CN114344278B - Nucleic acid delivery vectors and uses thereof - Google Patents

Abstract

The present invention is in the field of nucleic acid delivery technology. The present invention relates to a nucleic acid delivery vector, an immunotherapeutic agent and a vaccine containing the nucleic acid delivery vector, use of the nucleic acid delivery vector in the preparation of an immunotherapeutic agent and a vaccine, and the like.

Description

Nucleic acid delivery vectors and uses thereof

Technical Field

The present invention is in the field of nucleic acid delivery technology. The present invention relates to a nucleic acid delivery vector, an immunotherapeutic agent and a vaccine containing the nucleic acid delivery vector, use of the nucleic acid delivery vector in the preparation of an immunotherapeutic agent and a vaccine, and the like.

Background

Diseases caused by invasion of viruses into the body (such as related diseases caused by Covid-19, HIV, HPV, etc.) seriously threaten human health. Traditional vaccines have limited prophylactic capacity against viruses. Both new LNP-based mRNA crown vaccines developed by the company Moderna and Pfizer, bioNtech have met with great clinical success. As they are effective in eliciting innate and adaptive immunity.

The best place for such vaccines to function is the immune organ. This is determined by the way the immune system of the human body is activated. Lymphocytes, macrophages, and Antigen Presenting Cells (APCs) can perform their effector functions as single cells. But in order to be activated they must aggregate and interact in a specific environment (such as spleen and lymph nodes). The antigen concentration in these environments is greater. Thus, in these environments, specific encounters of cells with antigen are controllable and significantly enhanced. Also, because some APCs retain antigen throughout the time, stimulation of T and B cells by antigen can be greatly prolonged. Thus, secondary lymphoid organs such as lymph nodes and spleen present antigen in an optimal manner while increasing the chance of antigen interaction with specific cells. And lymphocytes encounter antigen anywhere else and activation is extremely inefficient and of no biological relevance. The presence of a large number of antigen presenting cells in close proximity to T and B cells in the spleen and lymph nodes makes it an ideal immune microenvironment for activation of T and B cells.

Similarly, APCs in the spleen and lymph nodes ingest nucleic acids (e.g., mRNA) when delivered by the vector to an immune organ. Nucleic acid (e.g., mRNA) that escapes endosomes will then express the antigen molecule it encodes in the cytoplasm. These antigen molecules expressed by nucleic acids (e.g., mRNA) are presented to MHC molecules on their surface after APC treatment. MHC and B7 on the APC surface bind to TCR and CD28 on the T cell surface, activating the T cell. BCR and CD40 on the B cell surface bind to antigen and CD40L on the T cell surface, respectively, or bind directly to certain specific antigen activations. Activating specific T and B cells can effectively boost adaptive immunity against and kill foreign pathogens. Delivery of antigen-encoding nucleic acids (e.g., mRNA) to the spleen and lymph nodes can induce a strong adaptive immune response, potentially becoming an immunotherapeutic or vaccine with great protective efficacy.

High dependence on the delivery vehicle is caused by poor mRNA stability, high negative charge density and poor endosome escape ability. An ideal vaccine vector should have the following characteristics: (1) The carrier is stable, so that mRNA is not easy to leak in storage, transportation and blood circulation, and mRNA is prevented from being degraded by ribozyme; (2) The enrichment of specific target organs is large, and the enrichment of other non-target organs is small, so that the functions of the specific target organs can be exerted to the maximum extent, and the systemic toxicity is reduced; (3) The particle size of the delivery vehicle should not be too large and the particle size distribution is uniform, so that the nano-preparation can be better transported in blood vessels and parenchymal organs and can efficiently deliver mRNA to target organs; (4) Achieving endosome escape while the delivery vehicle is to be taken up by the target cells, delivering mRNA into the cytoplasm, while the target organ is transfected with high efficiency; (5) The vector itself should not be immunogenic, as these immunogenic reactions may enhance the body's clearance of the vector, reduce transfection efficiency, and may produce adverse reactions. In addition to using mRNA encoding viral antigens, researchers have used mRNA encoding tumor antigens or tumor-associated antigens for immunotherapy of tumors. CN109152830a discloses a core-shell nanoparticle based tumor vaccine for delivering mRNA encoding tumor specific antigens to antigen presenting cells. Specifically, the invention comprises the steps of complexing positively charged polymers with mRNA encoding tumor antigens to form a stable core, and coating cationic lipid, neutral lipid and PEG lipid on the core through microfluidics. Such nucleocapsid structured RNA vaccines can enter dendritic cells by megapinocytosis. mRNA in the vaccine can also enhance the expression of interferon-alpha and interleukin-12 through Toll-like receptor 7/8 signal transduction, and simultaneously express the coded antigen, so that the tumor can be effectively treated. At present, various tumor immunotherapy based on mRNA encoding tumor antigen or tumor-related antigen is put into clinical study, and is used for immunotherapy of melanoma, non-small cell lung cancer, ovarian cancer, prostate cancer, colorectal cancer and other cancers. Meanwhile, mRNA immunotherapy for encoding bacterial antigens, yeast antigens, parasitic antigens, fungal antigens, degenerative disease antigens, atopic disease antigens, autoimmune disease antigens and the like has wide application potential.

US20190015330A1 discloses a core-shell structured nanoparticle of liposome/calcium phosphate nanoparticle (LCP), which is composed of a liposome-encapsulated calcium phosphate nano-precipitate. mRNA, caP and anionic lipid DOPA together form the core of LCP, which is suspended in water in nano-precipitated form after removal of the organic solvent, and PEG lipids and cationic lipids, neutral lipids are entrapped on the core to form stable LCP. LCP accumulates mainly in tumors, but is less abundant in the liver and other organs (paragraph 0166). It should be noted that the effect of DOPA in this patent is significantly different from the present invention. DOPA is in the core of LCP, and is packed in the core of CaP together with RNA, DOPA head is embedded in the core, and DOPA is only in the inner layer of double-layer lipid membrane. In the present invention, however, the anionic lipids (e.g., DOPA) and other lipids together form the shell of the nanoparticle, and are present in the inner and outer membranes of the bilayer lipid membrane. The function of DOPA in this US patent is on the one hand to stabilize the core and control the growth of the core; on the other hand, helps the shell wrap around the core, forming a stable LCP. Similarly, patent CN111643454a discloses a manganese-containing microprecipitated liposome consisting of a manganese ion-containing microprecipitated inner core and a phospholipid bilayer shell containing a cationic lipid. The tissue distribution in mice showed that the liposomes were distributed mainly in the liver and spleen and in small amounts in the lungs.

Cationic lipid is widely used in nanoparticle preparations because cationic lipid and neutral lipid are added into the shells of the nanoparticles to enable the shells and cores to be compounded to form core-shell-structure nanoparticles, so that hollow particles are reduced, and uniformity and stability of particle sizes are facilitated. However, cationic lipids (e.g., DOTAP and DOTMA, etc.) are inherently more toxic, resulting in limited applications of such core-shell structures.

In clear distinction from such patents, the shell of the nanoparticle of the present invention does not contain a cationic lipid, and in the absence of such cationic lipid, the inventors surprisingly found that the shell of the nanoparticle of the present invention (containing anionic lipid, PEG lipid and neutral lipid, but no cationic lipid) forms a uniform nanoparticle structure as does the core (containing the polymer and nucleic acid of formula I). Furthermore, it has surprisingly been found that the delivery vehicle of the present invention is capable of specifically targeting immune organs and thus is more suitable for use as an immunotherapeutic agent or vaccine. In addition, transfection efficiency is increased because anionic lipids such as DOPA also promote membrane fusion, destroying endosomes. In addition, the invention adopts the complex formed by the polymer shown in the formula I of the biodegradable and reduction-responsive polymer and the nucleic acid (such as mRNA) as a core, the polymer can protect the nucleic acid (such as mRNA) from degradation, improve the transfection efficiency of the nucleic acid (such as mRNA), prolong the transfection of the nucleic acid (such as mRNA), break disulfide bonds under the reduction condition in cells, and release the nucleic acid (such as mRNA) in the reduction response.

The invention is based on a polymer/nucleic acid (such as mRNA) compound core shown in a formula I, and anionic lipid, neutral lipid, PEG lipid and the like are coated on the compound core by a microfluidic technology to form a cation-free nanoparticle preparation. In particular, the lipopolymer nanoparticles of the present invention have the following advantages: (1) The nanoparticle specifically targets immune organs and continuously expresses the coded protein, so that enrichment in immune organs is remarkably improved. Delivery of antigen-encoding nucleic acids (e.g., mRNA) to the spleen and lymph nodes can induce a strong adaptive immune response, which greatly aids in the protective efficacy of the vaccine. To our current knowledge, there is no prescription similar to the present invention for vaccine studies. Most of the reported mRNA vaccines are based on cationic lipids, but the invention does not use cationic lipids, so that the safety is greatly improved. For mRNA vaccine preparations of non-targeted immune organs, one adopts modification of miR-122 on mRNA to degrade mRNA entering liver cells so as to reduce the expression of the mRNA in the liver and reduce toxicity. But this approach reduces the availability of mRNA drugs. In contrast, the present invention can increase the efficacy of drugs while reducing toxicity by targeted delivery of nucleic acids (e.g., mRNA); (2) The anionic lipid is used, so that potential toxicity risks brought by the cationic lipid are avoided, and the nanoparticle preparation with uniform particle size can be formed by wrapping the core; (3) The polymer used in the invention has the characteristics of biodegradability and good biocompatibility, can increase transfection efficiency, and can release nucleic acid (such as mRNA) in intracellular reduction response; (4) Compared with other core-shell structure nano particles, the micro-fluidic nano particle production technology is a mature preparation production technology, and related products enter clinic.

Disclosure of Invention

In one aspect of the present invention, there is provided a lipopolymer nanoparticle, wherein the molar ratio of ionizable nitrogen atoms in the lipopolymer nanoparticle to phosphorus atoms in a nucleic acid is from 3 to 40,

wherein the polymer nanoparticle comprises:

(1) A core comprising a polymer of formula I and a nucleic acid (preferably one or more nucleic acids);

and preferably, the core does not comprise an anionic lipid; and

(2) A lipid bilayer comprising:

(a) 20-50mol% of anionic lipid, and

(b) 25-75 mole% neutral lipid, and

(c) 1-4mol% of PEG lipid,

preferably, the lipid bilayer further comprises (d) 15-25mol% cholesterol or cholesterol analogue,

and the lipid bilayer is free of cationic lipids and each layer of the lipid bilayer contains anionic lipids.

Wherein preferably the lipid bilayer comprises:

(a) 25-35mol% (preferably 30 mol%) of an anionic lipid, and

(b) 62-72mol% (preferably 67 mol%) neutral lipid, and

(c) 2.5-3.5mol% (preferably 3 mol%) PEG lipid;

or alternatively

(a) 40-50mol% (preferably 45 mol%) of an anionic lipid, and

(b) 27-37mol% (preferably 32 mol%) neutral lipid, and

(c) 2.5 to 3.5mol% (preferably 3 mol%) PEG lipid, and

(d) 17-22mol% (preferably 20 mol%) cholesterol or cholesterol analogues.

More preferably, the lipid bilayer consists of:

(a) 30mol% of an anionic lipid (preferably DOPA), and

(b) 67mol% neutral lipid (preferably DSPC), and

(c) 3mol% PEG lipid (preferably PEG 2000-DMG);

or alternatively

(a) 45mol% anionic lipid (preferably DOPA), and

(b) 32mol% neutral lipid (preferably DSPC), and

(c) 3mol% PEG lipid (preferably PEG 2000-DSPE), and

(d) 20mol% cholesterol or cholesterol analogues.

The term "cationic lipid" or "charged lipid" refers to any of a variety of lipid materials that exist in positively or negatively charged form independent of pH over a useful physiological range (e.g., pH 3 to pH 9). Charged lipids may be of synthetic or natural origin. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dihydrocarbyl trimethylammonium propane (e.g., DOTAP, DOTMA), dihydrocarbyl dimethylaminopropane, ethyl phosphorylcholine, dimethylaminoethane carbamoyl sterols (e.g., DC-Chol). As previously mentioned, the lipopolymer nanoparticles of the present invention do not contain the aforementioned cationic lipids.

Wherein the term "anionic lipid" refers to a lipid that is negatively charged at physiological pH. Preferably, the anionic lipid may be 1, 2-dioleoyl-sn-glycero-3-phosphate (DOPA), dimyristoyl phosphatidic acid sodium salt (DMPA), dipalmitoyl phosphatidic acid sodium salt (DPPA), distearoyl phosphatidic acid sodium salt (DSPA), phosphatidylglycerol (POPG), distearoyl phosphatidylglycerol sodium salt (DSPG), 1, 2-palmitoyl phosphatidylglycerol sodium salt (DPPG), dimyristoyl phosphatidylglycerol sodium salt (DMPG), 1, 2-dioleoyl-sn-glycero-3-phosphoryl-rac- (1-glycero) sodium salt (DOPG).

Wherein, preferably, the neutral lipid may be distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), palmitoyl Oleoyl Phosphatidylethanolamine (POPE) and oleoyl phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1 carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE) and 1, 2-dipentamyl-sn-glycerol-3-phosphate ethanolamine (trans-DOPE).

Wherein, preferably, the cholesterol analogue can be cholesterol, beta-sitosterol, stigmasterol, ergosterol, campesterol, stigmasterol, brassicasterol, or tomato alkali, ursolic acid, alpha-tocopherol, etc.

Wherein, preferably, the PEG lipid can be PEG-DMG, PEG-dipalmitoyl glycerol, PEG-DSPE, PEG-dilauryl glyceramide, PEG-dimyristoyl glyceramide, PEG-dipalmitoyl glyceramide and PEG-distearoyl glyceramide, PEG-cholesterol (1- [8' - (cholest-5-ene-3 [ beta ] -oxy) carboxamido-3 ',6' -dioxaoctyl ] carbamoyl- [ omega ] -methyl-poly (ethylene glycol), PEG-DMB, PEG2k-DMG, PEG2k-DSPE, PEG2k-DSG, PEG2k-DMA or PEG2k-DSA, preferably PEG2k-DMG or PEG2k-DSPE.

In some embodiments, the term "nucleic acid" or "nucleic acid molecule" will be recognized and understood by one of ordinary skill in the art, for example, and is intended to mean a molecule that includes, preferably consists of, a nucleic acid component. The term nucleic acid molecule preferably refers to a DNA or RNA molecule. Preferably synonymously with the term polynucleotide. Preferably, the nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers covalently linked to each other by phosphodiester bonds of a sugar/phosphate backbone. The term "nucleic acid molecule" also encompasses modified nucleic acid molecules, such as base-modified, sugar-modified or backbone-modified DNA or RNA molecules as defined herein. Nucleic acids may include natural nucleic acids and artificial nucleic acids. The term "artificial nucleic acid" as used herein will be recognized and understood by one of ordinary skill in the art and is intended to represent, for example, non-naturally occurring artificial nucleic acids. The artificial nucleic acid may be a DNA molecule, an RNA molecule or a hybrid molecule comprising DNA and RNA portions. In general, artificial nucleic acids can be designed and/or generated by genetic engineering methods to correspond to a desired artificial nucleotide sequence (heterologous sequence). In the context of the present invention, an artificial sequence is generally a sequence that does not occur in nature, i.e. it differs from the wild-type sequence by at least one nucleotide. The term "wild-type" as used herein will be recognized and understood by one of ordinary skill in the art and is intended to represent, for example, naturally occurring sequences. Furthermore, the term "artificial nucleic acid" is not limited to meaning "single molecule" but is generally understood to include a collection of substantially identical molecules. Artificial RNA: the term "artificial RNA" as used herein is intended to mean non-naturally occurring RNA. In other words, an artificial RNA can be understood as a non-natural nucleic acid molecule. Such RNA molecules may be non-natural due to their individual sequences (which are not naturally occurring, e.g. G/C content modified coding sequences, UTRs) and/or due to other modifications, e.g. structural modifications which are not naturally occurring nucleotides. In general, artificial RNAs can be designed and/or generated by genetic engineering methods to correspond to desired artificial nucleotide sequences (heterologous sequences). In the context of the present invention, an artificial RNA sequence is typically a sequence that does not occur in nature, i.e. it differs from the wild-type sequence by at least one nucleotide. The term "artificial RNA" is not limited to refer to a "single molecule" but is generally understood to include a collection of substantially identical molecules. Thus, it may involve a plurality of substantially identical RNA molecules contained in an aliquot or sample. In the context of the present invention, the RNA of the invention is an artificial RNA as defined herein. For example, the nucleic acid suitable for use in the present invention may be mRNA, which may be 0.5-3kb in size.

In some embodiments, the mRNA comprises one or more non-standard nucleotide residues. Non-standard nucleotide residues may include, for example, 5-methyl-cytidine ("5 mC"), pseudouridine ("ψu"), and/or 2-thio-uridine ("2 sU"). See, e.g., U.S. Pat. No.8,278,036 or WO2011012316 for discussion of such residues and their incorporation into mRNAs. mRNA can be RNA, which is defined as RNA in which 25% of the U residues are 2-thio-uridine and 25% of the C residues are 5-methylcytidine. See also US20120195936 and international publication WO2011012316, both hereby incorporated by reference in their entirety. The presence of non-standard nucleotide residues may result in an mRNA that has higher stability and/or lower immunogenicity than a control mRNA having the same sequence but containing only standard residues. In further embodiments, the mRNA may comprise one or more non-standard nucleotide residues selected from the group consisting of: isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine cytosine, and combinations of these modifications with other nucleobase modifications. Certain embodiments may also include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more of 2' -O-alkyl modifications, locked Nucleic Acids (LNAs)). In some embodiments, the RNA can be complexed or hybridized to additional polynucleotides and/or peptide Polynucleotides (PNAs). In embodiments where the sugar modification is a2 '-O-alkyl modification, such modifications may include, but are not limited to, 2' -deoxy-2 '-fluoro modifications, 2' -O-methyl modifications, 2 '-O-methoxyethyl modifications, and 2' -deoxy modifications. In certain embodiments, any of these modifications may be present in 0-100% of the nucleotides-e.g., greater than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the component nucleotides, alone or in combination.

Messenger RNA (mRNA): as used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide encoding at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNAs. The mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems, and optionally purified, chemically synthesized, and the like. Where appropriate, for example, in the case of chemically synthesized molecules, the mRNA may include nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, and the like. Unless otherwise indicated, mRNA sequences are presented in the 5 'to 3' direction. In some embodiments, the mRNA is or comprises a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deadenosine, 7-deazaguanosine, 8-oxo-guanosine, O (6) -methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); an interlayer base; modified sugars (e.g., 2 '-fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioate and 5' -N-phosphoramidite linkages).

In some embodiments, the mRNA may contain RNA backbone modifications. In general, the backbone modification is a modification in which the phosphate of the backbone of a nucleotide contained in RNA is chemically modified. Exemplary backbone modifications generally include, but are not limited to, modifications from the group consisting of: methylphosphonate, phosphoramidate, phosphorothioate (e.g., cytidine 5' -O- (1-phosphorothioate)), borophosphate, positively charged guanidinium groups, etc., which means replacing the phosphodiester linkage with other anionic, cationic or neutral groups.

Thus, preferably, the nucleic acid is mRNA.

In one embodiment, the mRNA encodes an antigen selected from one or more of the following antigens: tumor antigens, tumor-associated antigens, viral antigens, bacterial antigens, yeast antigens, parasitic antigens, fungal antigens, degenerative disease antigens, atopic disease antigens, autoimmune disease antigens.

Thus, in one embodiment, the tumor antigen is selected from one or more of the following groups: OVA, 5T4, 707-AP,9D7,AFP,AlbZIPHPG1, α5β1-integrin, α5β6-integrin, α -methylacyl-CoA racemase, ART-4, B7H4, BAGE-1, BCL-2, BING-4, CA15-3/CA27-29, CA19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CEA, CLCA2, CML28, coactosin-like protein, collagen XXIII, COX-2, CT-9/BRD6, cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10/MAGE-B1, DAM-6/MAGE-B2, EGFR/Her1, MMPRIN, epCam, ephA2, ephA3, erbB3, EZH2, FGF-5, FN, fra-1, G250/CAIX, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GDEP, gnT-V, gp100, GPC3, HAGE, HAST-2, hepsin, her2/neu/ErbB2, HERV-K-MEL, HNE, homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HST-2, hTERT, iCE, IGF-1R, IL-13, IL-2, IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, KI-67, KI-1, KI-5, LAGE-1, livin, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B1, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin, MART-1/Melan-A, MART-C2, matrix protein 22, MC1R, M-CSF, mesothe MG, 50/PX, MMP11, MMR/CAIX-D1, MAGE-D2, MUX-A88, MUX-D2, N-acetylglucosamine transferase-V, neo-PAP, NGEP, NMP22, NPM/ALK, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP, OGT, OS-9, osteocalcin, osteopontin, p15, p190minorbcr-abl, p53, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, pim-1-kinase, pin1, POTE, PRAME, prostein, protease-3, PSA, PSCA, PSGR, PSMA, RAGE-1, RHAM/CD 168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEP-1, STEM-90, fbRP, TARP, TATGB-72, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP-2/INT2, trp-p8, tyrosinase, UPA, VEGF, VEGFR-2/FLK-1, WT1; preferably, the mutated antigen expressed in the cancer disease is selected from the group consisting of: alphA-Actin-4/m, ARTC1/m, bcr/abl, beta-catenin/m, BRCA1/m, BRCA2/m, CASP-5/m, CASP-8/m, CDC27/m, CDK4/m, CDKN2A/m, CML66, COA-1/m, DEK-CAN, EFTUD2/m, ELF2/m, ETV6-AML1, FN1/m, GPNMB/m, HLA-A 0201-R170I, HLA-A11/m, HLA-A2/m, HSP70-2M, KIAA0205/m, K-Ras/m, LDLR-FUT, MART2/m, ME1/m, MUM-1/m, MUM-2/m, MUM-3/m, myoglobin class I/m, neo-PAP/m, NFYC/m, N-Ras/m, OGT/m, OS-9/m, p53/m, pml/RARa, PRDX5/m, PTPRK/m, RBAF600/m, SIRT2/m, SYT-SSX-1, SYT-SSX-2, TEL-AML1, TGFbRII, TPI/m.

Thus, in one embodiment, the virus is selected from one or more of the following groups: SARS, covd 19, poxvirus, ebola virus, marburg virus, dengue virus, influenza virus, parainfluenza virus, respiratory syncytial virus, measles virus, human immunodeficiency virus, human papilloma virus, varicella-zoster virus, herpes simplex virus, cytomegalovirus, EB virus, JC virus, rhabdovirus, rotavirus, rhinovirus, adenovirus, papilloma virus, parvovirus, picornavirus, poliovirus, mumps-causing virus, rabies-causing virus, respiratory enterovirus, rubella virus, outer virus, myxovirus, retrovirus, hepadnavirus, coxsackievirus, venezuki encephalomyelitis virus, japanese encephalitis virus, yellow fever virus, rift valley fever virus, hepatitis a virus, hepatitis b virus, hepatitis c virus, hepatitis delta virus, hepatitis e virus, and the like.

Thus, in one embodiment, the infectious disease antigen is selected from one or more of the following groups: ADsA, antigens TbH9 of mycobacterium tuberculosis, DPV, 381, mtb41, mtb40, mtb32A, mtb9.9a, mtb9.8, mtb16, mtb72f, mtb59f, mtb88f, mtb71f, mtb46f, and Mtb31f.

Thus, in one embodiment, the degenerative disease antigen is selected from one or more of the following groups: aβ (1-42), tau protein, α -synuclein (α -Syn).

In one embodiment, the invention provides an immunotherapeutic agent or vaccine comprising the lipopolymer nanoparticles of the invention.

In one embodiment, the present invention provides a use of the lipopolymer nanoparticle of the invention in the preparation of an immunotherapeutic agent or vaccine.

The term "immunotherapy" means the treatment of a disease or disorder by inducing or enhancing an immune response.

Wherein, preferably, the immunotherapeutic agent or vaccine of the invention further comprises one or more pharmaceutically acceptable excipients.

In one embodiment, the invention provides a method of delivering a nucleic acid into a cell comprising delivering into the cell a lipopolymer nanoparticle of the invention,

wherein, preferably, the cell is a mammalian cell, more preferably the cell is a human cell;

wherein, preferably, the cell is an immune cell, more preferably the cell is a human immune cell.

In one aspect of the present invention, the present invention provides a method of preparing the lipopolymer nanoparticle of the present invention, the method comprising:

(1) Preparing a solution a which is a buffer containing nucleic acid (e.g., one or more nucleic acids), preferably mRNA, at a ph=3.0 to 6.0 (preferably ph=4.0);

(2) Preparing a solution B which is a buffer containing a polymer of formula I having a structure of ph=3.0 to 6.0 (preferably ph=4.0):

(3) A solution C is formulated comprising: anionic lipids, neutral lipids, and PEG lipids, preferably, the solution C also contains cholesterol or cholesterol analogues,

(4) Mixing the solution A and the solution B according to a volume ratio of 1:3 (preferably mixing in a microfluidic chip) to obtain a solution D,

(5) Mixing the solution C and the solution D according to a volume ratio of 1:3 (preferably mixing in a microfluidic chip) to obtain a solution E,

(6) The solution E is dialyzed and then concentrated (preferably ultrafiltration, such as in an ultrafiltration tube) to the desired volume. The dialysis may be in a buffer (e.g. PBS) at ph=7.0-8.0 (preferably ph=7.4) (preferably in a dialysis cartridge, preferably overnight). Thus, step (6) may be: the solution E is transferred to a dialysis cartridge (e.g. Slide-a-lyzer tm, mwco=20k) and dialyzed (preferably overnight) in PBS at ph=7.0-8.0 (preferably ph=7.4). After that, in ultrafiltration tube (e.g

Ultra, mwco=10k) to the desired volume。

Preferably, the content of the anionic lipid and the neutral lipid in the solution C, and the content of the PEG lipid are respectively: 20-50mol%, 25-75mol% and 1-4mol%; more preferably, the content of the anionic lipid, neutral lipid and PEG lipid in the solution C is respectively: 25-35mol%, 62-72mol%, 2.5-3.5mol%; more preferably, the content of the anionic lipid, neutral lipid and PEG lipid in the solution C is respectively: 30mol%, 67mol% and 3mol%;

when the solution C contains cholesterol or the like, the content thereof in the solution C is 15 to 25mol%, preferably 17 to 22mol%, more preferably 20mol%.

Thus, in some alternatives, the solution C comprises 25-35mol% (preferably 30 mol%) anionic lipid, and 62-72mol% (preferably 67 mol%) neutral lipid, and 2.5-3.5mol% (preferably 3 mol%) PEG lipid; or in some alternatives, the solution C comprises 40-50mol% (preferably 45 mol%) anionic lipid, and 27-37mol% (preferably 32 mol%) neutral lipid, and 2.5-3.5mol% (preferably 3 mol%) PEG lipid, and 17-22mol% (preferably 20 mol%) cholesterol or cholesterol analogue.

Thus, in some alternatives, the solution C consists of the following components: 30mol% anionic lipid, 67mol% neutral lipid, and 3mol% peg lipid; or 45mol% anionic lipid, 32mol% neutral lipid, 3mol% peg lipid, and 20mol% cholesterol or cholesterol analog.

Thus, in some alternatives, the solution C consists of the following components: 30mol% DOPA, 67mol% DSPC and 3mol% PEG2000-DMG; or 45mol% DOPA, 32mol% DSPC, 3mol% PEG2000-DSPE and 20mol% cholesterol.

Drawings

Fig. 1: flow chart of lipopolymer nanoparticle preparation.

Fig. 2: percent mRNA expression of luciferase mRNA in liver, spleen and lymph nodes at 6h and 24h for each formulation group of lipopolymer nanoparticles and MC3-LNP group.

Fig. 3: comparison of the expression levels of luciferase mRNA in the liver and spleen at 6h and 24h for each formulation group of lipopolymer nanoparticles and MC3-LNP group, where negative values are decreased expression and positive values are increased expression.

Fig. 4: expression of luciferase mRNA in spleen and lymph nodes at 6h and 24h for each formulation group.

Fig. 5: comparison of the lipopolymer nanoparticle G1 and lipopolymer nanoparticle G7 formulation group with the MC3-LNP formulation group.

Detailed Description

Example 1 materials and methods

The mRNA used in the experiment was Fluc mRNA (L-7202-1000) from Trilink, 1.9kb in length, containing 5'-cap and 3' -polyA tails. The lipid materials used for the experiments were as follows: DOPA (Avanti Polar Lipids, inc.), DSPC (sierra biotechnology limited), cholesterol (sigmA-Aldrich Shanghai trade limited), PEG2k-DMG (Avanti Polar Lipids, inc.).

The device used for preparing the lipid polymer nano-particles is a micro-fluidic device with a micro-scale of Maianan, and the model is INano L.

Particle size detection used dynamic light scattering laser particle sizer (Zetasizer Ultra, malvern Panalytical Ltd). At the time of detection, the sample was diluted 50-fold with 5mM NaCl solution and transferred to a DTS1070 cuvette. The detection mode was 173 ° back-scattered light, and each sample was equilibrated in the apparatus for 120s and detection was resumed after reaching 25 ℃.

The animal experiment mice are C57BL/6,6-8 week old female mice. After 6h and 24h intraperitoneal injection of luciferase, the mice were observed for fluorescence of isolated organs by the IVIS imaging system.

Example 2 preparation and characterization of lipopolymer nanoparticles

The flow of the preparation of the lipopolymer nanoparticle of the present example is shown in fig. 1, and specifically includes:

(1) Preparing a solution a, which is a malate buffer containing mRNA, ph=4.0;

(2) Solution B was formulated as a malate buffer comprising a polymer of formula I having the structure:

(3) Solution C was formulated with the lipids contained in groups G1 to G7 as shown in table 1 below:

(4) Mixing the solution A and the solution B according to a volume ratio of 1:3 (preferably mixing in a microfluidic chip) to obtain a solution D,

(5) Mixing the solution C and the solution D according to a volume ratio of 1:3 (preferably in a microfluidic chip) to obtain a solution E,

(6) The solution E was transferred to a dialysis cartridge (Slide-a-lyzer tm, mwco=20k) and dialyzed overnight in ph=7.4 PBS. Then in ultrafiltration tube [ ]

Ultra, mwco=10k) to the desired volume.

The product particle size, particle size distribution (PDI) and Zeta potential were measured by a dynamic light scattering laser particle sizer (Zetasizer Ultra, malvern).

The prescription, the particle size distribution (PDI) and the Zeta potential of the prepared lipopolymer nano particles are shown in table 1:

TABLE 1

From table 1, it can be seen that nanoparticles having a small, uniform particle size and negatively charged surface can be prepared by coating anionic lipid, neutral lipid and PEG lipid on a complex core formed by the polymer of formula I and mRNA.

Example 3 preparation and characterization of the MC3-LNP formulation.

MC3-LNP with Fluc mRNA entrapped was prepared as a control. The specific method comprises the following steps: the following two solutions were prepared separately: A. an ethanol solution of lipids, the molar ratio of each lipid being: dlin-MC3-DMA DSPC cholesterol PEG2 k-DMG=50:10:38.5:1.5; B. malate buffer containing mRNA, ph=4.0. Mixing the solution A and the solution B in a microfluidic chip according to a volume ratio of 1:3, and obtaining an LNP intermediate solution. The LNP intermediate solution was transferred to a dialysis cartridge (Slide-a-lyzer tm, mwco=20k), dialyzed in ph=7.4 PBS for 16h, and ethanol was removed to give the LNP product.

The product particle size, particle size distribution (PDI) and Zeta potential were measured by a dynamic light scattering laser particle sizer (Zetasizer Ultra, malvern) (table 2).

TABLE 2

Sample group	Particle size (nm)	PDI	Zeta potential (mV)
				MC3-LNP	70.83	0.07276	-4.966

EXAMPLE 4 biodistribution Studies

To examine immune organ targeting of lipid polymer nanoparticle formulations in vivo, we performed biodistribution studies.

The method comprises the following steps: the preparation method of the lipopolymer nanoparticle preparation is shown in example 2. The preparation of MC3-LNP is described in example 3.

Female C57BL/6 black mice of 6-8 weeks old were selected to evaluate the in vivo immune organ targeting effect of the lipopolymer nanoparticle formulation. Mice were randomly grouped (n=4), and the lipopolymer nanoparticle formulation and the MC3-LNP formulation (2 mg/kg) were injected into the mice via tail vein. Luciferase was injected intraperitoneally at 6h and 24h after injection of the lipopolymer nanoparticles, respectively. Mice were then sacrificed and major organs and inguinal lymph nodes were collected and their fluorescence was observed by an IVIS imaging system.

Results: as shown in fig. 2, the expression of lipopolymer nanoparticles was mainly concentrated in spleen and lymph nodes. Unlike the major metabolic organ, in which MC3-LNP is centrally expressed in the liver, the lipopolymer nanoparticle preparation is expressed in a small amount in the liver and in the spleen and two inguinal lymph nodes. And the expression of lipopolymer nanoparticles in spleen and lymph nodes did not decrease, and even increased, over time. This may be due to the presence of the polymer resulting in slow release of the mRNA, extending the duration of action of the mRNA. The results show that the immune organ targeting ability of the lipopolymer nanoparticle formulation is far superior to MC3-LNP.

EXAMPLE 5 biodistribution Studies

In order to examine the immune organ targeting of the lipopolymer nanoparticle preparation in vivo and screen to obtain the preferred lipopolymer nanoparticle preparation, we further compared the expression level of MC3-LNP and lipopolymer nanoparticle in mice for 6h and 24 h.

The method comprises the following steps: experimental methods are described in example 2.

Results: as shown in FIG. 3, MC3-LNP expression was reduced in both liver and spleen. Surprisingly, over time, the expression level of G1 in the lipid polymer nanoparticles decreased in the liver and increased in the immune organ, indicating that it was continuously expressed in the target organ and continuously decreased in the non-target organ, thus increasing the efficacy while reducing toxicity. Next we analyzed the expression of 7 groups of formulations 6h and 24h (fig. 4). It can be seen that groups G1 and G7 had higher transfection in both spleen and lymph nodes at both time points. And compared with MC3-LNP, G1 and G7 are mainly expressed in immune organs (figure 5), so the polymer nanoparticle, particularly G1 and G7, is an immune organ targeting lipid polymer nanoparticle preparation with great transformation prospect.

By comparing the expression of different lipopolymer nanoparticles and LNP in immune and major metabolic organs we screened two groups of preferred (G1 and G7) immune organ targeted lipopolymer nanoparticle formulations.

Claims (2)

1. A lipopolymer nanoparticle comprising:

(1) A core consisting of a polymer of formula I and one or more nucleic acids, wherein the nucleic acids are mRNA of 0.5-3kb in size,

and

(2) Lipid bilayer consisting of:

(a) 30mol% DOPA, and

(b) 67mol% DSPC, and

(c) 3mol% of PEG2000-DMG;

or alternatively

Lipid bilayer consisting of:

(a) 45mol% DOPA, and

(b) 32mol% DSPC, and

(c) 3mol% PEG2000-DMG, and

(d) 20mol% of cholesterol in the water-soluble polymer,

and wherein the molar ratio of ionizable nitrogen atoms in the lipopolymer nanoparticle to phosphorus atoms in the nucleic acid is from 3 to 40.

2. A method of preparing the lipopolymer nanoparticle of claim 1, the method comprising:

(1) Preparing a solution a, which is a buffer containing one or more mRNA, at ph=4.0;

(2) Preparing a solution B which is a buffer solution containing a polymer represented by formula I, wherein the polymer represented by formula I has a structure of ph=4.0:

(3) Preparing a solution C consisting of:

(a) 30mol% DOPA, and

(b) 67mol% DSPC, and

(c) 3mol% of PEG2000-DMG;

or alternatively

Preparing a solution C consisting of:

(a) 45mol% DOPA, and

(b) 32mol% DSPC, and

(c) 3mol% PEG2000-DMG, and

(d) 20mol% of cholesterol in the water-soluble polymer,

(4) Mixing the solution A and the solution B in a microfluidic chip according to a volume ratio of 1:3 to obtain a solution D, (5) mixing the solution C and the solution D in the microfluidic chip according to a volume ratio of 1:3 to obtain a solution E, (6) dialyzing the solution E, concentrating to a required volume,

wherein the dialysis is in a buffer solution with ph=7.4, dialyzed overnight in a dialysis cartridge, and

wherein the concentration is in an ultrafiltration tube.

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