Construction and use of environmentally adaptive co-regulated mRNA nanodelivery system

Abstract

A construction and use of an environmentally adaptive co-regulated mRNA nanodelivery system. By incorporating EACR molecules into mRNA nanoparticles, the microenvironment is remodeled to be suitable for robust and sustained mRNA expression, while tissue damage associated with the self-immunogenicity of mRNA drugs is avoided. The nanodelivery system is compatible with ionizable lipid nanoparticles, cationic liposomes, cationic nanoemulsions, and polymeric nanoparticles. The EACR molecules include: anti-inflammatory drugs, tyrosine kinases / adaptors, JAK / STAT and MAPK pathway inhibitors, nutrients and metabolites, membrane transporters / ion channels, phosphodiesterase and cellular stress inhibitors, and natural viral proteins. The therapeutic mRNAs may encode tumor, viral, or bacterial antigens, immunomodulatory factors, therapeutic antibodies, or functional proteins / enzymes, and can play a role in the fields of regenerative medicine, protein supplementation / replacement therapy, targeted gene editing, and immunotherapy.

Description

Construction and application of an environmentally adaptive synergistically regulated mRNA nanodelivery system TECHNICAL FIELD

[0001] The present application belongs to the field of pharmacy, and relates to the construction and application of an environmentally adaptive synergistically regulated mRNA nanodelivery system. The present application is mainly used for constructing an mRNA nanoplatform that can reshape the microenvironment to be suitable for robust and sustained expression of mRNA, while avoiding tissue damage related to the immunogenicity of mRNA drugs, and the medical application of the mRNA nanoplatform in the fields of regenerative medicine, protein supplement / substitute therapy, targeted gene editing, cancer immunotherapy, infectious disease vaccine, etc. BACKGROUND

[0002] As a platform for delivering therapeutic proteins, mRNA drugs have attracted widespread attention in the fields of protein supplement / substitute therapy, regenerative medicine, targeted gene editing, cancer immunotherapy, infectious disease vaccine, etc. Compared with traditional drugs based on recombinant proteins, plasmids and viral vectors, mRNA drugs have the advantages of non-genetic integration, controllable activity, endogenous expression, efficient production, etc., and thus become a safer and more economical alternative [Nat Med 28(11), 2273-2287 (2022)].

[0003] The therapeutic application of mRNA drugs needs to overcome the challenges from the expression amount of target proteins and the requirement for biological safety, especially in the field of non-immunotherapy. Specifically, the transient expression of nanogram to microgram amount of antigenic proteins is sufficient to induce an effective human immune response. In contrast, it is necessary to deliver milligram, even gram amount of therapeutic proteins such as growth factors, enzymes, hormones or monoclonal antibodies to reach the threshold of effective treatment; at the same time, it is necessary to maintain the in vivo level of therapeutic proteins for a long time. On the other hand, immune activation produces therapeutic benefits for mRNA vaccines, but limits the tolerance of mRNA drugs under high dose and repeated administration [Nat Biotechnol 40(11), 1586-1600 (2022)] [Nat Rev Drug Discov 21(10), 699-701 (2022)] [Biomedicines 9(5), 530 (2021)].

[0004] From a mechanistic perspective, the antagonistic microenvironment caused by the intrinsic immunogenicity of mRNA drugs is the main reason for limiting their non-immunogenic applications. The immunogenicity of mRNA and its delivery carriers can activate the innate immune system, induce an inflammatory, degrading, and pro-cell death microenvironment, thereby destroying the intracellular and extracellular stability of mRNA molecules, limiting the robustness and persistence of protein translation, and increasing the risk of local or systemic tissue damage. In mammalian cells, in vitro synthesized mRNA molecules themselves, immunostimulatory byproducts and contaminants generated during mRNA production, and delivery carriers all have certain immunogenicity, which can act as danger signals, stimulate host cell pattern recognition receptors (PRRs), and ultimately upregulate type I interferons, pro-inflammatory cytokines, and chemokines [Nanomedicine 10(4), 775-782 (2014)] [Cell Mol Life Sci 72(20), 3971-3982 (2015)] [Biomaterials 31(26), 6867-6875 (2010)] [Immunity 55(11), 1993-2005 (2022)]. Type I interferons terminate global mRNA translation, accelerate mRNA degradation, and destroy mRNA molecule stability by inducing potent antiviral active proteins such as protein kinase (PKR), 2'-5'-oligoadenylate synthetase (OAS), and RNA-specific adenosine deaminase (ADAR), respectively [Drug Discov Today 21(1), 11-25 (2016)]. Pro-inflammatory cytokines and chemokines cause local, even systemic inflammation, which on the one hand recruits phagocytes to promote the phagocytosis and degradation activity of these cells to mRNA, limiting the stability of mRNA outside the target cells; on the other hand, it causes inflammatory cell damage, even 'pyroptosis', to directly harm the target cells [Nature 526(7575), 660-665 (2015)].

[0005] To overcome or avoid the above antagonistic microenvironment to produce adverse effects on mRNA drug target protein yield and biosafety, current research mainly increases the stability and / or reduces the immunogenicity of mRNA drugs through two strategies of optimizing the properties of mRNA molecules and delivery systems. Innovations at the mRNA level include: structure and sequence optimization, incorporation of modified nucleosides, improved production and purification processes, etc. [Adv Drug Deliv Rev 176, 113900 (2021)] [Signal Transduct Target Ther 7(1), 166 (2022)]; progress in delivery vectors mainly through: developing new functional lipids, lipids or polymers to promote mRNA uptake, endosome escape, cytoplasmic release in target cells [J Nanobiotechnology 20(1), 276 (2022)], using faster-degrading lipids to reduce the toxicity of the delivery system [Nat Commun 12(1), 7101 (2021)], or using endogenous carriers such as cellular vesicles to reduce immunogenicity [Nat Biomed Eng 7(7), 887-900 (2023)] and so on. However, almost all these non-immunogenic attempts are currently in the preclinical development stage, suggesting the need for a more convenient, efficient, safe, and universal optimization strategy.

[0006] SUMMARY

[0007] One of the purposes of the present application is to provide a construction method of an environment-adaptive synergistically regulated mRNA nanodelivery system, the specific steps are as follows:

[0008] (1) Screening of environment-adaptive synergistically regulated (EACR) molecules:

[0009] The EACR molecules described in the present application are a series of drugs with potential to reshape the microenvironment to enhance mRNA expression, which are determined by high-throughput screening of FDA-approved natural drug libraries, traditional Chinese medicine compound libraries, and other environment-regulating molecules related to mRNA expression.

[0010] EACR molecules can be selected from glucocorticoids, non-steroidal anti-inflammatory drugs, etc. Inflammatory inhibitors, tyrosine kinase / adaptor, JAK / STAT and MAPK pathway-related inhibitors, sugars, lipids, amino acids, vitamins, nucleosides, etc. Nutrients and metabolites, inhibitors of membrane transporters / ion channels and phosphodiesterases, cell stress inhibitors, and natural viral proteins.

[0011] The inflammation-related molecules such as glucocorticoids, non-steroidal anti-inflammatory drugs, etc. include: dexamethasone, chloro betamethasone propionate, betamethasone, chlorfluazate, hydrocortisone, budesonide, fluticasone propionate, celecoxib, meloxicam, indomethacin, sulindac, phenylsulfonethylamide, acetylsalicylic acid, acetaminophen, fenoprofen calcium, meclofenamate sodium, mefenamic acid, 4-biphenyl acetic acid, flurbiprofen, minoxidil, salicylic acid, aminosalicylic acid, ibuprofen, tolfenamic acid and other anti-inflammatory drugs.

[0012] The inhibitors related to tyrosine kinase / adaptor, JAK / STAT and MAPK pathways include: sunitinib maleate, ruxolitinib phosphate, nintedanib and other drugs for inhibiting innate immune response.

[0013] The nutrients and metabolites such as sugars, lipids, amino acids, vitamins, nucleosides include: sodium gluconate, DL-xylose, glimepiride, saxagliptin hydrate, linagliptin, clofibrate, probucol, guanidine hydrochloride, uracil, vitamin E, vitamin K1, panthenol and other drugs for promoting cell viability and enhancing mRNA translation.

[0014] The inhibitors of membrane transporters / ion channels and phosphodiesterases include: etidronate sodium, caffeine, carvedilol, cinaldipine, bupivacaine hydrochloride, trichlormethiazide, felodipine, anagrelide, pantoprazole sodium hydrate, amlodipine besylate and other drugs for regulating ion transport, affecting cell viability and translation.

[0015] The cell stress inhibitors include: integrated stress response inhibitors (ISRIB), curcumin, 2-aminopurine and other drugs that can inhibit mRNA translation arrest or degradation.

[0016] The natural viral proteins include: vaccinia virus proteins B18R, E3 and K3, influenza A virus proteins NS1 and PB1-F2, alphavirus capsid proteins and non-structural proteins, Middle East respiratory syndrome coronavirus proteins ORF4a and PIV-5V, arenavirus nucleoprotein NP and matrix protein Z and other macromolecular drugs that can escape or inhibit innate immunity.

[0017] (2) Selection and construction of therapeutic mRNA:

[0018] The sequence of the therapeutic mRNA described in the application is determined by querying the NCBI database or using gene sequencing method, and the sequence and structure are further optimized by core algorithm technology.

[0019] The therapeutic mRNA described in the application can encode tumor, viral or bacterial antigens, immunomodulatory factors, therapeutic antibodies, or functional proteins / enzymes; the mRNA can be in a non-modified form, or in a form of sequence and structure optimization, incorporation of modified nucleosides.

[0020] (3) Construction of a nano delivery system co-loading therapeutic mRNA and EACR molecules:

[0021] The nano preparation co-loading therapeutic mRNA and EACR molecules, i.e., the mRNA nano platform for environment-adaptive synergistic regulation, is prepared by microfluidic technology, thin film hydration method, high-energy emulsification method, etc.

[0022] The environment-adaptive synergistic regulation mRNA nano delivery system described in the present application includes ionizable lipid nanoparticles, cationic liposomes, cationic nanoemulsions, polymer nanoparticles, etc.

[0023] The nano delivery system described in the present application co-loads therapeutic mRNA and EACR molecules through electrostatic interaction, hydrophobic interaction, chemical bonding, steric hindrance, etc.

[0024] The second object of the present application is to provide the application of the environment-adaptive synergistic regulation mRNA nano delivery system in the fields of regenerative medicine, protein supplement / substitute therapy, targeted gene editing, cancer immunotherapy, infectious disease vaccine, etc. The application is the application of the co-loaded therapeutic mRNA and EACR molecules obtained by using the delivery system in the preparation of drugs involved in regenerative medicine, protein supplement / substitute therapy, targeted gene editing, cancer immunotherapy, infectious disease vaccine. The defect genes, deficient proteins or antigen epitopes related to these diseases or pathological conditions have been identified and recognized, and can be treated by injecting mRNA drugs.

[0025] The environment-adaptive synergistic regulation mRNA nano delivery system constructed in the present application is administered by intramuscular injection, intravenous injection or subcutaneous injection, and after reaching the target tissue, releases EACR molecules to relieve the inflammatory, RNA-degrading and cell death-promoting microenvironment, thereby achieving robust and long-acting expression of mRNA, and reducing the risk of local or systemic tissue damage caused by the immunogenicity of mRNA itself.

[0026] The environment-adaptive synergistic regulation mRNA nano delivery system constructed in the present application can be used in the non-immunological treatment fields of regenerative medicine, protein supplement / substitute, targeted gene editing, etc., and can be expanded to immunological applications such as cancer immunotherapy and infectious disease vaccine, and has good production and application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0027] FIG. 1 is a mode diagram of different administration methods.

[0028] FIG. 2 is the expression of mRNA in BMDCs over time under different EACR molecules and different administration methods (n = 4). eGFP mRNA (5 moU) is used as a reporter gene, and CNE is used as a delivery system.

[0029] Figure 3 is the effect of non-steroidal anti-inflammatory drugs and prostaglandin analogs on mRNA in vitro expression. HEK293T cells were used as target cells, eGFP mRNA (5 moU) as reporter gene, LNP as delivery system, and transfection was performed for 24 h.

[0030] Figure 4 is the effect of different concentrations of CXB and ISRIB on mRNA transfection and cell viability (n = 5-6). HEK293T cells were used as target cells, Luciferase mRNA as reporter gene, LNP as delivery system, and transfection was performed for 24 h.

[0031] Figure 5 is the transcriptomic analysis of the effect of CXB on mRNA expression. BMDCs were used as target cells, eGFP mRNA (5 moU) as reporter gene, CNE as delivery system, and transfection was performed for 24 h. Groups: mRNA (A#), CXB + mRNA (B#), mRNA & CXB (C#), mRNA + CXB (D#). KEGG pathway enrichment analysis results are shown in the figure (n = 3).

[0032] Figure 6 is a comparison chart of the number of differentially expressed genes under different administration methods.

[0033] Figure 7 is the results of gene set enrichment analysis (GSEA) related to mRNA translation and innate immunity under different administration methods.

[0034] Figure 8 is the compatibility analysis of the EACR strategy for unmodified mRNA and 5-methoxyuridine (5moU) modified mRNA. BMDCs were used as target cells, eGFP mRNA as reporter gene, CNE as delivery system, and mRNA expression was analyzed after different formulations were treated for 24 h (n = 4-5).

[0035] Figure 9 is the results of flow cytometry to investigate the expression of H2Kb / SIINFEKL and costimulatory molecules CD80 / CD86 by BMDCs after being treated with different formulations for 24 h, with untreated cells and lipopolysaccharide (LPS) treated cells as negative and positive controls, respectively (n = 4). eGFP mRNA (5 moU) was used as the reporter gene, and CNE was used as the delivery system.

[0036] Figure 10 is the results of ELISA to investigate the secretion of pro-inflammatory factors IL-1β, TNF-α, and IL-6 by BMDCs after being treated with different formulations for 24 h, with untreated cells and lipopolysaccharide (LPS) treated cells as negative and positive controls, respectively (n = 4). eGFP mRNA (5 moU) was used as the reporter gene, and CNE was used as the delivery system.

[0037] Figure 11 is a heatmap of the temporal expression levels of eGFP mRNA (5 moU) delivered by CNE or LNP-based nanoplatforms in different cells (n = 4-7).

[0038] Figure 12 is a small animal live imaging analysis of the effect of CXB addition (co-encapsulated inside the nanoparticle or administered orally as a free form) on the in vivo expression kinetics of Luciferase mRNA following intramuscular (i.m.), intravenous (i.v.) or intradermal (i.d.) injection using CNE as the delivery vehicle (n = 4).

[0039] Figure 13 is a small animal live imaging analysis of the effect of CXB addition (co-encapsulated inside the nanoparticle or administered orally as a free form) on the in vivo expression kinetics of Luciferase mRNA following intramuscular (i.m.), intravenous (i.v.) or intradermal (i.d.) injection using LNP as the delivery vehicle (n = 4).

[0040] Figure 14 is a small animal live imaging analysis of the comparison of the fluorescence signal on the abdominal side of the mouse following intradermal (i.d.) injection of CNE or LNP-based Luciferase mRNA delivery systems on the back of the mouse for 4 h (n = 4).

[0041] Figure 15 is an ELISA analysis of the levels of IL-6 and TNF-a in the peripheral blood of mice following intradermal (i.d.) injection of CNE or LNP-based mRNA delivery systems on the back of the mouse for 24 h (n = 4).

[0042] Figure 16 is the appearance of different formulations after storage at 4 °C for 3 days. The dotted circle indicates lipid / drug sedimentation.

[0043] Figure 17 is the change in the particle size (A), polydispersity index (PDI, B) and surface potential (C) of LNP and CXB 1X Figure 17 is the change in the particle size (A), polydispersity index (PDI, B) and surface potential (C) of LNP and CXB 1X Figure 17 is the change in the particle size (A), polydispersity index (PDI, B) and surface potential (C) of LNP and CXB

[0044] Figure 18 is the change in the particle size (A), polydispersity index (PDI, B) and surface potential (C) of CXB 2X Figure 18 is the change in the particle size (A), polydispersity index (PDI, B) and surface potential (C) of CXB

[0045] Figure 19 is a transmission electron microscopy (TEM) observation of the fingerprint-like multilayer structure of CXB 2X Figure 19 is a transmission electron microscopy (TEM) observation of the fingerprint-like multilayer structure of CXB

[0046] Figure 20. Small animal in vivo imaging analysis of the effect of the addition of CXB (CXB incorporated into nanoparticles at different doses) on the in vivo expression kinetics of Luciferase mRNA after intramuscular injection (i.m.) (n = 4).

[0047] Figure 21. ELISA analysis of free mRNA, LNP and CXB 2X - Expression of the protein of interest after 24 h of in vitro transfection of HEK293T, C2C12, H9c2 and HL-1 cells with LNP (n = 4). 165 mRNA as reporter gene (n = 4).

[0048] Figure 22. Echocardiography analysis of the left ventricular ejection fraction (EF) and fractional shortening (FS) of mice at weeks 2 and 4 after different administration modalities (n = 5-7).

[0049] Figure 23. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatine kinase (CK) levels in the serum of the peripheral blood of mice at week 4 (n = 6).

[0050] Figure 24. Body weight change of mice in each group during the experiment (n = 5-7).

[0051] Figure 25. High-throughput screening of drugs acting on environmental-related pathways in the FDA library. eGFP mRNA (5 moU) as reporter gene, HEK293T as target cells, addition of 10 μΜ drug, administration for 12, 24, 48 or 72 h (n = 4-6).

[0052] Figure 26. Results of the secondary screening of candidate drugs at different concentrations. eGFP mRNA (5 moU) as reporter gene, HEK293T as target cells, addition of 2, 10, 20 μΜ drug, administration for 24 h (n = 6).

[0053] Figure 27. Results of the tertiary screening of candidate drugs with Luciferase mRNA as reporter gene. HEK293T as target cells, addition of 10 μΜ drug, administration for 24 h (n > 6).

[0054] Figure 28. Effect of different concentrations of ubiquinol, thioproline, ruxolitinib phosphate (INCB-18424) and cinaldopril on mRNA transfection and cell viability (n = 5-6). HEK293T cells as target cells, Luciferase mRNA as reporter gene, LNP as delivery system. DETAILED DESCRIPTION

[0055] The application is further illustrated in the accompanying drawings and examples.

[0056] Example 1 Preliminary screening of EACR molecules and their exposure timing to enhance mRNA expression

[0057] To verify the effect of environmental improvement strategies on mRNA drug expression in vitro, we first analyzed mouse bone marrow-derived dendritic cells (BMDCs), which are difficult to transfect and sensitive to the microenvironment. Based on the inventors' previous research [Sci Adv 8(47), eabo1827 (2022)], we prepared cationic nanoemulsions—CNEs. Using CNEs as delivery carriers and eGFP mRNA (5 moU) as a reporter gene, we analyzed the effects of several potential EACR molecules: steroidal steroids—dexamethasone (DEX) and clobetasol propionate (CP); non-steroidal anti-inflammatory drugs (NSAIDs)—the cyclooxygenase 2 (COX-2) inhibitor celecoxib (CXB); and cell stress inhibitors—integrated stress response inhibitor (ISRIB) on mRNA expression kinetics. Different drug exposure timings (see Figure 1) were set, including mRNA prior to (mRNA+Drug), simultaneous (mRNA&Drug), or after drug molecules (Drug+mRNA) exposure, to determine the optimal drug exposure timing.

[0058] It has been reported that the anti-inflammatory effects of DEX and CP can enhance the expression of self-amplifying mRNA by inhibiting type I interferons [Mol Ther 29(4), 1370-1381 (2021)], and ISRIB can block mRNA translation arrest by inhibiting eIF2a phosphorylation [Biol Pharm Bull 42(2), 299-302 (2019)]. The results confirmed (see Figure 2) that the addition of DEX, CP, or ISRIB did indeed promote mRNA expression in vitro, and the degree of promotion varied depending on the different APCs and different drug exposure timings. The exposure of CXB, especially when administered simultaneously or after mRNA administration, showed excellent expression-promoting effects, which had not been reported in other studies.

[0059] Further, using lipid nanoparticles (LNP) based on ionizable lipids as delivery system, eGFP mRNA (5 moU) as reporter gene, the engineered cell line HEK293T was transfected, and the effects of simultaneous exposure of 34 FDA-approved NSAIDs and 4 prostaglandin analogues (downstream inflammatory effectors of COX) on mRNA drug expression were screened. The preparation of LNP was based on the inventors' previous research foundation [Sci Adv 8(47), eabo1827 (2022)]. The results confirmed (see Figure 3) that most NSAIDs were beneficial to transfection, while prostaglandin analogues were harmful to transfection, showing that the EACR strategy targeting the inflammatory microenvironment might be a solution to insufficient mRNA drug expression. In addition, the results of analyzing the effects of cell viability and dosing showed again (see Figure 4) that CXB had good mRNA expression promotion effect at 0.2-10 μM, but as the dose further increased, the decrease in cell viability due to toxicity was not conducive to expression; ISRIB had little effect on cell viability at 0.2-50 μM, and the mRNA expression promotion effect was stable. These results show that the different effects of different EACR molecules on mRNA expression are derived from the drug itself, rather than the vector or cell line.

[0060] Next, the potential mechanism of the introduction of CXB and its exposure timing affecting mRNA expression was analyzed. With CNE as the delivery system and eGFP mRNA (5 moU) as the reporter gene, transcriptomic analysis was performed on BMDCs treated with mRNA (A#), CXB+mRNA (B#), mRNA&CXB (C#), and mRNA+CXB (D#) for 24 h. KEGG pathway enrichment analysis showed (see Figure 5) that the addition of CXB, regardless of the order of administration (B_Vs_A, C_Vs_A, D_Vs_A), could promote mRNA translation and protein synthesis by up-regulating the PI3K-AKT-mTOR signaling pathway, RNA and mitochondrial biosynthesis, ribosome biogenesis, endoplasmic reticulum protein processing, etc.; in addition, it could also inhibit inflammation by down-regulating the NF-kappa B, cytokine receptor, pattern recognition receptor (Toll-like receptor, C-type lectin receptor), complement activation, TNF, and JAK-STAT signaling pathways, thereby reducing RNA degradation and avoiding the destruction of mRNA stability by innate immune activation, maintaining the robustness and persistence of protein translation.

[0061] In addition, the effect of drug addition itself (B_Vs_A, C_Vs_A, D_Vs_A) on genes (transcriptome) is more significant (about 800 genes can be up-regulated and about 300 genes can be down-regulated), and the difference in drug exposure (C_Vs_B, D_Vs_B) can also induce differential expression of some genes (about 80 genes can be up-regulated and about 70 genes can be down-regulated) (see Figure 6). Analysis of these differentially expressed genes (C_Vs_B, D_Vs_B) can find that the reasonable addition of CXB (at the same time or after mRNA exposure) can further up-regulate the mTOR pathway to promote mRNA translation; or by inhibiting the activation of the complement pathway, phospholipase A2 activity (C_Vs_B), JAK-STAT signaling pathway (D_Vs_B) to limit the release of inflammatory mediators, reduce the mRNA translation restriction and stability destruction mediated by inflammatory factors (see Figure 7).

[0062] In summary, the inventors used a simultaneous administration scheme for further research.

[0063] Example 2 EACR molecule CXB promotes non-inflammatory expression of various mRNAs in immune and non-immune cells

[0064] To verify the universality of the EACR strategy, the compatibility of EACR molecules to different mRNA molecules and different cell lines was first investigated. First, CNE was used as a delivery carrier to transfect BMDCs, and unmodified nucleosides and 5-methoxyuridine (5moU) modified eGFP mRNA were set as controls. The results showed (see Figure 8) that CXB and ISRIB had significant mRNA expression promoting effects when different nucleic acids were used, especially CXB. In addition, the CXB expression promoting effect was stronger in non-modified mRNA than in modified mRNA, which may be because CXB plays a role by reducing the immunogenicity-related inflammation of mRNA, and the immunogenicity of modified nucleic acids is lower; while the ISRIB expression promoting effect was weaker in non-nucleoside modified mRNA than in modified mRNA, which may be because the action pathway of ISRIB (mediated by limiting eIF2α phosphorylation to block overall mRNA translation) is less related to inflammation, and the combination of modified mRNA has a more significant synergistic expression promoting effect.

[0065] Next, BMDCs were transfected with mRNA encoding chicken ovalbumin (OVA) (5 moU) as a reporter gene, and 24 h later the expression of OVA-specific antigenic peptide-major histocompatibility complex-pMHC (H2Kb / SIINFEKL) and costimulatory molecules CD80 / 86 on the cell surface was analyzed, and the secretion of proinflammatory cytokines IL-1β, TNF-α and IL-6 by the cells was detected. The results showed that the addition of CXB and ISRIB promoted the expression of H2Kb / SIINFEKL (see Figure 9), but did not cause inflammatory activation of the cells (see Figure 10), which can be beneficial for the non-immunogenic application of mRNA drugs that need to avoid innate and adaptive immune activation.

[0066] To control the simultaneous exposure of drugs and achieve the convenience of administration, the EACR molecule was encapsulated into nanoparticles to achieve the co-delivery of mRNA and EACR. Considering that ISRIB has poor "granulation" (insoluble in water, insoluble in ethanol, and difficult to be encapsulated into nanoparticles), CXB was selected to prepare CXB-loaded nanoparticles with the same amount of free drug (1X) 1X -CNE and CXB 1X -LNP. The effect of the addition of CXB on transfection efficiency was investigated in APCs - BMDCs and mouse myeloid macrophages (BMDMs), and non-immune cells - umbilical vein endothelial cells (HUVEC), keratinocytes (HaCaT), fibroblasts (NIH / 3T3, HSF and L929), skeletal muscle cells (C2C12), cardiac muscle cells (H9c2), embryonic kidney cells (HEK239T), hepatic stellate cells (LX-2), hepatocytes (L-O2 and AML-2), in free form or directly encapsulated in CNE or LNP. The results showed (see Figure 11) that the addition of CXB, whether in free form or co-encapsulated form, promoted the in vitro expression of mRNA in various cell lines, whether based on CNE or LNP mRNA drugs: but the promotion rate was different in different cell lines, and the sensitivity of different cells to different formulation treatments or to environmental changes may be different, and the underlying rules need to be further explored.

[0067] Example 3 Study on the improvement of mRNA in vivo expression kinetics by nanoparticles incorporating EACR molecule CXB under different administration modes

[0068] Encouraged by the above experimental results, the effect of the EACR strategy on the in vivo expression amount and duration of protein was further analyzed. Luciferase mRNA (5 moU) was used as a reporter gene, and CNE and LNP were used as delivery systems, respectively, to analyze the co-encapsulation of CXB inside the mRNA drug (CXB 1X -CNE and CXB 1XmRNA in vivo expression kinetics after intramuscular injection (i.m., bilateral hind leg thigh), intravenous injection (i.v.) or intradermal injection (i.d., back skin) for oral administration of mRNA in free form or in the form of LNP.

[0069] The results show (see Figure 12) that, compared with CNE alone, CXB 1X -CNE promotes mRNA in vivo expression under all three administration routes, while oral delivery of CXB does not help, and even significantly inhibits expression under i.m. delivery. In contrast (see Figure 13), compared with LNP alone, CXB 1X -LNP also promotes mRNA in vivo expression under all three administration routes, in addition, oral delivery of CXB does not inhibit, and in some cases (i.v.) even significantly promotes mRNA in vivo expression.

[0070] In the experiment, it was observed that the in vivo diffusion of different delivery systems was different. Taking i.d. of local administration as an example (see Figure 14), the mouse was administered on the back, and at 4h, the abdominal cavity side of the LNP group had obvious bioluminescence signal from luciferase, and the improvement of the overall expression made it more intuitive; while the CNE group did not observe, which may be related to the differences in structure and biochemical properties such as surface electrical properties and shielding lipid content of different nanoparticles. On the other hand, the results of the levels of IL-6 and TNF-α in the peripheral blood of mice at 24h show (see Figure 15) that the systemic inflammatory inducibility of the LNP system is indeed overall stronger than that of CNE; in addition, the introduction of CXB affects systemic inflammation, and the level of systemic inflammation is negatively correlated with mRNA in vivo expression. These results show that whether oral CXB is beneficial to mRNA drug in vivo expression depends on the systemic inflammatory inducibility of the delivery carrier; the in vivo diffusion and systemic inflammatory induction ability of LNP are greater than those of CNE, and inflammation relief can provide more benefits for mRNA expression.

[0071] Example 4 Formulation-optimized EACR nanoparticles (CXB 2X -LNP) have better mRNA expression-promoting ability

[0072] During the experiment, it was observed that CXB 1X -CNE has poor formulation stability, and sedimentation occurs after 3 days of storage at 4°C, and needs to be prepared fresh, which is not convenient for use (see Figure 16). In contrast, CXB 1XThe LNP has better stability, and after being stored at 4°C for one month, the particle size, polydispersity index (PDI) and surface potential of the nanoparticles do not change significantly, and the CXB in the nanoparticles can be slowly released (see Figure 17). In addition, considering that the non-immunotherapy of mRNA drugs requires a large amount of expression of target proteins, and the expression amount of the mRNA delivery system based on LNP is significantly higher than that of CNE, therefore, LNP is a more suitable delivery system for EACR.

[0073] The above CXB 1X The in vitro transfection effect of LNP is not as good as that of the free CXB group (CXB & LNP), and it is considered that the drug encapsulation rate may need to be optimized. The CXB 1X The encapsulation rate of CXB in LNP is found to be only 54.9548 ± 0.07368%, so the actual amount of drug used is only half. In order to achieve the expected dosage, 2 times the dosage of CXB 2X LNP, the drug encapsulation rate is 45.4481 ± 0.01537%, which meets the drug concentration. And the CXB 2X LNP can still maintain good particle size distribution, storage stability and in vitro release (see Figure 18), and the mRNA encapsulation rate has no significant difference compared with traditional LNP (LNP: 96.92 ± 8.87%, CXB 2X LNP: 95.67 ± 5.90%). Transmission electron microscopy (TEM) observation shows that, unlike the spherical structure of traditional LNP, CXB 2X LNP presents a fingerprint-like multi-layer structure (see Figure 19), which may be caused by the incorporation of CXB causing the rearrangement of the mRNA nanoparticle structure. The in vivo transfection (i.m.) results show (see Figure 20) that CXB 2X LNP indeed shows better mRNA expression kinetics optimization capability - CXB 1X The area under the curve (AUC) of the in vivo expression of the target protein of the LNP group is 2.48 times that of the LNP group (see Figure 13), and the CXB 2X The AUC of the LNP group is 3.71 times that of the LNP group. In addition, further increase of the dosage of CXB (i.e., CXB 4X LNP) has no benefit to the expression of mRNA drugs, which may be due to the adverse effects of this large dose on the structure of nanoparticles, the encapsulation and release of drugs, or the encapsulation of mRNA.

[0074] In summary, the present application designs and optimizes a kind of EACR molecules containing, can efficiently improve the mRNA in vitro and in vivo expression kinetics of nano-preparation - CXB 2X LNP, for subsequent non-immunotherapy of mRNA drugs.

[0075] Example 5 Formulation-optimized EACR nanoparticles (CXB 2X Delivery of hVEGF-A 165 mRNA improves prognosis of myocardial infarction in mice

[0076] Patients with myocardial infarction (MI) suffer from impaired cardiac function due to the loss of a large number of cardiomyocytes, which usually do not regenerate after ischemic injury. The myocardial region around the core of myocardial infarction is poorly perfused. Although the contractility of this myocardial tissue is reduced, it is still alive and can be restored by revascularization therapy. Vascular endothelial growth factor (VEGF) is a highly specific pro-vascular endothelial cell growth factor. VEGF-A of the mammalian VEGF family can promote neovascularization and increase vascular permeability. Delivery of VEGF-A to ischemic tissue to induce angiogenesis is considered a promising approach to treating myocardial or peripheral ischemic MI.

[0077] Splice variant VEGF-A 165 plays a key role in processes such as angiogenesis, pro-vascular, endothelial and cardiac cell survival, enhancement of epicardial progenitor cell proliferation to endothelium, etc. Clinical trials have shown (NCT03370887) that epicardial injection of VEGF-A 165 mRNA is safe in a physiological buffer, however, the delivery of this free form of mRNA is insufficient for the effectiveness of the treatment, which may be related to the insufficient transfection efficiency of free mRNA. The introduction of delivery systems such as LNP may improve the expression efficiency of mRNA, but the adverse reactions caused by the inflammatory microenvironment related to the immunogenicity of LNP may outweigh its therapeutic benefits. It is envisaged that the intramyocardial injection of EACR platform-mediated VEGF-A 165 mRNA can alleviate the inflammatory, RNA-degrading, pro-cell death microenvironment caused by the immunogenicity of mRNA drugs, achieve massive expression of therapeutic proteins, and thus efficiently and safely induce cardiovascular regeneration in MI patients to reduce or reverse myocardial damage.

[0078] First, hVEGF-A 165 as the target gene, the in vitro transfection results show (see Figure 21) that the transfection ability of free mRNA is very poor in HEK293T, C2C12, H9c2 and mouse myocardial cells HL-1, and the use of LNP as a delivery vehicle can significantly promote protein expression, and CXB 2X -LNP has a significantly higher promotion effect than the LNP group. Next, using a mouse permanent occlusive myocardial infarction model, randomly divided into infarction control group (MI control) receiving normal saline, free mRNA group (Free mRNA), LNP group and CXB 2X- LNP group, with non-surgery treated mice as negative control group (Sham). The drug was injected directly into the ischemic area of the myocardium of the mice at the same time of the coronary artery ligation. The mice were subjected to echocardiography at 2 and 4 weeks after the surgery. The results (see Figure 22) showed that the coronary artery ligation could cause the decrease of the left ventricular ejection fraction (EF) and the fractional shortening (FS) of the mice (indicators reflecting the impairment of heart function), which became more serious over time. In the experimental groups, the treatment effect of the free mRNA was poor, and there was no significant difference with the MI control group; the LNP group had certain ability to improve heart function; the CXB 2X group had the best ability to improve heart function, which might be related to the efficient in vivo expression of the therapeutic protein hVEGF-A 165 The analysis of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatine kinase (CK) and other indicators in the serum of the peripheral blood of the mice at 4 weeks (see Figure 23) found that the LNP group had certain inflammation inducibility (the body weight of the mice also showed significant decrease after administration, see Figure 24), while the CXB 2X group did not induce such inflammation related side effects.

[0079] In summary, the lipid nanoparticles containing the EACR molecule CXB used to deliver VEGF mRNA can efficiently and safely promote the in vivo expression of VEGF, and alleviate the pathological damage of the acute MI model mice.

[0080] Example 6 High-throughput screening of potential other EACR molecules in FDA drug library

[0081] The above studies mainly focus on the inflammation signaling pathway related to NSAIDs, so further investigate the potential of other related pathway molecules. First, with eGFP mRNA (5 moU) as a reporter gene and HEK293T as a target cell, high-throughput screening of FDA library drugs acting on other environment-related pathways was carried out, including: glucocorticoids, other inflammation-related molecule inhibitors; inhibitors of tyrosine kinase / adaptor, JAK / STAT pathway, MAPK; nutrients and metabolites (sugar, fat, amino acid, vitamin, nucleoside); commonly used pharmaceutical excipients, immunosuppressants; membrane transporters / ion channels, phosphodiesterase (PDE) inhibitors, etc. The signal of the target protein GFP was detected at 12, 24, 48 and 72 h after transfection. Compared with the control group (DMSO-Control) with mRNA-LNP and blank drug solvent, the molecules with increased GFP expression at each time point were used as the initial screening of candidate drugs (see Figure 25). Next, secondary screening of candidate drugs at different concentrations (2, 10, 20 μM) was carried out, and the fluorescence signal of GFP at 24 h after transfection was detected. It was found that the candidate drugs screened at 2 and 10 μM had overall better expression, and the results were reproducible; at high concentrations, some drugs may be toxic (see Figure 26). Finally, in order to exclude the interference of drug fluorescence and verify the universality of drug molecules in promoting the expression of different mRNAs, Luciferase mRNA was used as a reporter gene to re-screen the above candidate drugs, and finally several candidate molecules were determined: DL-xylose, trichlormethiazide, glimepiride, saxagliptin, uracil, dexpanthenol, panthenol, benzyl alcohol, benzalkonium chloride, thioproline, phosphorus rosuvastatin (INCB-18424), etidronate sodium, caffeine, cilnidipine (see Figure 27). Among them, dose investigation was carried out on panthenol, thioproline, phosphorus rosuvastatin (INCB-18424) and cilnidipine (see Figure 28), and it was found that these molecules had good mRNA expression effect at appropriate concentrations.

[0082] These candidate drugs may have the potential to be EACR molecules, by remodeling the microenvironment, to achieve large-scale and long-term expression of mRNA drug target proteins, thereby providing guidance for the non-immune application of mRNA drugs and providing a reference for the design of immune application of mRNA drugs. More research is needed to investigate the overall regulation of the immune system by these drug molecules, suitable delivery vectors for different EACR molecules, delivery techniques, and specific clinical indications, in order to customize a series of therapeutic mRNA drugs based on the EACR platform.

[0083] Example 7 Delivery of mRNA encoding collagen and fibrin by EACR nanoplatform to alleviate skin aging

[0084] Skin aging is inevitable and is affected by both intrinsic and extrinsic factors. Intrinsic factors are determined by individual genetic background, while extrinsic factors are affected by environment and individual behavior. In addition to natural aging, long-term sunlight exposure can also cause skin aging. The medium and long-wave ultraviolet rays in sunlight can directly damage the epidermis and dermis of the skin, leading to the breaking of collagen and elastin in the dermis, presenting disordered and abnormal cross-linking, causing skin relaxation and wrinkles, which is called skin photoaging. With cationic nanoparticles (such as liposomes, nanoemulsions, etc.) as delivery systems, mRNA encoding human collagen (Collagen I, sequence reference: https: / / www.ncbi.nlm.nih.gov / CCDS / CcdsBrowse.cgi?REQUEST=NUCID&DATA=NM_000088&ORGANISM=9606&BUILDS=CURRENTBUILDS), mRNA encoding human fibroin (Elastin, sequence reference: https: / / www.ncbi.nlm.nih.gov / CCDS / CcdsBrowse.cgi?REQUEST=NUCID&DATA=NM_000501&ORGANISM=9606&BUILDS=CURRENTBUILDS), or mRNA encoding human TGF-β (sequence reference: https: / / www.ncbi.nlm.nih.gov / nuccore / NM_000660.7) are used as therapeutic genes, and panthenol is selected as an EACR molecule to construct the corresponding nanoparticles. By intradermal injection of the nanoparticles, skin fibroblasts are induced to express collagen and fibroin, thereby alleviating the problem of soft tissue loss and decreased elasticity of the skin caused by natural aging or photoaging.

[0085] Example 8 EACR nanoparticle platform delivers mRNA encoding uric acid oxidase to treat hyperuricemia and gout

[0086] Urate oxidase is an oxidase that can directly oxidize and decompose uric acid into soluble allantoin. Because the human body lacks uric acid oxidase, it cannot oxidize and eliminate uric acid as quickly as mammals, and uric acid becomes the end product of purine metabolism, so hyperuricemia and gout are prone to occur. Introducing exogenous uric acid oxidase can reduce the level of uric acid in the body to treat hyperuricemia and relieve gout. Using LNP as a delivery system, mRNA encoding mouse (or other mammals close to human genotype) uric acid oxidase (sequence reference: https: / / www.uniprot.org / uniprotkb / P25688 / entry#sequences) that is codon-optimized and nucleotide-modified is used as a therapeutic gene, and phosphoric acid ruxolitinib (INCB-18424) and vaccinia virus protein B18R are selected as EACR molecules, and the corresponding nanoparticles are constructed by the film hydration method. By intravenous injection of the nanoparticles into hyperuricemia patients, the accumulation of nanoparticles in the liver is promoted, thereby facilitating the expression of uric acid oxidase in the liver, followed by oxidation of uric acid, and relieving the symptoms.

[0087] Example 9 EACR nanoparticle platform delivers mRNA encoding viral antigens for infectious disease vaccines

[0088] Antiviral immunity relies on the linkage of virus-specific cellular and humoral immunity, especially humoral immunity, which can clear viruses through virus neutralization, complement activation, etc. Using polymeric nanoparticles as a delivery system, mRNA encoding the receptor binding domain (RBD, sequence reference [Cell 182, 1271-1283 (2020)]) of the SARS-COV-2 spike protein of the novel coronavirus is used as a therapeutic gene, and ISRIB is selected as an EACR molecule, and MPLA is added as an adjuvant, and the corresponding nanoparticles are constructed by high-energy emulsification. By intramuscular injection (once every 2 weeks, for 2-3 times) of the nanoparticles, efficient expression of the antigen is induced, thereby activating humoral and cellular immunity and playing a role in virus prevention.

Claims

1. A method for constructing an mRNA nano-delivery system with coordinated regulation of environmental adaptability, characterized in that: This is achieved through the following scheme: (1) Screening of environmental adaptability co-regulatory (EACR) molecules: Through high-throughput screening of natural medicine libraries, traditional Chinese medicine compound libraries, and other environmental regulatory molecules related to mRNA expression, we identify EACR molecules with the potential to reshape the microenvironment to enhance mRNA expression; (2) Selection and construction of therapeutic mRNA molecules: Determine the mRNA sequence by querying the NCBI database or using gene sequencing methods, and optimize the mRNA sequence and structure through core algorithm technology; (3) Construction of a nano-delivery system co-loading therapeutic mRNA and EACR molecules: Nanoformulations co-loaded with therapeutic mRNA and EACR molecules were prepared using microfluidics, thin film hydration, and high-energy emulsification methods.

2. The construction method according to claim 1, characterized in that The EACR molecules in step (1) are selected from glucocorticoids, nonsteroidal anti-inflammatory drugs, inhibitors of tyrosine kinase / aptamers, JAK / STAT and MAPK pathways, sugars, lipids, amino acids, vitamins, nucleosides, inhibitors of membrane transporters / ion channels and phosphodiesterases, cell stress inhibitors, and natural viral proteins.

3. The construction method according to claim 2, characterized in that The glucocorticoids and non-steroidal anti-inflammatory drugs in the EACR molecule include: dexamethasone, clobetasol propionate, betamethasone, clofluosol, hydrocortisone, budesonide, fluticasone propionate, celecoxib, meloxicam, indomethacin, sulindac, phenethylamine, acetylsalicylic acid, acetaminophen, fenoprofen calcium, meclofenamic acid sodium, mefenamic acid, 4-felbinac, flurbiprofen, minoxidil, salicylic acid, aminosalicylic acid, ibuprofen, and tolfenamic acid.

4. The construction method according to claim 2, characterized in that Inhibitors related to tyrosine kinase / aptamer, JAK / STAT and MAPK pathways in the EACR molecule include: sunitinib maleate, ruxolitinib phosphate, and nintedanib.

5. The construction method according to claim 2, characterized in that The sugars, lipids, amino acids, vitamins, nucleoside nutrients and metabolites in the EACR molecule include sodium gluconate, DL-xylose, glimepiride, saxagliptin hydrate, linagliptin, clofibrate, probucol, guanidine hydrochloride, uracil, vitamin E, vitamin K1, and panthenol.

6. The construction method according to claim 2, characterized in that The inhibitors of membrane transporters / ion channels and phosphodiesterases in the EACR molecule include: etidronate sodium, caffeine, carvedilol, cilnidipine, bupivacaine hydrochloride, trichlorothiazide, felodipine, anagrelide, pantoprazole sodium hydrate, and amlodipine besylate.

7. The construction method according to claim 2, characterized in that: The cell stress inhibitors in the EACR molecule include: comprehensive stress response inhibitor, curcumin, and 2-aminopurine.

8. The construction method according to claim 2, characterized in that: The natural viral proteins in the EACR molecule include: vaccinia virus proteins B18R, E3 and K3, influenza A virus proteins NS1 and PB1-F2, alphavirus capsid protein and non-structural protein, Middle East respiratory syndrome coronavirus proteins ORF4a and PIV-5V, and arenavirus nucleoprotein NP and matrix protein Z.

9. The construction method according to claim 1, characterized in that: The therapeutic mRNA described in step (2) encodes tumor, viral or bacterial antigens, immunomodulatory factors, therapeutic antibodies, or functional proteins / enzymes; the mRNA is in an unmodified form, or in a form that has been optimized in sequence and structure and incorporated with modified nucleosides.

10. The construction method according to claim 1, characterized in that: The nano-delivery system for co-loading therapeutic mRNA and EACR molecules described in step (3) includes: ionizable lipid nanoparticles, cationic liposomes, cationic nanoemulsions, and polymer nanoparticles.

11. The construction method according to claim 1, characterized in that: The nano-delivery system described in step (3) co-loads therapeutic mRNA and EACR molecules through electrostatic interaction, hydrophilic-hydrophobic interaction, chemical bonding, and steric hindrance.

12. Application of the environmentally adaptable and co-regulated mRNA nano-delivery system prepared by the method of claim 1 in regenerative medicine, protein supplementation / replacement therapy, targeted gene editing, cancer immunotherapy, and infectious disease vaccines, characterized in that: The application is the use of co-loaded therapeutic mRNA and EACR molecules obtained by the delivery system in the preparation of drugs involved in regenerative medicine, protein supplementation / replacement therapy, targeted gene editing, cancer immunotherapy, and infectious disease vaccines.

13. The use according to claim 12, characterized in that The drug is in the form of an injection.

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