC chemistry.

[0063] According to a third object, the present invention discloses the double-stranded RNA, the mature tumor-suppressing miR-126-3p sequence, and the carrier system of the present invention for medical use.

[0064] In particular, the aforementioned medical uses are disclosed with respect to the mounted chitosan nanoparticles disclosed above and with respect to the mounted and targeted chitosan nanoparticles.

[0065] In one embodiment, the double-stranded RNA of the present invention, the mature tumor-suppressing miR-126-3p sequence of the present invention, the nanocarrier system of the present invention, the mounted nanocarrier system, the mounted and targeted nanocarrier system, and in particular the chitosan nanoparticles and mounted and targeted chitosan nanoparticles disclosed above, are used to treat tumors expressing the tumor marker CSPG4.

[0066] In a preferred embodiment, the medical use of the present invention is used in the treatment of tumors, including melanoma, ovarian cancer, and lung cancer.

[0067] In a preferred embodiment, the double-stranded RNA of the present invention, the mature tumor-suppressing miR-126-3p sequence of the present invention, the nanocarrier system of the present invention, the mounted nanocarrier system, the mounted and targeted nanocarrier system, and in particular the chitosan nanoparticles and mounted and targeted chitosan nanoparticles disclosed above are disclosed for medical use in the treatment of melanoma.

[0068] In a more preferred embodiment, the chitosan nanoparticles of the present invention are disclosed for medical use in the treatment of metastatic melanoma resistant to targeted therapy (precision therapy).

[0069] The terms "targeted therapy" or "precision therapy" refer to a specific target, namely BRAFV. 600E This refers to treatments that target [a specific target].

[0070] In particular, the nanocarrier system of the present invention, especially the chitosan nanoparticles, is disclosed for medical use in the treatment of metastatic melanoma resistant to treatment with vemurafenib or dabrafenib.

[0071] In certain embodiments, the nanocarrier system of the present invention, particularly the chitosan nanoparticles, is disclosed for medical use in combination with a PI3K / AKT inhibitor in the treatment of metastatic melanoma.

[0072] In a preferred embodiment, such a PI3K / AKT inhibitor is represented as PIK-75.

[0073] The hydrochloride salt (CAS 372196-77-5) is shown below. [ka]

[0074] The present invention is further disclosed in the following experimental section.

[0075] Chemical modification of miR126-3p and its stability in human plasma All pyrimidines in the sense strand and one uridine in the antisense strand of miR126 in the present invention are modified by adding a methyl group (CH3) to the 2'-O position of the pentose (2'-OMe). Furthermore, to protect this molecule from nuclease cleavage, a 2'-deoxythymidine nucleotide overhang is added to each 3' end (OMe-miR126) (Figure 1A). The negative control sequence (OMe-CTRL) is similarly modified (not shown).

[0076] To verify the functional efficiency of the OMe-miR126 sequence, we evaluated its ability to inhibit the expression of the PI3KR2(p85β) subunit of PI3K, a key molecule in the PI3K / AKT signaling pathway. PI3KR2 has been reported as a direct target of miR126-3p (Guo C, 2008; Felli N, 2013). Specifically, OMe-miR126 or a relative negative control was introduced into the A375M-VR cell line, and commercially available miR126 sequences, Dharm-miR126-3p(Invitrogen) and Dharm-CTRL, were used as controls (Felli N, 2013). Immunoblot analysis showed comparable reductions in p85β levels (50-40%) (Figure 1B, left panel). Time-course evaluation showed that the inhibitory effect of OMe-miR126 remained very strong even 72 hours after transfection (Figure 1B, right panel).

[0077] Another important issue to consider before selecting sequences for in vivo experiments was the stability of RNA molecules in plasma. Therefore, the sequences of OMe-miR126 and Dharm-miR126 were compared in PBS containing 50% human plasma at 37°C. The results showed that the degradation of OMe-miR126 was slower than that of Dharm-miR126. Specifically, the time course shown in Figure 1C shows that starting with the same amounts of OMe-miR126 and Dharm-miR126 oligos, after 6 hours of incubation, neither decreased significantly, while the other decreased to one-third of its original volume. Furthermore, Dharm-miR126 was completely degraded after 24 hours, while OMe-miR126 remained clearly visible (approximately 80% of the initial amount). To verify the lifetime of OMe-miR126, the incubation time was extended to 48 hours to confirm its complete disappearance (not shown). Based on evidence of efficacy regarding the suppression of target gene expression and stability in plasma, further experiments have focused on OMe-miR126. Transient transfection of A375M-VR with this oligonucleotide revealed a significant inhibition of cell proliferation after OMe-miR126 overexpression (Figure 1D).

[0078] Previous results have shown that the chemical modification introduced into the miR126-3p standard sequence to obtain OMe-miR126 does not alter its functionality and significantly improves its stability in the presence of plasma enzymes.

[0079] OMe-miR126 sequence and BRAF inhibitor combination The cell proliferation inhibitory effect of the OMe-miR126 sequence was evaluated in metastatic melanoma cell line (A375M) and its derivative, the dabrafenib-resistant sub-cell (A375M-DR). These cell lines were generated by exposing parental cells to gradually increasing concentrations of the drug.

[0080] As shown in Figure 2A, ICs of vemurafenib (704.7 nM), dabrafenib (41 nM), and PIK-75 (73.5 nM) 50 The values ​​were halved when treated in combination with OMe-miR126 (329.6 nM, 22.3 nM, and 40.3 nM, respectively). Dharm-miR126 was used as a positive control because it reproduces sequences in which a synergistic effect has already been demonstrated (Pedini F, 2019). In addition, in combination with other drugs (vemurafenib + PIK-75 or dabrafenib + PIK-75), IC was also obtained with OMe-miR126. 50 The value decreased (see upper panel of Figure 2A).

[0081] The purpose of this study was to evaluate therapeutic strategies for metastatic melanoma resistant to standard therapy using BRAF inhibitors. Therefore, we evaluated the inhibitory effect of OMe-miR126 on A375M-DR and its potential synergistic effect with PIK-75. As shown in Figure 2B, the IC of PIK-75 50The value decreased from 153.3 nM to 120.5 nM. To calculate potential synergistic effects, the Excess Over Bliss (EOB) score (Liu Q et al. 2018) was used. This parameter is used to explain the phenomenon where the combined effect of two or more drugs is greater than the sum of the individual effects (EOB>0: synergistic effect, EOB=0: independent effect, EOB<0: antagonistic effect). The obtained values ​​suggested that OMe-miR126 showed synergistic effects with a single drug (vemurafenib, dabrafenib, or PIK-75) or in combination thereof (vemurafenib + PIK-75, or dabrafenib + PIK-75) (Figure 3).

[0082] The same experimental design was replicated using a different melanoma cell line, SKMEL28, and its dabrafenib-resistant strain, SKMEL28-DR, yielding similar results (data not shown).

[0083] The results described demonstrate that OMe-miR126 exhibits synergistic effects with BRAF inhibitors and PI3K inhibitors, and that this combination inhibits the proliferation of susceptible and resistant metastatic melanoma cell lines.

[0084] Synthesis and analysis of single-chain variable fragment 9.2.27 (scFv-9.2.27) Chitosan nanoparticles have been used for the direct delivery of OMe-miR126 into metastatic melanoma cells. In particular, chitosan-based nanoparticles (CS) are conjugated with scFv designed to have variable heavy chain (VH) and variable light chain (VL) of mouse mAb 9.2.27, which is specific to the melanoma marker CSPG4. scFv-9.2.27 was produced and purified according to previously published data (Flego et al., 2021; Ascione et al. 2019). Purified scFv-9.2.27 was detected by Western blotting with HRP-conjugated anti-6HismAb (Figure 4A). To evaluate the binding ability of scFv-9.2.27 to its target antigen, flow cytometry-based immunofluorescence assays were performed using A375M melanoma cell lines and 293FT cell lines expressing high and low levels of the surface antigen CSPG4, respectively. The results showed that this antibody can selectively interact with the CSPG4 membrane receptor (Figure 4B). To evaluate the direct toxic effects of the scFv-9.2.27 protein, A375M-DR was treated in vitro while increasing the amount of this soluble protein. As shown in Figure 4C, no toxic effects were observed up to 1 μg / ml.

[0085] The data above demonstrates that scFv-9.2.27 can effectively and specifically recognize the melanoma-specific marker CSPG4. Furthermore, it did not affect the proliferation of A375M-DR at concentrations up to 1 μg / ml.

[0086] Synthesis and characterization of nanoparticles Synthesis of antibody-conjugated nanoparticles to direct OMe-miR126 delivery The preparation of CS, the capture of OMe-miR126 within CS to obtain CS@miR126s, and the final conjugation of scFv-9.2.27@CSs@126s complex (hereinafter abbreviated as Ab-CS126s) were carried out as reported in Figure 5A (left panel). Briefly, chitosan nanoparticles were prepared by a polymer-electrolyte complexing method by mixing two polymer solutions with opposite charges in a 1:1 (v / v) ratio. The net charge and size of the polyplex were mainly determined by the N / P ratio, and by increasing the N / P ratio from 5 to 10 (at a fixed degree of CS deacetylation), with lower molecular weights required to achieve efficient stabilization of the complex. A 1 mg / mL chitosan solution was prepared by dissolving the polymer in a 0.1 M aqueous acetic acid solution (pH=4). Next, 80 μg of oligonucleotide was diluted with an appropriate amount of sterile redistilled water to achieve an N / P ratio of 10. The nucleic acid solution was added dropwise to 500 μL of chitosan solution while magnetically agitated. The mixture was stirred at a constant speed for 15 minutes, left at room temperature for 30 minutes to allow for the spontaneous formation of nanoparticles, and stored at 4°C for 24 hours before use.

[0087] Capture efficiency of mounted nucleic acids The amount of unbound nucleic acids was measured using a UV-Vis spectrophotometer. The sample was centrifuged at 14,000 rpm at 4°C for 30 minutes to promote polyplex precipitation. The supernatant was analyzed using a spectrophotometer. A capture efficiency of 70% ± 9.3 was obtained according to the following formula. [ka]

[0088] The EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) / NHS (N-hydroxysuccinimide) reaction was used to conjugate chitosan nanoparticles and antibody fragments. 500 μg of chitosan nanoparticles were precipitated by centrifugation (14000 rpm, 30 minutes, 4°C). The pellet was suspended in 500 μl of PBS (pH=7.4). Subsequently, 50 μg of antibody was diluted with an equal volume of MES buffer (pH=6). EDC and NHS were added to the reaction mixture in a molar ratio of 2:5 and stirred with a magnetic stirrer for 15 minutes. After adjusting the pH to 7, the NP solution was added, and the reaction mixture was incubated at room temperature for 2 hours with continuous stirring with a magnetic stirrer. The solution was transferred to a dialysis membrane and then to a beaker containing sterile distilled water. Dialysis was performed for 5 days. The amount of bound antibody was evaluated using a spectrophotometer. The antibody / CS weight ratio was 1:10, and the total amount of bound antibody exceeded 80% (Figure 5A, right panel).

[0089] Characterization of nanoparticles First, the dimensional distribution and polydispersity index (PdI) of CS126s and Ab-CS126 were measured by DLS (Figure 5B). Due to the small size of the antibodies used, slight differences in dimensional values ​​were observed. These are actually protein fragments with a molecular weight of approximately 30 kDa and relatively low steric envelope. The average PdI of all nanoparticle samples was 0.2. A low PdI indicates good system quality.

[0090] Chitosan nanoparticles generally possess a positive surface charge due to the presence of amino groups that become positively charged at acidic pH. Since cell membranes are negatively charged, this positive charge is optimal for the internalization of nanovectors within cells (Dowaidar M, 2018; Faust JJ, 2014). As shown in Figure 5B, the average zeta potential was +37.8±4.3mV for CS126s and +33.3±1.2mV for Ab-CS126s. The size distributions of CS-126s and Ab-CS126s were characterized by NanoSight analysis, and DLS data was confirmed. The average concentrations of the CS-126s and Ab-CS126s preparations were 2.2 x 10⁻¹⁰, respectively. 11particles / ml and 1.7x10 10 It was revealed that the number of particles per ml was [number] (Figure 5C, data not shown).

[0091] The experimental data described above demonstrates that the conditions used for nanoparticle synthesis efficiently capture OMe-miR126 molecules, enabling the expression of antibody molecules useful for targeting melanoma cells on their surface.

[0092] Physicochemical characterization of CS126 and Ab-CS126 nanoparticles The morphology of chitosan nanoparticles was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). From SEM / TEM analysis, the polyplex appeared as highly aggregated spherical nanostructures (Figure 6A). Nanoparticles after conjugation with antibody fragments exhibit greater dispersibility in an aqueous environment and show spherical shape. Since SEM / TEM analysis is performed on dry samples, the micrographs usually show nanoparticles of smaller dimensions than those obtained from analysis in aqueous solution. Under these conditions, the nanoparticles are strongly hydrated, so the dimensions detected by DLS are larger. To improve the characterization of the nanocarrier of this application, the amount of scFv-9.2.27 bound to Ab-CS126 was evaluated by WES analysis (Figure 6B). Direct comparison with a positive control (soluble scFv-9.2.27) revealed that 250 pg of scFv-9.2.27 / 6.8X10 7 It is possible to estimate the amount of Ab-CS126. As the final step before conducting functional experiments, the stability of CS126 in human plasma was evaluated. Figure 6C shows that CS nanoparticles extended the stability of OMe-miR126 after 48 hours of incubation, at which point complete disappearance of the sequence was observed as shown by gel electrophoresis.

[0093] The reported data demonstrate that both CS-126 and Ab-CS126 possess spherical morphological characteristics. scFv-9.2.27 is conjugated to Ab-CS126, and the captured OMe-miR126 exhibits improved stability in 50% serum medium compared to the free oligo.

[0094] In Vitro Internalization of CS126 and Ab-CS126 To evaluate the internalization efficiency of CS126 and Ab-CS126, the melanoma cell line A375M-DR was incubated while increasing the amount of particles conjugated with FITC molecules. As shown in Figure 7A (left panel), immunofluorescence microscopy showed significant internalization of the FITC signal in both CS126 and Ab-CS126 cell incubations. However, CS126 formed free aggregates and showed a dispersed distribution stained at the cell periphery, while AbCS126 diffusely stained the cells, suggesting internalization of the complex. In flow cytometry analysis, after incubating 1.6×10 8 particles for 20 minutes, approximately 100% of the cells were FITC positive (Figure 7A, right panel). To actually evaluate the intracellular translocation ability of OMe-miR-126, the amount of OMe-miR-126 captured in CS126 and Ab-CS126 was measured by qRT-PCR, and it was found to be 1.2x10 -8 and 1.7x10 -10 pMole / nanoparticle, respectively (Figure 7B). Next, the amount of OMe-miR-126 after incubating the same number of CS126 and Ab-CS126 nanoparticles with A375M-DR was evaluated by qRT-PCR, and an increase of approximately 56-fold and approximately 7.3-fold was observed for CS126 and Ab-CS126, respectively (Figure 7C). Considering that the amount of miR126 molecules captured in CS126 is different from that in Ab-CS126, it can be concluded that Ab-CS126 shows a 9.5-fold uptake efficiency compared to CS126 in the receptor A375M-DR melanoma (Figure 7D). The same experiment was also conducted on the SKMEL28 cell line (data not shown).

[0095] The above data indicate that both CS126 nanoparticles and Ab-CS126 nanoparticles improve the internalization efficiency.

[0096] Stability Evaluation of CSFITC and Ab-CSFITC: In Vitro and In Vivo Experiments The in vivo stability dynamics of CSFITC and Ab-CSFITC were evaluated by analyzing mouse plasma collected at different time points after injection of fluorescent particles. The results showed that in both CSFITC and Ab-CSFITC, the fluorescent particles rapidly disappeared after 60 minutes, becoming below the detection limit (Figure 8A, top panel). However, in in vitro experiments, both CSFITC and Ab-CSFITC maintained fluorescence up to 6 days of incubation in plasma, with Ab-CSFITC being more stable (Figure 8A, bottom panel). This suggests that the in vivo disappearance of fluorescence is due to particle distribution / removal, not degradation. To clarify this important aspect, major organs of mice injected with CSFITC and Ab-CSFITC were analyzed by immunofluorescence microscopy. As shown in Figure 8B, strong FITC signals were observed in the lungs, liver, kidneys, and spleen. As a final step before initiating in vivo treatment, the potential toxic effects of CS126 and Ab-CS126 were evaluated after repeated administration to mice for 3 weeks. Since the weight and health status of the administered mice remained within the normal range, no toxic effects were observed (data not shown).

[0097] The data above ultimately suggest that when CSFITC and Ab-CSFITC are administered in vivo, they are not degraded by plasma enzymes and are distributed to animal organs such as the liver and lungs, which are extremely important as frequent sites of melanoma metastasis.

[0098] In vivo experiment Since metastasis cannot be induced by intravenous injection from melanoma cells resistant to dabrafenib treatment (A375M-DR), a reliable animal model was developed that allows for the measurement of metastatic tumor spread over time. This model involved administering 5x10⁶ dabrafenib dissolved in 200 μL of PBS to the spleen of 5-week-old female severely immunodeficient NSG mice. 4These cells were obtained by injecting A375M-DR-LucGFP cells at a dose of one cell (Figure 9A). To avoid dominant tumor growth at the injection site, the spleen was removed 30 minutes later. Metastatic progression was monitored weekly by bioluminescence imaging of inoculated cells using the IVIS optical imaging system. Approximately two weeks after inoculation, when the signal was barely detectable, the mice were randomly assigned to one of six treatment groups. 1) Control group (9 mice), 2) PIK-75 (6 mice), 3) Ab-CS126 (6 mice), 4) PIK-75 + CS126 (4 mice), 5) PIK-75 + Ab-CS(-) (5 mice), 6) PIK-75 + Ab-CS126 (8 mice). All treatment groups received dabrafenib intraperitoneally three times a week, PIK-75 daily, and Ab-CS126 intravenously three times a week.

[0099] Statistical analysis comparing the slopes of tumor growth curves in each group showed significant suppression in the PIK-75 + Ab-CS126 group compared to the other groups (Figure 9B). After euthanasia, organ analysis confirmed a decrease in tumor growth in the liver and a very significant reduction in lung metastasis formation. Figure 9C shows representative images from each group.

[0100] Overall, these findings suggest that the combination of PI3K / AKT inhibitors and Ab-CS126 may be a promising treatment option for patients who have shown resistance to conventional therapy.

[0101] Detection of apoptosis in cryopreserved mouse liver and lung tissue sections To evaluate the efficacy of the PIK-75+Ab-CS126 combination, terminal deoxyribonucleotide transferase (dUTP) nick-end labeling (TUNEL) assays were performed on liver and lung sections of NSG-treated mice, and the results were compared with those of the control group. The control group consisted of tissues taken from untreated healthy animals and mice that received PIK-75+Ab-CS126 combination therapy but were not injected with A375M-DR cells. As shown in Figure 10, strong positive signals were observed in both liver and lung tissue sections of NSG-treated mice, while no apoptotic signals were observed in the control group. Interestingly, although the lungs of PIK-75+Ab-CS126-treated mice did not show visible metastasis in the in vivo Imaging System (IVIS) (Figure 9C, right panel), TUNEL staining, which suggests apoptosis, was strongly positive. This suggests that most of the cells infiltrating the lungs died after PIK-75+Ab-CS126 administration. To confirm the actual presence of nanoparticles in mouse tissue, staining was performed using an antibody that can recognize scFv-9.2.27. As shown in Figure 10B, kidney sections from mice administered with anti-scFv-9.2.27 were stained positively.

[0102] These results strongly support the idea that treatment with modified miR126 encapsulated in CS nanoparticles carrying an anti-CPSG4 antibody leads to cell death of metastatic melanoma cells in both the liver and lungs.

[0103] Application to tumors expressing CSPG4 Preliminary data (see Figure 11) show elevated expression levels of the membrane marker CSPG4 in ovarian (A2780 and SK-OV3) and lung (H1975 and HCC827) tumor cell lines. Furthermore, introduction of the OMe-miR126 sequence into the SK-OV3 cell line via lipofectamine demonstrated a synergistic effect between this sequence and PI3K / AKT pathway inhibitors (PIK-75 and BKM120), which is consistent with previous observations in melanoma (see Figure 12).

[0104] Liposomes as a Carrier System LPNs in the 100-150 nm range can be prepared by a microfluidic reactor in a multiple lipidic composition having multiple lipid components that can be complexed with nucleic acids to provide a hydrated layer that improves colloidal stability and protein adsorption. Available techniques enable the incorporation of OMe-miR126 into appropriately designed liposome particles, including the use of the miRNA (OMe-miR126) of the present invention in the therapy of CPSG4-expressing cancers, and the functionalization of the liposome particles with an anti-CSPG4scFv antibody.

[0105] reference TIFF2026509509000006.tif251164 TIFF2026509509000007.tif248164 TIFF2026509509000008.tif163164

Claims

1. A double-stranded RNA comprising a mature tumor suppressor miRNA sequence represented by miR-126-3p having a sense strand sequence corresponding to SEQ ID NO: 6, and an antisense strand sequence corresponding to SEQ ID NO: 7, Table 1 Modified double-stranded RNA in which all pyrimidine residues in the sense strand and the fourth uridine residue in the antisense strand are modified by 2'-O-pentose O-methylation (2'-OMe).

2. The double-stranded RNA according to claim 1, wherein the sense strand and the antisense strand each contain two 2'-deoxythymidine residues at their respective 3' ends.

3. The double-stranded RNA according to claim 1 or 2, delivered via a suitable nanocarrier system.

4. The double-stranded RNA according to claim 3, wherein the nanocarrier system is represented by chitosan nanoparticles or liposomes.

5. The double-stranded RNA according to claim 3 or 4, wherein the nanocarrier system is equipped with the double-stranded RNA.

6. The double-stranded RNA according to any one of claims 3 to 5, wherein the nanocarrier system is linked to the targeting portion.

7. The double-stranded RNA according to any one of claims 3 to 5, wherein the nanocarrier system is covalently bonded to the targeting portion.

8. The double-stranded RNA according to claim 6 or 7, wherein the targeting portion is represented by an antibody or an antibody fragment.

9. The double-stranded RNA according to claim 8, wherein the antibody is selected from the group including human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, or multispecific antibodies.

10. The double-stranded RNA according to claim 8 or 9, wherein the antibody fragment is selected from the group comprising Fv, Fab, F(ab')2, Fab', dsFv, scFv, or sc(Fv)2.

11. The double-stranded RNA according to any one of claims 8 to 10, wherein the targeting portion is represented by scFv.

12. The double-stranded RNA according to any one of claims 6 to 11, wherein the targeted portion is represented by scFv-9.2.27 having the amino acid VH sequence of SEQ ID NO: 3 and the amino acid VL sequence of SEQ ID NO:

5.

13. A double-stranded RNA according to any one of claims 1 to 12, for medical use.

14. Double-stranded RNA for medical use according to claim 13 in the treatment of tumors expressing the tumor marker CSPG4.

15. The double-stranded RNA for medical use according to claim 14, wherein the tumor expressing the tumor marker CSPG4 includes melanoma, ovarian cancer, and lung cancer.

16. The double-stranded RNA according to claim 15 for medical use in the treatment of melanoma resistant to targeted therapy.

17. The double-stranded RNA for medical use according to claim 15 or 16, wherein the melanoma is a melanoma resistant to treatment with vemurafenib or dabrafenib.

18. Double-stranded RNA for medical use according to any one of claims 14 to 17, for medical use in the treatment of tumors in combination with a PI3K / AKT inhibitor.

19. Double-stranded RNA for medical use according to claim 18, for medical use in the treatment of metastatic melanoma in combination with a PI3K / AKT inhibitor.

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