An nqo1-responsive mrna translation masker and uses, products, and methods thereof

Abstract

This invention belongs to the field of mRNA drugs, specifically relating to an NQO1-responsive mRNA translation masking agent and its applications, products, and methods. The molecular formula of the mRNA translation masking agent is C0. 21 H 28 The mRNA translation masking agent described above (N4O4) can covalently bind to mRNA via acylation, forming steric hindrance to inhibit translation, and efficiently restore translational ability in an NQO1 high-expression environment. This not only prolongs the intracellular half-life of mRNA but also increases the cumulative yield of the target protein. The mRNA translation masking agent and its masking method provided by this invention can be used for tumor-targeted therapy, achieving not only specific clearance of tumor cells but also reducing hepatotoxicity. The technical solution of this invention contributes to the development of safe, convenient, and responsive tumor-targeted therapeutic mRNA drugs.

Description

Technical Field

[0001] This invention belongs to the field of mRNA drugs, specifically relating to an NQO1-responsive mRNA translation masking agent and its applications, products, and methods. Background Technology

[0002] Cancer is a major disease threatening human life and health. Although targeted therapy and immunotherapy have made significant progress in recent years, chemotherapy remains one of the most important treatment methods. However, traditional chemotherapy drugs suffer from poor water solubility, low tumor specificity, and short blood circulation time, leading to significant toxic side effects, high dosages, and long treatment cycles. In recent years, cell-selective technology based on mRNA translation regulation has become a core challenge in achieving targeted cancer therapy in the biomedical field.

[0003] Currently developed innovative strategies exhibit different technical paths and limitations: In Cell access through RNA sensing by Endogenous ADAR (CellREADR) technology, a sequence containing a UAG stop codon is designed upstream of the region of mRNA encoding the target protein. Tumor-specifically highly expressed RNA can hybridize with this sequence and activate ADAR-mediated A-to-I base editing, enabling the AUG codon to be recognized as the AGG codon, thereby translating the target protein. Similar to the CellREADR strategy, GEMS (genetically encoded m6A sensor) technology can achieve m6A modification-dependent protein translation. After expressing a fusion protein of the m6A-binding protein YTH and the C-to-U base editing enzyme APOBEC1 in cells, the CGA codon downstream of the m6A site of the mRNA is edited into a UGA stop codon, preventing the translation of downstream degradation codons of the target protein. Thus, the target protein can only be expressed in cells with high levels of dynamic m6A modification. Another strategy relies on abnormal pre-mRNA splicing events in tumor cells. By designing synthetic introns that can only be spliced ​​in specific tumor cells, mRNA drugs can be prevented from undergoing intron excision in normal cells, thus preventing the expression of therapeutic proteins. However, not all tumors allow for the design of sufficiently specific A-to-I base editing elements or the creation of synthetic introns; m6A modification also exists in non-tumor cells. Therefore, leaky expression in normal cells is common, limiting the wider application of these strategies. Furthermore, small molecule-mediated selective splicing strategies can activate mRNA protein expression under the influence of specific small molecules. However, this strategy relies on exogenous small molecules as regulatory switches and cannot achieve tumor selectivity.

[0004] Given that strategies such as CellREADR and GEMS either rely on the presence of tumor-specific RNA sequences or are limited by the widespread distribution of modification markers in normal tissues, leading to a significant risk of leakage expression in normal cells; and that aberrant splicing strategies are difficult to apply broadly due to the heterogeneity of splicing factor expression in tumors. Therefore, there is an urgent need to develop new tumor-specific mRNA translation control technologies. Summary of the Invention

[0005] To address the aforementioned shortcomings, this invention provides an NQO1-responsive reversible masking agent for mRNA translation, CR-3. Using CR-3, in vitro transcribed mRNA can be blocked through a simple and efficient process. This blocked mRNA cannot translate into protein on its own, but it can efficiently restore its translational ability under NQO1-mediated regulation, and its intracellular half-life is significantly increased.

[0006] The technical solution of this invention is as follows:

[0007] On one hand, this invention provides an NQO1-responsive mRNA translation masking agent, named CR-3; the molecular formula of CR-3 is C 21 H 28 N4O4, structural formula as follows:

[0008] .

[0009] Specifically, the general route of the synthesis method is as follows:

[0010] ;

[0011] The overall route is as follows:

[0012] S1, compound 1 and compound 1-A undergo a substitution reaction under protic acid conditions to generate compound 2;

[0013] S2 and compound 2 react with the halogenating agent in a polar solvent to form compound 3;

[0014] S3, compound 3 and compound 3-B undergo a condensation reaction in an organic solvent under the catalysis of a condensing agent to generate compound 4;

[0015] S4 and compound 4 undergo a deprotection reaction under acidic conditions to generate compound 5;

[0016] S5 and compound 5 react with a carbonylating agent in the presence of a base to undergo a carbonylation reaction to generate compound 6;

[0017] S6, compound 6, and a nitrogen-containing heterocyclic compound undergo a substitution reaction under inert gas protection to generate compound CR-3.

[0018] Specifically, the protic acid mentioned in step S1 can be methanesulfonic acid, sulfuric acid, or p-toluenesulfonic acid;

[0019] The halogenating agent in step S2 can be N-bromosuccinimide or bromine, and the polar solvent is a mixture of acetonitrile and water.

[0020] The condensing agent in step S3 can be DCC / DMAP or EDC / HOBt, and the organic solvent is tetrahydrofuran or dichloromethane (DCM).

[0021] The acidic condition described in step S4 can be a 3-4 M HCl / EtOAc solution;

[0022] The carbonylating agent in step S5 can be triphosgene or phosgene, and the base can be triethylamine or diisopropylethylamine;

[0023] The nitrogen-containing heterocyclic compound in step S6 can be an imidazole or a 1,2,4-triazole, and the inert gas can be nitrogen or argon.

[0024] More specifically, step S6 may also include the addition of triethylamine and DMAP.

[0025] Preferably, the protic acid in step S1 can be methanesulfonic acid;

[0026] The halogenating agent mentioned in step S2 may be N-bromosuccinimide (NBS);

[0027] The condensing agent in step S3 can be DCC / DMAP, and the organic solvent is tetrahydrofuran (THF).

[0028] The acidic condition described in step S4 can be a 4 M HCl / EtOAc solution;

[0029] The carbonylating agent in step S5 can be triphosgene, and the base can be triethylamine;

[0030] The nitrogen-containing heterocyclic compound in step S6 can be an imidazole, and the inert gas can be nitrogen.

[0031] Preferably, in step S1, the equivalent ratio of compound 1-A to compound 1 is 0.8-1.2;

[0032] In step S2, compared to compound 2, the equivalent ratio of N-bromosuccinimide is 0.9-1.1, the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 5:1, and the volume ratio is 10-15.

[0033] In step S3, compared to compound 3, the equivalence ratio of compound 3-B is 0.9-1.1, the equivalence ratio of DCC is 1-1.2, the equivalence ratio of DMAP is 0.9-1.1, and the volume ratio of tetrahydrofuran is 8-12.

[0034] In step S4, the volume ratio of HCl / EtOAc solution added is 8-12 compared to compound 4;

[0035] In step S5, compared to compound 5, the equivalence ratio of triphosgene is 0.2-0.6, the equivalence ratio of triethylamine is 1.8-2.2, and the volume ratio of dichloromethane is 8-12.

[0036] In step S6, compared to compound 6, the equivalent ratio of imidazole is 1-2, the equivalent ratio of triethylamine is 1-1.5, and the equivalent ratio of DMAP is 0.8-1.2; step S6 also includes THF, with an input volume ratio of 8-12.

[0037] Preferably, the conditions for the substitution reaction in step S1 are: 70-80℃ for 8-14 hours; the conditions for the halogenation reaction in step S2 are: 15-25℃ for 3-5 hours; the conditions for the condensation reaction in step S3 are: 15-25℃ for 8-14 hours; the conditions for the deprotection reaction in step S4 are: 15-25℃ for 1-3 hours; the conditions for the carbonylation reaction in step S5 are: 0-25℃ for 8-14 hours; and the conditions for the substitution reaction in step S6 are: 15-25℃ for 8-14 hours.

[0038] Preferably, the conditions for the substitution reaction in step S1 are: 70-80℃ for 12 hours; the conditions for the halogenation reaction in step S2 are: 15-25℃ for 4 hours; the conditions for the condensation reaction in step S3 are: 15-25℃ for 12 hours; the conditions for the deprotection reaction in step S4 are: 15-25℃ for 2 hours; the conditions for the carbonylation reaction in step S5 are: 0-25℃ for 12 hours; and the conditions for the substitution reaction in step S6 are: 15-25℃ for 12 hours.

[0039] In another aspect, the present invention provides a method for mRNA translation masking, wherein the aforementioned mRNA translation masking agent CR-3 is reacted with mRNA under the catalysis of an organic base to achieve mRNA translation masking.

[0040] Specifically, the concentration of the mRNA translation masking agent CR-3 is 0.1-1 M.

[0041] Preferably, the concentration of the mRNA translation masking agent is 0.1 M, 0.5 M, or 1 M.

[0042] Specifically, the organic base is DMAP or DBU.

[0043] Preferably, the organic base is DMAP.

[0044] Specifically, the reaction conditions are: 25-37°C for 0.5-2 hours.

[0045] Preferably, the reaction is carried out at 37°C for 2 hours with 1 M CR-3 reagent; at 30°C for 1 hour with 0.5 M CR-3 reagent; and at 25°C for 0.5 hours with 0.1 M CR-3 reagent.

[0046] Preferably, the sequence of the mRNA is as shown in SEQ ID NO.1-SEQ ID NO.4.

[0047] In another aspect, the present invention provides a drug comprising the aforementioned mRNA translation masking agent.

[0048] Specifically, the drug also includes a pharmaceutically acceptable carrier.

[0049] Preferably, the pharmaceutically acceptable carrier is selected from lipid nanoparticles, polymer nanoparticles, exosomes, inorganic nanoparticles, virus-like particles, or cell membrane-coated particles.

[0050] Specifically, the dosage form of the drug is selected from injections, lyophilized powder injections, nano-suspensions, oral dosage forms, topical drug delivery preparations, inhalants, or implants.

[0051] In another aspect, the present invention provides the application of the aforementioned mRNA translation masking agents, methods, or drugs in the preparation of antitumor products.

[0052] Specifically, the product in question is a medicine.

[0053] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0054] More preferably, the pharmaceutically acceptable carrier is selected from lipid nanoparticles, polymer nanoparticles, exosomes, inorganic nanoparticles, virus-like particles, or cell membrane-coated particles.

[0055] Preferably, the dosage form of the drug is selected from injections, lyophilized powder injections, nano-suspensions, oral dosage forms, topical drug delivery preparations, inhalants, or implants.

[0056] Specifically, the tumor is an NQO1-high expression type tumor.

[0057] Preferably, the tumor is a variety of solid tumors such as liver cancer, cervical cancer, lung cancer, and breast cancer.

[0058] The beneficial effects of this invention are as follows:

[0059] (1) This invention provides an NQO1-responsive mRNA translation masking agent and develops a universal RNA masking technology that is compatible with long-chain mRNA (>1000 nt) transcribed in vitro and short-chain mRNA (~20 nt) synthesized chemically.

[0060] (2) The RNA masking provided by the present invention can prolong the functional half-life of mRNA in cells and thus increase the expression level of protein encoded by mRNA.

[0061] (3) The technical solution provided by the present invention uses NQO1 as a tumor-specific target. This strategy can achieve selective activation of mRNA in various tumor types, avoid expression leakage in normal tissues, and provide a new path for broad-spectrum anti-tumor therapy.

[0062] (4) CR-3 is a trifunctional synergistic acylation masking agent: (a) the quinone propionate module acts as an NQO1-specific recognition element, achieving an enzyme-triggered site-specific response through two-electron reduction; (b) the dynamic self-eliminating linker endows the masking group with structural stability while ensuring efficient removal of the masking group under NQO1 enzyme induction; (c) the reaction-optimized leaving group enables rapid substitution reaction at the RNA 2'-OH site. Through one-step polyacylation modification, the masking agent CR-3 is covalently linked to the mRNA molecule, and the resulting steric hindrance significantly inhibits ribosome binding efficiency (in vitro translation inhibition rate >98%). At the same time, the masking group enhances the chemical stability of RNA by blocking the nucleophilic attack of the 2'-OH group on the adjacent 3'-phosphate group. When the mRNA is delivered to tumor cells, the NQO1 enzyme, which is highly expressed in the tumor cells, initiates a continuous reduction reaction of the quinone propionate module through redox catalysis, driving the dynamic linker to break and release the original mRNA, thereby achieving specific regulation of translation activity. Attached Figure Description

[0063] Figure 1 This is the nuclear magnetic resonance (NMR) spectrum of compound 2.

[0064] Figure 2 This is the nuclear magnetic resonance (NMR) spectrum of compound 3.

[0065] Figure 3 This is the nuclear magnetic resonance (NMR) image of compound 4.

[0066] Figure 4 This is the mass spectrum (MS) of crude compound 5.

[0067] Figure 5 This is the mass spectrum (MS) of crude compound 6.

[0068] Figure 6 This is the nuclear magnetic resonance (NMR) image of CR-3.

[0069] Figure 7 This is the mass spectrum (MS) of CR-3.

[0070] Figure 8 The in vitro translation recovery efficiency is calculated under different masking reaction conditions.

[0071] Figure 9 The cumulative yield of d2GFP fluorescent protein in real time and the total yield at the experimental endpoint are given.

[0072] Figure 10 To analyze the recovery translation efficiency of eGFP mRNA in cell lines with different NQO1 contents using fluorescence imaging.

[0073] Figure 11 To quantitatively analyze the recovery translation efficiency of eGFP mRNA in five cell lines 5 days after transfection using flow cytometry.

[0074] Figure 12 Photographs of mice and tumor locations to mask hBAX (left), untreated hBAX (middle), and blank treatment (right) groups (red / black arrows indicate SH-SY5Y and HepG2 xenografts, respectively).

[0075] Figure 13 To mask the relative HepG2 tumor clearance efficiency of treatment with hBAX and untreated hBAX.

[0076] Figure 14 Histological analysis diagrams of organs (heart, liver, spleen, lungs, and kidneys).

[0077] Figure 15 To assess the impact of serum ALT and AST levels on liver damage using ELISA analysis. Detailed Implementation

[0078] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0079] Example 1 Synthesis and Characterization of CR-3

[0080] 1.1 Overall Synthesis Route of CR-3

[0081]

[0082] 1.2 Synthesis steps and characterization of CR-3

[0083] (1) Synthesis of compound 2 (substitution reaction)

[0084]

[0085] A solution of compound 1 (10.0 g, 72.4 mmol) in MsOH (100 mL) was mixed with compound 1-A (9.92 g, 86.9 mmol). The mixture was stirred at 70–80 °C for 12 hours. Thin-layer chromatography (TLC) showed that reactant 1 was completely consumed and a new spot was formed (commercial hexane / ethyl acetate = 3:1, Rf = 0.45). The reaction mixture was quenched at 15–25 °C by adding H₂O (40.0 mL), diluted with EtOAc (40.0 mL), and extracted with EtOAc (40.0 mL × 2). The combined organic layers were washed with NaHCO₃ (40.0 mL), filtered, and concentrated under reduced pressure to give the residue. A crude product, compound 2 (10.0 g), was given as a brown solid.

[0086] 1 H NMR: (400 MHz, CDCl3)

[0087] δ 6.63 (s, 1H), 3.92 (s, 1H), 2.57 (s, 2H), 2.14 - 2.28 (m, 6H), 1.28- 1.32 (m, 6H).

[0088] nuclear magnetic resonance imaging Figure 1 According to the analysis of the nuclear magnetic resonance (NMR) results, it can be seen that the target product compound 2 has been successfully synthesized, and its structural characteristics are consistent with the expected design.

[0089] (2) Synthesis of compound 3 (halogenation reaction)

[0090]

[0091] N-bromosuccinimide (NBS, 8.49 g, 47.7 mmol) was added to a mixture of acetonitrile (ACN, 100 mL) and water (H2O, 20.0 mL) containing compound 2 (10.0 g, 45.4 mmol). The mixture was stirred at 15–25 °C for 4 hours. Thin-layer chromatography (TLC) showed that compound 2 was completely consumed and a new spot was formed (commercial hexane / ethyl acetate = 1:1, Rf = 0.20). The reaction mixture was quenched at 15–25 °C by adding water (H2O, 100 mL), diluted with ethyl acetate (EtOAc, 100 mL), and extracted with ethyl acetate (EtOAc, 100 mL × 2). The combined organic layers were washed with sodium bicarbonate (NaHCO3, 100 mL), filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, commercial hexane:ethyl acetate = 20 / 1 to 1 / 1) to give brown oily compound 3 (5.00 g, 16.1 mmol, 75.9% purity, 35.4% yield).

[0092] 1 H NMR: (400 MHz, CDCl3)

[0093] δ 6.53 (s, 1H), 2.94 (s, 2H), 2.76 (s, 3H), 2.00 (d, J = 2.13 Hz, 6H), 1.32 (s, 6H).

[0094] nuclear magnetic resonance imaging Figure 2 According to the analysis of the nuclear magnetic resonance (NMR) results, the target product compound 3 has been successfully synthesized, and its structural characteristics are consistent with the expected design.

[0095] (3) Synthesis of compound 4 (condensation reaction)

[0096]

[0097] A mixture of compound 3 (2.00 g, 8.46 mmol), compound 3-B (1.59 g, 8.46 mmol), DCC (1.92 g, 9.31 mmol), and DMAP (1.03 g, 8.46 mmol) in THF (1.00 mL) was stirred at 15–25 °C for 12 hours. Thin-layer chromatography (TLC) showed that reactant 3 was completely consumed and a new spot was formed (commercial hexane / ethyl acetate = 1:1, Rf = 0.42). The reaction mixture was filtered and concentrated under reduced pressure to give a residue. This residue was purified by column chromatography (SiO2, commercial hexane:ethyl acetate = 20 / 1 to 1 / 1) to give a brown, oily crude product, compound 4 (2.00 g).

[0098] 1 H NMR: (400 MHz, CDCl3)

[0099] δ 6.43 - 6.58 (m, 1H), 3.13 - 3.60 (m, 4H), 2.69 - 3.08 (m, 10H), 1.97 (s, 6H), 1.46 (d, J = 13.6 Hz, 9H), 1.26-1.31 (m, 6H).

[0100] nuclear magnetic resonance imaging Figure 3 According to the nuclear magnetic resonance (NMR) results, the target product compound 4 has been successfully synthesized, and its structural characteristics are consistent with the expected design.

[0101] (4) Synthesis of compound 5 (deprotection reaction)

[0102]

[0103] Compound 4 (2.00 g, crude) was dissolved in a mixture of HCl / EtOAc (4 M, 20.0 mL) and stirred at 15–25 °C for 2 hours. Thin-layer chromatography (TLC) showed that reactant 4 was completely consumed and a new spot was formed (commercial hexane / ethyl acetate = 1:1, Rf = 0.10). The reaction mixture was filtered and concentrated under reduced pressure to give the residue. Crude compound 5 (1.50 g) was given as a brown solid.

[0104] MS: m / z: 307. Mass spectrometry results are available in [link to mass spectrometry data]. Figure 4 According to the mass spectrometry (MS) results, the crude product contains compound 5, which is consistent with the expected design.

[0105] (5) Synthesis of compound 6 (carbonylation reaction)

[0106]

[0107] Triphosgene (1.16 g, 3.91 mmol) and triethylamine (Et3N, 990 mg, 9.79 mmol) were added to a solution of compound 5 (1.50 g, 4.90 mmol) in dichloromethane (DCM, 30.0 mL). The mixture was stirred at 0–25 °C for 12 hours. LC-MS analysis showed that the target product had been formed. The reaction mixture was concentrated under reduced pressure to obtain a residue, yielding crude compound 6 (1.80 g) as a brown solid.

[0108] MS: m / z: 370. (See mass spectrometry data...) Figure 5 According to the mass spectrometry (MS) results, the crude product contains compound 6, which is consistent with the expected design.

[0109] (6) Synthesis of CR-3 (substitution reaction)

[0110]

[0111] The mixture of compound 6 (1.80 g, 4.88 mmol), imidazole (498 mg, 7.32 mmol), triethylamine (Et3N, 493 mg, 4.88 mmol), and DMAP (596 mg, 4.88 mmol) in THF (18.0 mL) was degassed three times by nitrogen (N2) purging, followed by stirring at 15–25 °C for 12 h under N2 atmosphere. LC-MS analysis showed that the target product had been formed. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by column chromatography (SiO2, commercial n-hexane:ethyl acetate = 2 / 1 to 0 / 1) to give a brown oily product CR-3 (4-nitrophenyl 3-(4,5-dimethyl-3,6-dioxocyclohex-1,4-dien-1-yl)-3-methylbutyrate) (240 mg, 80.0% purity).

[0112] 1 H NMR: (400 MHz, CDCl3)

[0113] δ 7.87 (s, 1H), 7.69 (s, 3H), 7.20 (t, J = 1.31 Hz, 1H), 7.08 (s,1H), 3.59 (s, 3H), 3.07 (s, 2H), 3.03 (d, J = 5.13 Hz, 3H), 2.95 (s, 2H), 1.90-2.00 (m, 6H), 1.26 (d, J = 1.13 Hz, 6H).

[0114] LC-MS for C 21 H 28 N4O4, [M+H] + Theoretical value: 400.5, experimental value: 401.2.

[0115] nuclear magnetic resonance imaging Figure 6 Mass spectrometry Figure 7 Based on the analysis of the nuclear magnetic resonance (NMR) and mass spectrometry (MS) results, it can be seen that the target product CR-3 has been successfully synthesized, and its structural characteristics are consistent with the expected design.

[0116] Example 2: Masking RNA using CR-3

[0117] 2.1 Masking and Characterization of Short RNAs

[0118] For the masking reaction of short RNA (~20 nt), 5 µg of RNA (SEQ ID NO.1: 5'-AUCCUGCCGACUACGCCAAA-3', 20 nt) was dissolved in 5 µL of RNase-free water. Then, 2.5 μL of 80 mMDMAP and 2.5 μL of CR-3 reagent (dissolved in DMSO, concentration 0.1–5 M) were added sequentially, gently vortexed, and incubated at the specified temperature (4–37 °C) with intermittent mixing. The short RNA was then purified by ethanol precipitation: 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volume of pre-cooled ethanol were added, thoroughly mixed, and incubated at −20 °C for 1 h, followed by centrifugation at 16,000 × g for 20 min at 4 °C. The precipitate was washed with 70% ethanol, dried, and resuspended in RNase-free water. The amount of remaining 2′-acyl adducts was determined by MALDI–TOF MS analysis to assess the masking efficiency. The results are shown in Table 1.

[0119] Table 1. Effects of reaction temperature, reagent concentration, and reaction time on the efficiency of CR-3 masking RNA.

[0120]

[0121] As shown in Table 1, CR-3 can achieve different degrees of acylation and blocking of short RNA by adjusting the reaction concentration, time and temperature.

[0122] 2.2 Masking reaction of long mRNA

[0123] For the masking reaction of long eGFP mRNA (~1000 nt, sequence shown in Table 2) obtained from in vitro transcription, the reaction system was scaled up proportionally: 10 µg of RNA was diluted with RNase-free water to a total volume of 10 µL. Then, 5 µL of 80 mM DMAP and 5 µL of DMSO of a specified concentration were added to the RNA mixture to dissolve the CR-3 reagent, and the reaction was carried out under specified conditions. Note: To achieve mRNA acylation under different conditions, the degree of acylation under different conditions was inferred based on the trends in CR-3 reagent concentration, reaction time, and temperature. Specifically, the following three reaction degrees were defined: strong masking (1 M CR-3 reagent, 37°C for 2 hours), medium masking (0.5 M CR-3 reagent, 30°C for 1 hour), and weak masking (0.1 M CR-3 reagent, 25°C for 0.5 hours). After the reaction, the RNA was purified using RNA Clean Beads (N243, novoprotein) according to the manufacturer's protocol and stored at -80°C until transfection. The concentration of purified RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo).

[0124] Table 2 eGFP mRNA sequence information

[0125]

[0126] Example 3: In vitro testing of the translational ability of masked mRNA

[0127] Purified eGFP mRNA treated with different masking conditions as described in Example 2, Section 2.2 above was used for in vitro translation experiments in 384-well microplates. The steps were as follows: 2 µL of eGFP mRNA (1 μg / μL) was aliquoted into each well, followed by the addition of components from the PURExpress In Vitro Protein Synthesis Kit (E6800S, NEB) according to the manufacturer's instructions. The reaction mixture was incubated at 37°C for 2 or 4 hours. After incubation, the GFP fluorescence intensity was monitored using a Varioskan LUX multi-functional microplate reader (Thermo), with excitation / emission filters set to 488 nm and 519 nm, respectively.

[0128] Depend on Figure 8 It was found that the masking agent CR-3 covalently linked to the mRNA molecule, and the resulting steric hindrance significantly inhibited ribosome binding efficiency. Compared with untreated eGFP mRNA, the in vitro translation inhibition rate of eGFP mRNA treated under different masking conditions all reached over 98%. The in vitro translation inhibition rate was calculated as (1 - fluorescence intensity of the masked group / fluorescence intensity of the untreated group) × 100%.

[0129] Example 4: Cell model testing of tumor-specific translation ability of masked mRNA

[0130] 4.1 Acylation masking modification enhances intracellular RNA expression

[0131] HepG2 cells (provided by the National Experimental Cell Resource Sharing Platform) (5 × 10⁻⁶) 4 Cells per well were seeded into black-walled 96-well plates, and 100 μL of FluoroBrite DMEM medium (A1896701, Thermo) containing 10% FBS was added to each well. After 24 hours, 200 ng of d2GFP mRNA (~1000 nt, sequence shown in Table 3) obtained from in vitro transcription, either weakly masked with CR-3 as described in Example 2, 2.2, or untreated, was transfected using NanoLNP™ transfection reagent (CT0010, CYTOCH) according to the manufacturer's instructions. Six hours after transfection, 180 μL of fresh FluoroBrite DMEM medium was added to remove excess transfection reagent. GFP fluorescence intensity was dynamically monitored using a Varioskan LUX multi-plate reader (Thermo), with 488 nm excitation and 519 nm emission filters set.

[0132] Table 3 d2GFP mRNA sequence information

[0133]

[0134] Depend on Figure 9 It can be seen that the masking agent CR-3 prolongs the half-life of mRNA by enhancing the structural stability of mRNA, and ultimately increases the yield of the target protein by 49%.

[0135] 4.2 Reversible RNA masking regulates selective translation recovery in tumor cells

[0136] Five common cell lines with varying NQO1 expression levels (HEK293T, SH-SY5Y, HeLa, HepG2, and A549, all provided by the National Experimental Cell Resource Sharing Platform) were selected for experimental use. The first two cell lines expressed almost no NQO1 protein, while the latter three cell lines highly expressed NQO1 protein. For mRNA transfection, the five cell lines were transfected at a rate of 5 × 10⁻⁶ cells / year. 5Cells were seeded at a density of 100 cells / well in 6-well plates and cultured to 70-80% confluence. Following the manufacturer's instructions, 1 μg of eGFP mRNA transcribed in vitro with either CR-3 weak masking treatment or no treatment as described in Example 2.2 above, along with 1 μg of unmodified internal control mCherry mRNA (~1000 nt, sequence shown in Table 4), were co-transfected using NanoLNP™ RNA transfection reagent (CT0010, CYTOCH). The relative recovery expression of eGFP protein in different cell lines was continuously observed using cellular fluorescence imaging, and the relative recovery efficiency of eGFP mRNA was quantitatively characterized using flow cytometry.

[0137] Table 4 mCherry mRNA sequence information

[0138]

[0139] Cell fluorescence imaging ( Figure 10 ) and flow cytometry ( Figure 11 The results showed that the translation level of masked mRNA in tumor cells was more than 100 times that in normal cells.

[0140] Example 5: Testing the tumor-specific killing ability of masked mRNA in a mouse model

[0141] 5.1 Acylation masks pro-apoptotic RNA, specifically inhibiting tumor growth.

[0142] Female BALB / c nude mice (6-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in an independently ventilated cage (IVC) system at the Laboratory Animal Center of Tsinghua University, meeting the specific pathogen-free (SPF) standard. During tumor transplantation, 1×10⁶ mice were used. 7One SH-SY5Y (NQO1-deficient) or HepG2 (NQO1-highly expressed) cell line (both provided by the National Experimental Cell Resource Sharing Platform) was resuspended in 150 μL of PBS and injected subcutaneously into the left (SH-SY5Y) and right (HepG2) groin areas of each mouse, respectively. Tumor volume was monitored every 3 days using calipers and calculated using the formula V = (major diameter × minor diameter²) / 2. On day 9 (when the tumor volume reached approximately 100 mm³), mice were randomly divided into three groups (n=6 per group) and administered the drugs via tail vein injection: Group 1 received 10 μg of hBAX mRNA (~1000 nt, sequence shown in Table 5) weakly masked as described in Example 2, Section 2.2, encapsulated in 100 μL of in vivo-jetRNA® (101000021, Polyplus), transfected with the aforementioned in vivo transfection reagent; Group 2 received 10 μg of untreated hBAX mRNA encapsulated with the same amount of transfection reagent; and Group 3 received 100 μL of sterile PBS as a blank treatment. Booster injections were given weekly for 3 weeks (4 times in total). On day 28, mice were sacrificed by CO2 asphyxiation and photographed.

[0143] Table 5 hBAX mRNA sequence information

[0144]

[0145] Collect tumor images at the treatment endpoint ( Figure 12 Furthermore, the relative clearance efficiency of HepG2 (highly expressed NQO1) tumors during treatment was continuously monitored. Figure 13 It is known that masked BAX mRNA can specifically kill HepG2 tumors.

[0146] 5.2 Biosafety Evaluation of Acylation Masked RNA Drugs

[0147] After reaching the experimental endpoint, tumors and major organs (heart, liver, spleen, lung, and kidney) of mice treated with different regimens in 5.1 were harvested, fixed in 10% formalin, embedded in paraffin, sectioned (5 μm), and stained with hematoxylin and eosin (H&E) for histopathological analysis. Serum samples were collected via retroorbital hemorrhage. Liver function impairment was assessed by quantifying alanine aminotransferase (ALT, EM0351, Finetest) and aspartate aminotransferase (AST, EM0857, Finetest) levels using ELISA kits according to the manufacturer's instructions. Absorbance values ​​were read at 450 nm using a Varioskan LUX microplate reader (Thermo).

[0148] Histological analysis ( Figure 14 ) and serum biochemical analysis ( Figure 15The results showed that, compared with the untreated group, treatment with acylated masked RNA drugs did not show significant pathological damage to major organs, and effectively reduced liver function damage (P<0.0001, according to two-way ANOVA with Dunnett correction).

[0149] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. An NQOl-responsive mRNA translation masking agent, characterized in that, The mRNA translation masking agent is named as CR-3; the molecular formula of the CR-3 is C 21 H 28 N4O4, and the structural formula is as follows: 。 2. The method for synthesizing the mRNA translation masking agent according to claim 1, characterized in that, The general route of the synthesis method is as follows: ； The overall route is as follows: S1, compound 1 and compound 1-A undergo a substitution reaction under protic acid conditions to generate compound 2; S2 and compound 2 react with the halogenating agent in a polar solvent to form compound 3; S3, compound 3 and compound 3-B undergo a condensation reaction in an organic solvent under the catalysis of a condensing agent to generate compound 4; S4 and compound 4 undergo a deprotection reaction under acidic conditions to generate compound 5; S5 and compound 5 react with a carbonylating agent in the presence of a base to undergo a carbonylation reaction to generate compound 6; S6, compound 6 and nitrogen-containing heterocyclic compounds undergo a substitution reaction under inert gas protection to generate compound CR-3; In step S5, the carbonylating agent is triphosgene, and the base is triethylamine or diisopropylethylamine. The nitrogen-containing heterocyclic compound in step S6 is imidazole, and the inert gas is nitrogen or argon.

3. The synthesis method according to claim 2, characterized in that, The protic acid mentioned in step S1 is methanesulfonic acid, sulfuric acid, or p-toluenesulfonic acid; The halogenating agent in step S2 is N-bromosuccinimide or bromine; the polar solvent is an aqueous solution of acetonitrile. The condensation reagent in step S3 is DCC / DMAP or EDC / HOBt; the organic solvent is tetrahydrofuran or dichloromethane. The acidic condition described in step S4 is a 3-4 M HCl / EtOAc solution; The base mentioned in step S5 is triethylamine; The inert gas mentioned in step S6 is nitrogen.

4. The synthesis method according to claim 3, characterized in that, The halogenating agent in step S2 is N-bromosuccinimide; The organic solvent in step S3 is tetrahydrofuran.

5. The synthesis method according to claim 3, characterized in that, Step S6 also includes the addition of triethylamine and DMAP.

6. The synthesis method according to claim 5, characterized in that, In step S1, the equivalent ratio of compound 1-A to compound 1 is 0.8-1.2; In step S2, compared to compound 2, the equivalent ratio of N-bromosuccinimide is 0.9-1.1, the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 5:1, and the volume ratio is 10-15. In step S3, compared to compound 3, the equivalence ratio of compound 3-B is 0.9-1.1, the equivalence ratio of DCC is 1-1.2, the equivalence ratio of DMAP is 0.9-1.1, and the volume ratio of tetrahydrofuran is 8-12. In step S4, the volume ratio of HCl / EtOAc solution added is 8-12 compared to compound 4; In step S5, compared to compound 5, the equivalence ratio of triphosgene is 0.2-0.6, the equivalence ratio of triethylamine is 1.8-2.2, and the organic solvent for the carbonylation reaction is dichloromethane, with a volume ratio of 8-12. In step S6, compared to compound 6, the equivalent ratio of imidazole is 1-2, the equivalent ratio of triethylamine is 1-1.5, and the equivalent ratio of DMAP is 0.8-1.2; step S6 also includes THF, with an input volume ratio of 8-12.

7. The synthesis method according to claim 2, characterized in that, The conditions for the substitution reaction in step S1 are: 70-80℃ for 8-14 hours; the conditions for the halogenation reaction in step S2 are: 15-25℃ for 3-5 hours; the conditions for the condensation reaction in step S3 are: 15-25℃ for 8-14 hours; the conditions for the deprotection reaction in step S4 are: 15-25℃ for 1-3 hours; the conditions for the carbonylation reaction in step S5 are: 0-25℃ for 8-14 hours; and the conditions for the substitution reaction in step S6 are: 15-25℃ for 8-14 hours.

8. A method for masking mRNA translation, characterized in that, The mRNA translation masking agent described in claim 1 is reacted with mRNA under the catalysis of an organic base to achieve mRNA translation masking.

9. The method according to claim 8, characterized in that, The concentration of the mRNA translation masking agent is 0.1-1 M.

10. The method according to claim 8, characterized in that, The organic base is DMAP or DBU.

11. The method according to claim 8, characterized in that, The reaction conditions are: 25-37℃ for 0.5-2 hours.

12. The method for mRNA translation masking according to any one of claims 8-11, characterized in that, The sequence of the mRNA is shown in SEQ ID NO.1-SEQ ID NO.

4.

13. A drug, characterized in that, The drug includes the mRNA translation masking agent of claim 1.

14. The medicament according to claim 13, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

15. The medicament according to claim 14, characterized in that, The pharmaceutically acceptable carrier is selected from lipid nanoparticles, polymer nanoparticles, exosomes, inorganic nanoparticles, virus-like particles, or cell membrane-coated particles.

16. The medicament according to claim 13, characterized in that, The dosage form of the drug is selected from injections, lyophilized powder injections, nano-suspensions, oral dosage forms, topical administration preparations, inhalants, or implants.

17. The use of the mRNA translation masking agent of claim 1 or the medicament of any one of claims 13-16 in the preparation of an antitumor product; The tumor was an NQO1-overexpressing tumor.

18. The application according to claim 17, characterized in that, The tumor is liver cancer, cervical cancer, lung cancer, or breast cancer.

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