Lipid nanoparticle compositions and uses thereof

Abstract

Disclosed herein are lipid nanoparticles (LNPs) and pharmaceutical compositions and vaccines comprising the same. Also disclosed herein are methods of administering the LNP compositions, pharmaceutical compositions, and vaccines described herein for preventing infectious diseases and / or for immunizing a subject or for delivering a payload to cells in the central nervous system (CNS) of a subject. Methods for treating and / or preventing CNS diseases are also provided.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Applications Nos. 63 / 605,185, 63 / 605,386, and 63 / 605,392, filed December 1, 2023, and U.S. Provisional Patent Application No. 63 / 618,062, filed January 5, 2024, which are incorporated herein by reference in their entirety.

[0003] Incorporate into the sequence list by reference

[0004] This application contains a sequence list submitted via EFS-WEB in .XML format, which is incorporated herein by reference in its entirety. The .XML file named 061529-506001WO.xml was created on November 26, 2024, and is 328KB in size. Background Technology

[0005] There is an unmet need for lipid nanoparticle compositions for the specific delivery of antigens and polynucleotides encoding antigens. This disclosure provides lipid nanoparticle compositions and pharmaceutical compositions, as well as methods for using them as vaccines. Summary of the Invention

[0006] In one aspect, this disclosure provides a lipid nanoparticle (LNP) composition comprising an antigen or a polynucleotide encoding an antigen, an accessory lipid, a sterol and / or a polyethylene glycol-conjugated lipid (PEG lipid), an ionizable cationic lipid, and a selective organ-targeting (SORT) lipid.

[0007] In some embodiments, the LNP composition comprises a polynucleotide encoding an antigen, and the polynucleotide is mRNA. In some embodiments, the polynucleotide is chemically modified mRNA. In some embodiments, the polynucleotide encoding the antigen is DNA. In some embodiments, the mRNA or DNA encodes the antigens shown in Tables 3, 4, and 5.

[0008] One aspect of this disclosure is a lipid nanoparticle (LNP) composition comprising: pathogen-associated antigen or polynucleotide encoding a pathogen-associated antigen; and selective organ-targeting (SORT) lipids.

[0009] Another aspect of this disclosure is a lipid nanoparticle (LNP) composition comprising: a pathogen-associated antigen or a polynucleotide encoding a pathogen-associated antigen; and a permanent cationic lipid. In embodiments, the LNP composition further comprises selective organ-targeting (SORT) lipids; auxiliary lipids; sterols; and / or polyethylene glycol-conjugated lipids (PEG lipids). In some embodiments, the LNP composition further comprises selective organ-targeting (SORT) lipids.

[0010] A further aspect of this disclosure is a lipid nanoparticle (LNP) composition comprising: a pathogen-associated antigen or a polynucleotide encoding a pathogen-associated antigen; and an ionizable dendritic polymer lipid. In various embodiments, the LNP composition further comprises selective organ-targeting (SORT) lipids; auxiliary lipids; sterols; and / or polyethylene glycol-conjugated lipids (PEG lipids). In many embodiments, the ionizable dendritic polymer lipids have the formula D-IV. In embodiments, the LNP composition further comprises selective organ-targeting (SORT) lipids.

[0011] Another aspect of this disclosure is a lipid nanoparticle (LNP) composition comprising: pathogen-associated antigens or polynucleotides encoding pathogen-associated antigens; selective organ-targeting (SORT) lipids; ionizable cationic lipids; and helper lipids; sterols; and / or polyethylene glycol-conjugated lipids (PEG lipids).

[0012] The LNP composition contains a polynucleotide encoding a pathogen-associated antigen, for example, the polynucleotide being chemically modified.

[0013] In some implementations, the polynucleotide is mRNA.

[0014] In various implementations, the polynucleotide is DNA.

[0015] In many implementations, the polynucleotide encodes the pathogen-associated antigens shown in Tables 3, 4, or 5.

[0016] In the implementation scheme, the polynucleotide encodes a pathogen-associated antigen that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the antigens shown in Tables 3, 4, or 5.

[0017] In some implementations, the polynucleotide encodes an influenza virus antigen, a coronavirus antigen, an RSV antigen, a PIV3 antigen, or an hMPV antigen.

[0018] In one aspect, this disclosure provides a lipid nanoparticle (LNP) composition comprising a cancer antigen or a polynucleotide encoding a cancer antigen, an accessory lipid, a sterol and / or a polyethylene glycol-conjugated lipid (PEG lipid), an ionizable cationic lipid, and a selective organ-targeting (SORT) lipid.

[0019] In some embodiments, the LNP composition comprises a polynucleotide encoding a cancer antigen, and the polynucleotide is mRNA. In some embodiments, the polynucleotide is chemically modified mRNA. In some embodiments, the polynucleotide encoding the cancer antigen is DNA. In some embodiments, the mRNA or DNA encodes the antigens shown in Tables 3, 4, and 5.

[0020] One aspect of this disclosure is a lipid nanoparticle (LNP) composition comprising: a cancer antigen or a polynucleotide encoding a cancer antigen; and a selective organ-targeting (SORT) lipid.

[0021] Another aspect of this disclosure is a lipid nanoparticle (LNP) composition comprising: a cancer antigen or a polynucleotide encoding a cancer antigen; and a permanent cationic lipid. In embodiments, the LNP composition further comprises selective organ-targeting (SORT) lipids; auxiliary lipids; sterols; and / or polyethylene glycol-conjugated lipids (PEG lipids). In some embodiments, the LNP composition further comprises selective organ-targeting (SORT) lipids.

[0022] A further aspect of this disclosure is a lipid nanoparticle (LNP) composition comprising: a cancer antigen or a polynucleotide encoding a cancer antigen; and an ionizable dendritic polymer lipid. In various embodiments, the LNP composition further comprises selective organ-targeting (SORT) lipids; auxiliary lipids; sterols; and / or polyethylene glycol-conjugated lipids (PEG lipids). In many embodiments, the ionizable dendritic polymer lipids have the formula D-IV. In embodiments, the LNP composition further comprises selective organ-targeting (SORT) lipids.

[0023] Another aspect of this disclosure is a lipid nanoparticle (LNP) composition comprising: a cancer antigen or a polynucleotide encoding a cancer antigen; a selective organ-targeting (SORT) lipid; an ionizable cationic lipid; and an accessory lipid; a sterol; and / or a polyethylene glycol-conjugated lipid (PEG lipid).

[0024] The LNP composition contains a polynucleotide encoding a cancer antigen, for example, the polynucleotide being chemically modified.

[0025] In some implementations, the polynucleotide is mRNA.

[0026] In various implementations, the polynucleotide is DNA.

[0027] In many implementations, the polynucleotide encodes the cancer antigens shown in Table 6, Table 7, or Table 8.

[0028] In the implementation scheme, the polynucleotide encodes a cancer antigen that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the antigens shown in Tables 6, 7, or 8.

[0029] In some implementations, the polynucleotide encodes a lung cancer antigen, a prostate cancer antigen, or a neoantigen.

[0030] In various embodiments, the SORT lipid is a permanent cationic lipid, for example, the SORT lipid is 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). In some cases, the SORT lipid is dimethyl dioctadecylammonium (DDAB), or the SORT lipid is ethylphosphocholine. In various cases, ethylphosphocholine is 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (16:0 EPC) or 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:0 EPC). In some cases, DOTAP is present in the composition at a molar percentage of about 30% to about 50%. In various cases, ethylphosphocholine is present in the composition at a molar percentage of about 30% to about 50%.

[0031] In many embodiments, the SORT lipid is 1,2-dioleoyl-3-dimethylammonium propane (DODAP). In some cases, DODAP is present in the composition at a molar percentage of about 5% to about 30%.

[0032] In the embodiments, the SORT lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate (18:1 PA). In some cases, the 18:1 PA is present in the composition at a molar percentage of about 5% to about 30%.

[0033] In some embodiments, SORT lipids are present in the composition at a molar percentage of about 5% to about 50%.

[0034] In various embodiments, the ionizable cationic lipid is a dendritic polymer lipid. In some cases, the ionizable cationic lipid is a dendritic polymer lipid according to formula (I) or formula (X). In various cases, the dendritic polymer is: (4A3-SC7) or (5A2-SC8).

[0035] In many embodiments, the ionizable cationic lipid is present in the composition at a molar percentage of about 10% to about 25%.

[0036] In some embodiments, the auxiliary lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), or the auxiliary lipid is 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC). In some cases, the auxiliary lipid is present in the LNP composition at a molar percentage of about 7.5% to about 30%.

[0037] In some embodiments, the sterol is cholesterol or sitosterol. In some cases, cholesterol is present in the LNP composition at a molar percentage of about 20% to about 50%.

[0038] In various embodiments, PEG lipids are present in the LNP composition at a molar percentage of about 0.5% to about 10%.

[0039] In many embodiments, the LNP composition comprises mRNA with a lipid:mRNA ratio (weight / weight) between about 5:1 and about 40:1.

[0040] In the implementation scheme, the lipid:mRNA ratio (weight / weight) is approximately 40:1, approximately 36:1, or approximately 30:1.

[0041] In some embodiments, the LNP composition comprises: DOPE in a molar percentage of about 5% to about 25%; cholesterol in a molar percentage of about 20% to about 50%; DMG-PEG in a molar percentage of about 0.5% to about 10%; ionizable cationic lipids in a molar percentage of about 10% to about 25%; and 16:0 EPC in a molar percentage of about 30% to about 40%.

[0042] In various embodiments, the LNP composition comprises: DOPE in a molar percentage of about 5% to about 25%; cholesterol in a molar percentage of about 20% to about 50%; DMG-PEG in a molar percentage of about 0.5% to about 10%; ionizable cationic lipids in a molar percentage of about 10% to about 25%; and DODAP in a molar percentage of about 10% to about 30%.

[0043] In many embodiments, the LNP composition comprises: DOPE in a molar percentage of about 5% to about 25%; cholesterol in a molar percentage of about 20% to about 50%; DMG-PEG in a molar percentage of about 0.5% to about 10%; ionizable cationic lipids in a molar percentage of about 10% to about 25%; and 18:1 PA in a molar percentage of about 5% to about 20%.

[0044] In an embodiment, the LNP composition comprises: DOPE in a molar percentage of about 5% to about 25%; cholesterol in a molar percentage of about 20% to about 50%; DMG-PEG in a molar percentage of about 0.5% to about 10%; ionizable cationic lipids in a molar percentage of about 10% to about 25%; and DOTAP in a molar percentage of about 30% to about 50%.

[0045] In some embodiments, the LNP composition comprises: about 15% DOPE in molar percentage; about 30% cholesterol in molar percentage; about 3% DMG-PEG in molar percentage; about 15% ionizable cationic lipids in molar percentage; and about 30% 16:0 EPC in molar percentage.

[0046] In various embodiments, the LNP composition comprises: about 20% DOPE in molar percentage; about 40% cholesterol in molar percentage; about 3% DMG-PEG in molar percentage; about 15% ionizable cationic lipids in molar percentage; and about 15% DODAP in molar percentage.

[0047] In many embodiments, the LNP composition comprises: about 20% DOPE in molar percentage; about 40% cholesterol in molar percentage; about 5% DMG-PEG in molar percentage; about 20% ionizable cationic lipids in molar percentage; and about 5% 18:1 PA in molar percentage.

[0048] In an embodiment, the LNP composition comprises: about 10% DOPE in molar percentage; about 20% cholesterol in molar percentage; about 3% DMG-PEG in molar percentage; about 10% ionizable cationic lipids in molar percentage; and about 50% DOTAP in molar percentage.

[0049] In some embodiments, the ionizable cationic lipid is a dendritic polymer lipid, such as 4A3-SC7.

[0050] In various embodiments, the LNP composition comprises a Tris buffer, optionally with a pH of about 6 to about 9; the LNP composition comprises sucrose, optionally with a concentration (weight / weight) or molar percentage of about 5% to about 15%; the LNP composition comprises a citrate buffer, optionally with a pH of about 4 to about 6; the LNP composition comprises: (a) 15 mM Tris buffer, optionally with a pH of about 6 to about 9; and / or (b) sucrose with a concentration (weight / weight) or molar percentage of about 5% to about 15%; the LNP composition comprises 10 mM citrate buffer, optionally with a pH of about 4 to about 6.

[0051] In one aspect, this disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the LNP composition of any aspect or embodiment herein.

[0052] In another aspect, this disclosure provides a pharmaceutical composition comprising an LNP composition of any aspect or embodiment herein and a pharmaceutically acceptable excipient.

[0053] In a further aspect, this disclosure provides a vaccine comprising an LNP composition or a pharmaceutical composition of any aspect or embodiment herein. In some cases, the vaccine comprises an adjuvant, for example, squalene.

[0054] In another aspect, this disclosure provides a method for delivering an LNP composition to a subject in need, the method comprising administering to the subject in need an LNP composition, a pharmaceutical composition, or a vaccine of any aspect or embodiment herein, or any embodiment thereof, in any aspect or embodiment thereof.

[0055] In another aspect, this disclosure provides a method for preventing infectious diseases caused by pathogens in a subject of need, the method comprising administering to the subject of need an LNP composition, a pharmaceutical composition, and / or a vaccine of any aspect or embodiment herein.

[0056] In a further aspect, this disclosure provides a method for immunizing a subject in need against pathogen infection and / or disease or condition caused by a pathogen, the method comprising administering to the subject a vaccine of any aspect or implementation thereof herein.

[0057] In many implementations, the pathogen is a virus, which may optionally include influenza virus, coronavirus, RSV, PIV3, hMPV, or MeV.

[0058] In the implementation plan, the pathogen is bacteria.

[0059] In some implementations, administration to subjects in need may include intravenous, intrathecal, intramuscular, intradermal, subcutaneous, or intranasal administration.

[0060] In various embodiments, the method induces an immune response in a subject in need, optionally wherein the immune response is greater than that following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG).

[0061] In many embodiments, the method induces an antibody response in a subject in need, optionally wherein the antibody response is greater than that following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG).

[0062] In the implementation scheme, the method induces T cell proliferation in a desired subject, optionally wherein the T cell proliferation is greater than that following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG). In some cases, the T cells are helper T cells or cytotoxic T cells.

[0063] In some embodiments, the method activates T cells in a subject in need, optionally wherein T cell activation is greater than that following administration of the composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG). In some cases, the T cells are helper T cells or cytotoxic T cells.

[0064] In various implementation schemes, the method induces a cytokine response in the desired subject.

[0065] One aspect of this disclosure is the use of any LNP composition, any pharmaceutical composition, or any vaccine disclosed herein for the manufacture of a medicament for the vaccination of subjects in need against pathogen infection or for the manufacture of a medicament for the treatment of a disease or condition caused by a pathogen.

[0066] Another aspect of this disclosure is that any LNP composition disclosed herein, any pharmaceutical composition disclosed herein, or any vaccine disclosed herein is used to manufacture a medicine for vaccinating subjects in need against pathogen infection, or for treating a disease or condition caused by a pathogen.

[0067] A further aspect of this disclosure is that any LNP composition, any pharmaceutical composition, or any vaccine disclosed herein may be used to vaccinate subjects in need against pathogen infection or to treat a disease or condition caused by a pathogen.

[0068] Another aspect of this disclosure is a kit comprising any LNP composition disclosed herein, any pharmaceutical composition disclosed herein, or any vaccine disclosed herein, along with instructions for use thereof.

[0069] In some embodiments, the SORT lipid is a permanent cationic lipid. In some embodiments, the SORT lipid is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In some embodiments, the SORT lipid is dimethylbis(octadecylammonium) (DDAB). In some embodiments, the permanent cationic lipid is ethylphosphocholine. In some embodiments, ethylphosphocholine is 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (16:0 EPC). In some embodiments, ethylphosphocholine is 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:0 EPC). In some embodiments, the SORT lipid is 1,2-dioleoyl-3-dimethylammonium-propane (DODAP). In some embodiments, the SORT lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate (18:1 PA). In some embodiments, the SORT lipid is present in the LNP composition at a molar percentage of about 5% to about 50%. In some embodiments, DOTAP is present in the LNP composition at a molar percentage of about 30% to about 50%. In some embodiments, ethylphosphocholine is present in the LNP composition at a molar percentage of about 30% to about 50%. In some embodiments, DODAP is present in the LNP composition at a molar percentage of about 5% to about 30%. In some embodiments, 18:PA is present in the LNP composition at a molar percentage of about 5% to about 30%.

[0070] In some embodiments, the method induces an immune response in the subject, optionally wherein the immune response is greater than the immune response following administration of composition 2H. In some embodiments, the method induces an antibody response in the subject, optionally wherein the antibody response is greater than the antibody response following administration of composition 2H. In some embodiments, the method induces T cell proliferation in the subject, optionally wherein T cell proliferation is greater than T cell proliferation following administration of composition 2H. In some embodiments, the method activates T cells in the subject, optionally wherein T cell activation is greater than T cell activation following administration of composition 2H. In some embodiments, the T cells are helper T cells and cytotoxic T cells. In some embodiments, the method induces a cytokine response in the subject.

[0071] In another aspect, this disclosure provides a method for preventing cancer in a subject of need, the method comprising administering to the subject of need an LNP composition, a pharmaceutical composition, and / or a vaccine of any aspect or embodiment thereof, as described herein or in any embodiment thereof.

[0072] In another aspect, this disclosure provides a method for treating cancer or reducing the severity of cancer in a subject in need, the method comprising administering to the subject in need an LNP composition, a pharmaceutical composition, and / or a vaccine of any aspect or embodiment thereof, as described herein or in any embodiment thereof.

[0073] In a further aspect, this disclosure provides a method for inhibiting the growth, proliferation, progression, and / or metastasis of cancer cells in a subject in need, the method comprising administering to the subject in need an LNP composition, a pharmaceutical composition, and / or a vaccine of any aspect or embodiment thereof, as described herein or in any embodiment thereof.

[0074] In a further aspect, this disclosure provides a method for immunizing a subject in need against cancer and / or cancerous diseases or conditions, the method comprising administering to the subject a vaccine of any aspect or implementation thereof herein.

[0075] In some implementations, administration to subjects in need may include intravenous, intrathecal, intramuscular, intradermal, subcutaneous, or intranasal administration.

[0076] In various embodiments, the method induces an immune response in a subject in need, optionally wherein the immune response is greater than that following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG).

[0077] In many embodiments, the method induces an antibody response in a subject in need, optionally wherein the antibody response is greater than that following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG).

[0078] In the implementation scheme, the method induces T cell proliferation in a desired subject, optionally wherein the T cell proliferation is greater than that following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG). In some cases, the T cells are helper T cells or cytotoxic T cells.

[0079] In some embodiments, the method activates T cells in a subject in need, optionally wherein T cell activation is greater than that following administration of the composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG). In some cases, the T cells are helper T cells or cytotoxic T cells.

[0080] In the implementation plan, cancer is one or more of the following: acute myeloid leukemia (LAML), adrenocortical carcinoma (ACC), bladder cancer, bladder urothelial carcinoma (BLCA), brain cancer, breast cancer, invasive breast cancer (BRCA), Burkitt lymphoma, cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), chronic lymphocytic leukemia (CLL), colonic adenocarcinoma (COAD), colon cancer, colorectal cancer (CRC), diffuse large B-cell lymphoma (DLBCL), Ewing's sarcoma, glioblastoma multiforme (GBM), glioma, head and neck cancer, head and neck squamous cell carcinoma (HNSC), hepatocellular carcinoma, human cancer (including colorectal cancer, gastric cancer, kidney cancer and ovarian cancer), chromophobe renal cell carcinoma (KICH), clear cell renal cell carcinoma (KIRC), and papillary renal cell carcinoma (KI). RP), leukemia, hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung cancer, lung squamous cell carcinoma (LUSC), melanoma, multiple myeloma (MM), non-small cell lung cancer, ovarian adenocarcinoma, ovarian tumor, ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), primary adenocarcinoma, prostate adenocarcinoma (PRAD), prostate cancer (including high-grade prostatic intraepithelial neoplasia (PIN), androgen-dependent and androgen-independent prostate tumors), rectal adenocarcinoma (READ), renal cell carcinoma, salivary gland carcinoma, sarcoma, seminoma, cutaneous melanoma (SKCM), small bowel cancer, gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thyroid adenocarcinoma (THCA), thyroid cancer, uterine carcinosarcoma (UCS), and uterine endometrioid carcinoma (UCEC).

[0081] One aspect of this disclosure is the use of any LNP composition disclosed herein, any pharmaceutical composition disclosed herein, or any vaccine disclosed herein for the following purposes: to manufacture a medicine for vaccinating subjects in need against cancer and / or cancerous diseases or conditions, or for the prevention of cancer; for treating cancer or reducing the severity of cancer; and / or for inhibiting the growth, proliferation, progression, and / or metastasis of cancer cells.

[0082] Another aspect of this disclosure is that any LNP composition disclosed herein, any pharmaceutical composition disclosed herein, or any vaccine disclosed herein is used to manufacture a medicine for vaccinating subjects in need against cancer and / or cancerous diseases or conditions, or to manufacture a medicine for preventing cancer; for treating cancer or reducing the severity of cancer; and / or for inhibiting the growth, proliferation, progression, and / or metastasis of cancer cells.

[0083] A further aspect of this disclosure is that any LNP composition, any pharmaceutical composition, or any vaccine disclosed herein may be used to vaccinate subjects in need against cancer and / or cancerous diseases or conditions, or to prevent cancer; to treat cancer or reduce the severity of cancer; and / or to inhibit the growth, proliferation, progression, and / or metastasis of cancer cells.

[0084] Another aspect of this disclosure is a kit comprising any LNP composition disclosed herein, any pharmaceutical composition disclosed herein, or any vaccine disclosed herein, along with instructions for use thereof.

[0085] In some embodiments, the method induces an immune response in the subject, optionally wherein the immune response is greater than the immune response following administration of composition 2H. In some embodiments, the method induces an antibody response in the subject, optionally wherein the antibody response is greater than the antibody response following administration of composition 2H. In some embodiments, the method induces T cell proliferation in the subject, optionally wherein T cell proliferation is greater than T cell proliferation following administration of composition 2H. In some embodiments, the method activates T cells in the subject, optionally wherein T cell activation is greater than T cell activation following administration of composition 2H. In some embodiments, the T cells are helper T cells and cytotoxic T cells. In some embodiments, the method induces a cytokine response in the subject.

[0086] Another aspect is lipid nanoparticle compositions for specific delivery to the central nervous system (CNS).

[0087] In one aspect, this disclosure provides a lipid nanoparticle (LNP) composition comprising a payload, an ionizable cationic lipid, a selective organ-targeting (SORT) lipid and / or an accessory lipid, a sterol and / or a polyethylene glycol-conjugated lipid (PEG lipid), wherein the composition delivers the payload to cells in the central nervous system (CNS) of a subject.

[0088] In some embodiments, the payload comprises a polynucleotide, optionally mRNA, shRNA, or microRNA. In some embodiments, the mRNA encodes a polypeptide or protein selected from the groups shown in Tables 9, 10, and / or SEQ ID NO: 201-207. In some embodiments, the mRNA encodes a gene editing system or a component thereof. In some embodiments, the payload comprises a polypeptide or protein.

[0089] In some embodiments, the ionizable cationic lipid is a dendritic polymer lipid. In some embodiments, the ionizable cationic lipid is a dendritic polymer lipid according to formula (I) or formula (X). In some embodiments, the ionizable cationic lipid is 4A3-SC7. In some embodiments, the ionizable cationic lipid is 5A2-SC8. In some embodiments, the ionizable cationic lipid is present in the composition at a molar percentage of about 10% to about 35%.

[0090] In some embodiments, the SORT lipids are selected from the groups shown in Tables 16A and 16B. In some embodiments, the SORT lipids are present in the composition at a molar percentage of about 5% to about 65%. In some embodiments, the SORT lipids are present in the composition at a molar percentage of about 5% to about 35%.

[0091] In some embodiments, the cofactor lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE). In some embodiments, the cofactor lipid is 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC). In some embodiments, the cofactor lipid is present in the composition at a molar percentage of about 5% to about 35%.

[0092] In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is sitosterol. In some embodiments, cholesterol is present in the composition at a molar percentage of about 20% to about 50%.

[0093] In some embodiments, PEG lipids are present in the composition at a molar percentage of about 0.5% to about 10%.

[0094] In some embodiments, the composition comprises mRNA with a lipid:mRNA ratio (weight / weight) between 5:1 and 40:1.

[0095] In some embodiments, the composition comprises 4A3-SC7 in a molar percentage of about 5% to about 30%, SORT lipids in a molar percentage of about 5% to about 30%, DOPE in a molar percentage of about 8% to about 23%, cholesterol in a molar percentage of about 15% to about 46%, and / or DMG-PEG in a molar percentage of about 0.5% to about 10%.

[0096] In some embodiments, the composition comprises 4A3-SC7 in a molar percentage of about 13% to about 20%, SORT in a molar percentage of about 10% to about 30%, DOPE in a molar percentage of about 13% to about 20%, cholesterol in a molar percentage of about 30% to about 40%, and / or DMG-PEG in a molar percentage of about 3% to about 5%, wherein the SORT lipid is ethylphosphocholine.

[0097] In some embodiments, the composition comprises 4A3-SC7 in a molar percentage of about 13% to about 20%, SORT in a molar percentage of about 5% to about 30%, DOPE in a molar percentage of about 13% to about 20%, cholesterol in a molar percentage of about 30% to about 40%, and / or DMG-PEG in a molar percentage of about 3% to about 5%, wherein the SORT lipid is an anionic lipid.

[0098] In some embodiments, the composition comprises about 19% 4A3-SC7, about 20% 14:0 EPC, about 19% DOPE, about 38% cholesterol, and / or about 4% DMG-PEG.

[0099] In some embodiments, the composition comprises about 17% 4A3-SC7, about 30% 16:0 EPC, about 17% DOPE, about 33% cholesterol, and / or about 3% DMG-PEG.

[0100] In some embodiments, the composition comprises about 17% 4A3-SC7, about 30% 14:0 EPC, about 17% DOPE, about 33% cholesterol, and / or about 3% DMG-PEG.

[0101] In some embodiments, the composition comprises about 23% 4A3-SC7, about 5% 18:1 PA, about 23% DOPE, about 45% cholesterol, and / or about 5% DMG-PEG.

[0102] In some embodiments, the composition is capable of delivering mRNA to cells in the central nervous system (CNS) of a subject in an amount that effectively increases the expression and / or function of genes encoded by the mRNA. In some embodiments, the cells are endothelial cells. In some embodiments, the cells are pericytes. In some embodiments, the cells are smooth muscle cells (SMA).

[0103] In another aspect, this disclosure provides a method for treating and / or preventing CNS diseases in a subject of need, wherein the method comprises administering the composition described herein to the subject via intrathecal injection.

[0104] In some embodiments of the methods disclosed herein, the composition comprises a payload, wherein the payload is messenger RNA (mRNA), and wherein the method causes the payload to be delivered to the CNS in an amount that effectively increases the expression and / or function of a gene encoded by the mRNA.

[0105] In some embodiments, the method results in the expression of the peptide in the subject's CNS. In some embodiments, the method results in the expression of the peptide in the subject's CNS from 1 hour to 72 hours after administration of the composition to the subject.

[0106] In some embodiments of the methods disclosed herein, CNS diseases include acid lipase disorders, acid maltase deficiency, acid storage disorders, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Addie pupils, Addie syndrome, adrenoleukodystrophy, agnosia, Eckhardt syndrome, Eckhardt-Gutier syndrome, Alexander disease, Alpert disease, and alternating hemiplegia. Hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, hemangioma, hypoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, aromatic L-amino acid decarboxylase deficiency (AADC deficiency), aspartate glucosamineuria, Asperger's syndrome, ataxia, ataxia-telangiectasia (Lewis-Barr syndrome), ataxia and cerebellar or spinocerebellar degeneration, attention deficit hyperactivity disorder (ADHD). Disorders, autism, autonomic dysfunction, Bass syndrome, Barten disease, Becker's myotonia, Behçet's disease, Bell's palsy, Bernhardt-Rose syndrome, Binswanger disease, Bloch-Sulzberger syndrome, Bradbury-Egelston syndrome, Brown-Seckal syndrome, medullary spinal muscular atrophy, CADASIL, Canavan disease, burning neuralgia, cavernous malformation, cavernous hemangioma, central cervical spinal cord syndrome, central spinal cord syndrome, central pontine myelinolysis, ceramide deficiency, cerebellar degeneration Cerebellar hypoplasia, cerebral beriberi, cerebral gigantism, cerebral palsy, brain-ocular-facial-skeletal syndrome (COFS), cholesterol ester storage disease, chorea, chorea-acanthosis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, cavitary brain, congenital myasthenia gravis, corticobasal degeneration, cranial arteritis, Klebsiella encephalitis, Creutzfeldt-Jakob disease, Cushing's syndrome, cystinemia, cytomegalovirus infection. Inclusion body disease), oculoclonus-myoclonus syndrome, Dandy-Walker syndrome, Danon disease, Dawson's disease, Demosier syndrome, Degerina-Klempke palsy, dementia, dentate nucleus-cerebellar ataxia, dentate nucleus-red nucleus atrophy, dermatomyositis, developmental motor disorders, Dweck syndrome, diffuse sclerosis, autonomic dysfunction, writing disorders, reading disorders, dysphagia, motor disorders, myoclonic cerebellar dyssynergia, progressive cerebellar dyssynergia, epilepsy (e.g., Amish infantile epilepsy syndrome [AIES], benign familial infantile epilepsy [BFIS]),Benign familial neonatal epilepsy [BFNS], childhood absence epilepsy [CAE], childhood-onset epileptic encephalopathy [COEE], Dravet syndrome [DS], early infantile epileptic encephalopathy [EIEE], familial adult myoclonic epilepsy [FAME], familial febrile seizures [FFS], familial focal epilepsy with variable lesions [FFEVF], familial infantile myoclonic epilepsy [FIME], familial temporal lobe epilepsy [FTLE], focal epilepsy and speech disorders with or without intellectual disability [FESD], generalized epilepsy with paroxysmal motor disorders [GEPD], generalized epilepsy with febrile seizures plus [GEFS+], idiopathic generalized epilepsy [IGE], juvenile absence epilepsy [JAE], juvenile myoclonic epilepsy [JME], myopathic epilepsy ... Clonic-atonic epilepsy [MAE], nocturnal frontal lobe epilepsy [NFLE], progressive myoclonic epilepsy [PME], pyridoxine oxidase deficiency [PNPOD], pyridoxine-dependent epilepsy [EPD], and severe myoclonic epilepsy of infancy [SMEI], Fabry disease, Fall syndrome, familial autonomic dysfunction, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Fabry disease, fibromuscular dysplasia, Fisher syndrome, infantile relaxation syndrome, Friedreich ataxia, frontotemporal dementia, fucoside storage disease, galactosialidosis, Gaucher disease, systemic ganglioside storage disease, Göstadmann syndrome, GSS disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion body disease (giant Cell inclusion disease, globular cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, GM1 ganglioside storage disease, GM2 ganglioside storage disease (Tai-Sachs disease), Guillain-Barré syndrome, Hallewarden-Scholes disease, persistent unilateral headache, alternating hemiplegia (hemiplegia) Alterans syndrome, hereditary spastic paraplegia, hereditary ataxic polyneuritis, Holmes-Addie syndrome, holohemorrhagic malformation, Hughes syndrome, Huntington's disease, hydrocephalus, hydrocephalus, Cushing's syndrome, immune-mediated encephalomyelitis, inclusion body myositis, incontinence pigmentosa, infantile hypotonia, infantile axonal dystrophy, occipital cleft with exposed head, Isaac syndrome, Jubert syndrome, Karns-Sell syndrome, Kennedy disease, Kingsburn syndrome, Klein-Levin syndrome, Krippel-Fair syndrome, Krippel-Trenone syndrome (KTS), Klüfer-Busy syndrome, Korsakov amnesia syndrome, Crabbe disease, Kugelberg-Weyland disease, Lambert-Eton myasthenia gravis syndrome, Landau-Cleven syndrome, lateral femoral cutaneous nerve entrapment syndrome, lateral medullary syndrome, Leigh's disease, Lennox-Gasstor syndrome, Lesch-Niehan syndrome, Levin-Critchley syndromeLewy body dementia, lipoprotein storage disease, lissencephaly, locked-in syndrome, Luger's disease, lupus, Lyme disease, Machado-Joseph disease, megalencephaly, alpha-mannoside storage disease, beta-mannoside storage disease, Méridov-Robbes syndrome, Menkes disease, lateral femoral cutaneous neuralgia, metachromatic leukodystrophy, microcephaly, Miller-Fischer syndrome, Möbius syndrome, mucopolysaccharidosis type IH (Heller syndrome), mucopolysaccharidosis type IH / S (Heller-Shee syndrome), mucopolysaccharidosis type IS (Shee syndrome), mucopolysaccharidosis type II (Hunter syndrome), mucopolysaccharidosis type III-A (Samfylbo syndrome type A), mucopolysaccharidosis type III-B (Samfylbo syndrome type A). The following are listed as examples of diseases: Samuelleybo syndrome type B, mucopolysaccharidosis type III-C (Samuelleybo syndrome type C), mucopolysaccharidosis type III-D (Samuelleybo syndrome type D), mucopolysaccharidosis type IV-B (Mochio syndrome type B), mucopolysaccharidosis type VI (Maroto-Lamy syndrome), mucopolysaccharidosis type VII (Slay syndrome), mucopolysaccharidosis type IX (Natovich syndrome), multiple sclerosis, muscular dystrophy, myasthenia gravis, myelinolytic diffuse sclerosis, narcolepsy, neuroacanthocytosis, neurofibromatosis, neuroleptic malignant syndrome, sarcoidosis, Niemann-Pick disease, Ōtahara syndrome, olivopontocerebellar atrophy, oculoclonus myoclonus, and Ossa Levin-McLeod syndrome, pantothenic acid kinase-related neurodegenerative diseases, paraneoplastic syndromes, sensory abnormalities, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal unilateral headache, Parvo-Ros syndrome, Peyrenia, Pena-Shokel syndrome type II, periventricular leukomalacia, phytanoic acid storage disease, Pick's disease, piriformis syndrome, polymyositis, Pompe disease, post-poliomyelitis syndrome, posterior cortical atrophy, primary dentate nucleus atrophy, primary lateral sclerosis, primary progressive aphasia, prion diseases, progressive hemifacial atrophy, progressive motor ataxia, progressive multifocal leukoencephalopathy, progressive sclerotic gray matter dystrophy, progressive supranuclear palsy, prosopagnosia, Ramsey Hunt syndrome Syndrome type I, Ramsey-Hunter syndrome type II, Rasmussen encephalitis, Reifsum disease, Rett syndrome, Reye's syndrome, Riley-Day syndrome, Sandhoff disease, Hilde disease, Setterberg disease, Shay-Dreg syndrome, Sjögren's syndrome, spasticity, spina bifida, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, Still-Richardson-Olshefsky syndrome, striatal substantia nigra degeneration, Sturge-Weber syndrome, tardive dyskinesia, Tau proteinosis, Ty-Sachs disease, thoracic outlet syndrome, thyrotoxic myopathy, painful tic, Todd's paralysis, trigeminal neuralgia, tropical spastic paraplegia, Troy syndrome, vascular dementia, von Ecknomer disease.Von Hippel-Lindau disease (VHL), von Recklinghausen disease, Wallenberg syndrome, Wernig-Hoffmann disease, Wernicke-Korsakov syndrome, West syndrome, Whipple's disease, Williams syndrome, Wilson's disease, Wollman's disease, X-linked spinal-bulbar muscular atrophy, Zelweg syndrome, multiple sclerosis with brain atrophy, Lewis body dementia (LBD), or Anglemann syndrome.

[0107] In another aspect, this disclosure provides a method for delivering a payload to cells in the CNS of a subject, wherein the method includes administering the composition described herein to the subject via intrathecal injection.

[0108] In another aspect, this disclosure provides a kit comprising the compositions described herein.

[0109] In another aspect, this disclosure provides a pharmaceutical composition comprising the composition described herein and pharmaceutically acceptable excipients and / or diluents.

[0110] In another aspect, this disclosure provides for the use of the compositions described herein for the treatment of CNS diseases. In yet another aspect, this disclosure provides for the use of the compositions described herein for the treatment of CNS diseases by intrathecal administration.

[0111] In another aspect, this disclosure provides for the use of the compositions or pharmaceutical compositions described herein for the treatment of CNS diseases in subjects of need.

[0112] In some embodiments of the use or the composition used for that use, the CNS disease is acid lipase disorder, acid maltase deficiency, acid storage disorder, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Addie pupils, Addie syndrome, adrenoleukodystrophy, agnosia, Eckardie syndrome, Eckardie-Gutier syndrome, Alexander disease, Alpert disease, alternating hemiplegia. Hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, hemangioma, hypoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, aromatic L-amino acid decarboxylase deficiency (AADC deficiency), aspartate glucosamineuria, Asperger's syndrome, ataxia, ataxia-telangiectasia (Lewis-Barr syndrome), ataxia and cerebellar or spinocerebellar degeneration, attention deficit hyperactivity disorder (ADHD). Hyperactivity disorder, autism, autonomic dysfunction, Bass syndrome, Barten disease, Becker's muscle rigidity, Behçet's disease, Bell's palsy, Bernhardt-Rose syndrome, Binswanger disease, Bloch-Sulzberger syndrome, Bradbury-Egelston syndrome, Brown-Seckal syndrome, medullary spinal muscular atrophy, CADASIL, Canavan disease, burning neuralgia, cavernous malformation, cavernous hemangioma, central cervical spinal cord syndrome, central spinal cord syndrome, central pontine myelinolysis, ceramide deficiency Cerebellar degeneration, cerebellar hypoplasia, cerebral beriberi, cerebral gigantism, cerebral palsy, brain-ocular-facial-skeletal syndrome (COFS), cholesterol ester storage disease, chorea, chorea-acanthosis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, cavitary brain, congenital myasthenia gravis, corticobasal degeneration, cranial arteritis, Klebsiella encephalitis, Creutzfeldt-Jakob disease, Cushing's syndrome, cystinosis, giant cell inclusion body disease. Inclusion body disease), oculoclonus-myoclonus syndrome, Dandy-Walker syndrome, Danon disease, Dawson's disease, Demosier syndrome, Degerina-Klempke palsy, dementia, dentate nucleus-cerebellar ataxia, dentate nucleus-red nucleus atrophy, dermatomyositis, developmental motor disorders, Dweck syndrome, diffuse sclerosis, autonomic dysfunction, writing disorders, reading disorders, dysphagia, motor disorders, myoclonic cerebellar dyssynergia, progressive cerebellar dyssynergia, epilepsy (e.g., Amish infantile epilepsy syndrome [AIES], benign familial infantile epilepsy [BFIS]),Benign familial neonatal epilepsy [BFNS], childhood absence epilepsy [CAE], childhood-onset epileptic encephalopathy [COEE], Dravet syndrome [DS], early infantile epileptic encephalopathy [EIEE], familial adult myoclonic epilepsy [FAME], familial febrile seizures [FFS], familial focal epilepsy with variable lesions [FFEVF], familial infantile myoclonic epilepsy [FIME], familial temporal lobe epilepsy [FTLE], focal epilepsy and speech disorders with or without intellectual disability [FESD], generalized epilepsy with paroxysmal motor disorders [GEPD], generalized epilepsy with febrile seizures plus [GEFS+], idiopathic generalized epilepsy [IGE], juvenile absence epilepsy [JAE], juvenile myoclonic epilepsy [JME], myopathic epilepsy ... Clonic-atonic epilepsy [MAE], nocturnal frontal lobe epilepsy [NFLE], progressive myoclonic epilepsy [PME], pyridoxine oxidase deficiency [PNPOD], pyridoxine-dependent epilepsy [EPD], and severe myoclonic epilepsy of infancy [SMEI], Fabry disease, Fall syndrome, familial autonomic dysfunction, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Fabry disease, fibromuscular dysplasia, Fisher syndrome, infantile relaxation syndrome, Friedreich ataxia, frontotemporal dementia, fucoside storage disease, galactosialidosis, Gaucher disease, systemic ganglioside storage disease, Göstadmann syndrome, GSS disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion body disease (giant Cellinclusion disease, globular leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, GM1 ganglioside storage disease, GM2 ganglioside storage disease (Tai-Sachs disease), Guillain-Barré syndrome, Hallewarden-Scholes disease, persistent unilateral headache, alternating hemiplegia (hemiplegia) Alterans syndrome, hereditary spastic paraplegia, hereditary ataxic polyneuritis, Holmes-Addie syndrome, holohemorrhagic malformation, Hughes syndrome, Huntington's disease, hydrocephalus, hydrocephalus, Cushing's syndrome, immune-mediated encephalomyelitis, inclusion body myositis, incontinence pigmentosa, infantile hypotonia, infantile axonal dystrophy, occipital cleft with exposed head, Isaac syndrome, Jubert syndrome, Karns-Sell syndrome, Kennedy disease, Kingsburn syndrome, Klein-Levin syndrome, Krippel-Fair syndrome, Krippel-Trenone syndrome (KTS), Klüfer-Busy syndrome, Korsakov amnesia syndrome, Crabbe disease, Kugelberg-Weyland disease, Lambert-Eton myasthenia gravis syndrome, Landau-Cleven syndrome, lateral femoral cutaneous nerve entrapment syndrome, lateral medullary syndrome, Leigh's disease, Lennox-Gasstor syndrome, Lesch-Niehan syndrome, Levin-Critchley syndromeLewy body dementia, lipoprotein storage disease, lissencephaly, locked-in syndrome, Luger's disease, lupus, Lyme disease, Machado-Joseph disease, megalencephaly, alpha-mannoside storage disease, beta-mannoside storage disease, Méridov-Robbes syndrome, Menkes disease, lateral femoral cutaneous neuralgia, metachromatic leukodystrophy, microcephaly, Miller-Fischer syndrome, Möbius syndrome, mucopolysaccharidosis type IH (Heller syndrome), mucopolysaccharidosis type IH / S (Heller-Shee syndrome), mucopolysaccharidosis type IS (Shee syndrome), mucopolysaccharidosis type II (Hunter syndrome), mucopolysaccharidosis type III-A (Samfylbo syndrome type A), mucopolysaccharidosis type III-B (Samfylbo syndrome type A). The following are listed as examples of diseases: Samuelleybo syndrome type B, mucopolysaccharidosis type III-C (Samuelleybo syndrome type C), mucopolysaccharidosis type III-D (Samuelleybo syndrome type D), mucopolysaccharidosis type IV-B (Mochio syndrome type B), mucopolysaccharidosis type VI (Maroto-Lamy syndrome), mucopolysaccharidosis type VII (Slay syndrome), mucopolysaccharidosis type IX (Natovich syndrome), multiple sclerosis, muscular dystrophy, myasthenia gravis, myelinolytic diffuse sclerosis, narcolepsy, neuroacanthocytosis, neurofibromatosis, neuroleptic malignant syndrome, sarcoidosis, Niemann-Pick disease, Ōtahara syndrome, olivopontocerebellar atrophy, oculoclonus myoclonus, and Ossa Levin-McLeod syndrome, pantothenic acid kinase-related neurodegenerative diseases, paraneoplastic syndromes, sensory abnormalities, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal unilateral headache, Parvo-Ros syndrome, Peyrenia, Pena-Shokel syndrome type II, periventricular leukomalacia, phytanoic acid storage disease, Pick's disease, piriformis syndrome, polymyositis, Pompe disease, post-poliomyelitis syndrome, posterior cortical atrophy, primary dentate nucleus atrophy, primary lateral sclerosis, primary progressive aphasia, prion diseases, progressive hemifacial atrophy, progressive motor ataxia, progressive multifocal leukoencephalopathy, progressive sclerotic gray matter dystrophy, progressive supranuclear palsy, prosopagnosia, Ramsey Hunt syndrome Syndrome type I, Ramsey-Hunter syndrome type II, Rasmussen encephalitis, Reifsum disease, Rett syndrome, Reye's syndrome, Riley-Day syndrome, Sandhoff disease, Hilde disease, Setterberg disease, Shay-Dreg syndrome, Sjögren's syndrome, spasticity, spina bifida, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, Still-Richardson-Olshefsky syndrome, striatal substantia nigra degeneration, Sturge-Weber syndrome, tardive dyskinesia, Tau proteinosis, Ty-Sachs disease, thoracic outlet syndrome, thyrotoxic myopathy, painful tic, Todd's paralysis, trigeminal neuralgia, tropical spastic paraplegia, Troy syndrome, vascular dementia, von Ecknomer disease.Von Hippel-Lindau disease (VHL), von Recklinghausen disease, Wallenberg syndrome, Wernig-Hoffmann disease, Wernicke-Korsakov syndrome, West syndrome, Whipple's disease, Williams syndrome, Wilson's disease, Wollman's disease, X-linked spinal-bulbar muscular atrophy, Zelweg syndrome, multiple sclerosis with brain atrophy, Lewis body dementia (LBD), or Anglemann syndrome.

[0113] In some implementation schemes, CNS disorders include: lack of will, achromatopsia, acid lipase disorder, acid maltase deficiency, acid storage disorder, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Addie pupils, Addie syndrome, adrenoleukodystrophy, agnosia, agraphia, Eckhardt syndrome, Eckhardt-Gutier syndrome, motor blindness, Alexander disease, Alpert disease, and alternating hemiplegia. Hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), amnesia, anencephaly, aneurysm, Angelman syndrome, hemangioma, hypoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, Aromatic L-amino acid decarboxylase deficiency (AADC deficiency), aspartate glucosamineuria, Asperger's syndrome, ataxia, ataxia-telangiectasia (Lewis-Barr syndrome), ataxia and cerebellar or spinocerebellar degeneration, attention deficit hyperactivity disorder (ADHD). Disorders, autism, autonomic dysfunction, Bass syndrome, Barten disease, Becker's muscle rigidity, Behçet's disease, Bell's palsy, Bernhardt-Rose syndrome, bipolar disorder, Binswanger disease, Bloch-Sulzberger syndrome, Bradbury-Eggleston syndrome, Brown-Seckal syndrome, medullary spinal muscular atrophy, CADASIL, Canavan disease, carpal tunnel syndrome, burning neuralgia, cavernous malformation, cavernous hemangioma, central cervical spinal cord syndrome, central spinal cord syndrome, central pontine myelinolysis, ceramide deficiency, cerebellar degeneration, cerebellum Developmental disorders, cerebral beriberi, cerebral gigantism, cerebral palsy, brain-ocular-facial-skeletal syndrome (COFS), Charles Bonner syndrome, cholesterol ester storage disease, chorea, chorea-acanthosis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic fatigue syndrome, chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, cavitary brain, congenital myasthenia gravis, corticobasal degeneration, cranial arteritis, Kernel encephalitis, Creutzfeldt-Jakob disease, Cushing's syndrome, cystinosis, cytomegalovirus infection. Inclusion body disease), oculoclonus-myoclonus syndrome, Dandy-Walker syndrome, Danon disease, Dawson's disease, Demosier syndrome, Degerina-Klempke palsy, dementia, dentate nucleus-cerebellar ataxia, dentate nucleus-red nucleus atrophy, scratching, dermatomyositis, developmental motor disorders, Dweck syndrome, diffuse sclerosis, Down syndrome, Duchenne muscular dystrophy, autonomic dysfunction, writing disorders, dyslexia, dysphagia, motor disorders.Myoclonic cerebellar dyssynergia, progressive cerebellar dyssynergia, epilepsy (e.g., Amish infantile epilepsy syndrome [AIES], benign familial infantile epilepsy [BFIS], benign familial neonatal epilepsy [BFNS], childhood absence epilepsy [CAE], childhood-onset epileptic encephalopathy [COEE], Dravet syndrome [DS], early infantile epileptic encephalopathy [EIEE], afro syndrome, familial adult myoclonic epilepsy [FAME], familial febrile seizures [FFS], familial focal epilepsy with variable lesions [FFEVF], familial infantile myoclonic epilepsy [FIME], familial temporal lobe epilepsy [FTLE], focal epilepsy and speech disorders with or without intellectual disability [FESD], generalized epilepsy with paroxysmal motor disorders [GEPD], generalized epilepsy with febrile seizures plus [GEFS+], idiopathic generalized epilepsy [IGE], juvenile absence epilepsy Epilepsy [JAE], Juvenile Myoclonic Epilepsy [JME], Myoclonic-Atonic Epilepsy [MAE], Nocturnal Frontal Lobe Epilepsy [NFLE], Progressive Myoclonic Epilepsy [PME], 5'-Pyridoxamine Oxidase Deficiency [PNPOD], Pyridoxine-Dependent Epilepsy [EPD], and Severe Myoclonic Epilepsy of Infancy [SMEI], Fabry Disease, Fall Syndrome, Familial Autonomic Dysfunction, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Periodic Paralysis, Familial Spastic Paralysis, Fabry Disease, Fetal Alcohol Syndrome, Fibromuscular Dysplasia, Fisher Syndrome, Infantile Relaxatosis, Fauville Syndrome, Friedreich Ataxia, Frontotemporal Dementia, Fucoidoside Storage Disease, Galactosylsialic Acid Storage Disease, Gaucher Disease, Systemic Ganglioside Storage Disease, Gostelmann Syndrome, GSS Disease, Giant Axonal Neuropathy, Giant Cell Arteritis, Giant Cell Inclusion Disease Inclusion disease, spheroid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, GM1 ganglioside storage disease, GM2 ganglioside storage disease (Tai-Sachs disease), gray matter heterotopia, Guillain-Barré syndrome, Hallewarden-Scholes disease, persistent unilateral headache, alternating hemiplegia, hereditary spastic paraplegia, hereditary ataxic polyneuritis, herpes zoster (herpes zoster). Zoster, Hirayama disease, Holmes-Addie syndrome, holohemorrhagic forebrain, Hughes syndrome, Huntington's disease, hydrocephalus, hydrocephalus, Cushing's syndrome, immune-mediated encephalomyelitis, inclusion body myositis, incontinence pigmentosa, infantile hypotonia, infantile axonal dystrophy, occipital cleft with exposed head, Isaac syndrome, chromosome 15 isochromosomal abnormality, Jubert syndrome, Karnke syndrome, Kearns-Sell syndrome, Kennedy disease, Kingsburn syndrome, Klein-Levin syndrome, Krippel-Fair syndromeKrippel-Trenone syndrome (KTS), Klüffer-Busy syndrome, Korsakov amnesia syndrome, Crabbe disease, Kugelberg-Weyland disease, Lafra disease, Lambert-Eton myasthenia gravis syndrome, Landau-Clevenner syndrome, lateral femoral cutaneous nerve entrapment syndrome, lateral medullary syndrome, Lewy body dementia. Dementia, lipoprotein storage diseases, lissencephaly, locked-in syndrome, Lughlee's disease, lupus, Lyme disease, Machado-Joseph disease, megalencephaly, alpha-mannosin storage disease, beta-mannosin storage disease, M.R. Roe syndrome, Menkes disease, lateral femoral cutaneous neuralgia, metachromatic leukodystrophy, microcephaly, Miller-Fischer syndrome, misophonia, Möbius syndrome, Moyamoya disease, mucopolysaccharidosis type IH (Heller syndrome), mucopolysaccharidosis type IH / S (Heller-Shee syndrome), mucopolysaccharidosis type IS (Shee syndrome), mucopolysaccharidosis type II (Hunter syndrome) Mucopolysaccharidosis (MPS) type III-A (Samfilepo syndrome type A), MPS type III-B (Samfilepo syndrome type B), MPS type III-C (Samfilepo syndrome type C), MPS type III-D (Samfilepo syndrome type D), MPS type IV-B (Mochio syndrome type B), MPS type VI (Maroto-Lamy syndrome), MPS type VII (Slay syndrome), MPS type IX (Natovich syndrome), multiple sclerosis, muscular dystrophy, myasthenia gravis, and myelinolytic diffuse sclerosis. Narcolepsy, neuroacanthocytosis, neurofibromatosis, neuroleptic malignant syndrome, neurosarcoidosis, Niemann-Pick disease, Otahara syndrome, olivopontocerebellar atrophy, oculoclonus myoclonus, Osullivan-McLeod syndrome, otosclerosis, overuse syndrome, pantothenic kinase-associated neurodegenerative disease, paraneoplastic syndrome, paroxysmal sensory abnormalities, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal unilateral headache, Parfum-Ros syndrome, Peyrenia, Pena-Shokel syndrome type II, periventricular leukomalacia, phytanoic acid storage disease, Pick's disease, piriformis syndrome, polymyositis, Pang Bezoar disease, post-poliomyelitis syndrome, posterior cortical atrophy, Pred. Willie syndrome, primary dentate nucleus atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive hemifacial atrophy, progressive motor ataxia, progressive multifocal leukoencephalopathy, progressive sclerotic gray matter dystrophy, progressive supranuclear palsy, prosopagnosia, quadriplegia, rabies, Ramsey Hunt syndrome type I, Ramsey Hunt syndrome type II, Rasmussen encephalitis, Rifsum disease, Rett syndrome, Reye's syndrome, Riley-Day syndrome, Sandhof disease, Hilde disease, Setterberg disease.Herpes zoster, Shay-Dreg syndrome, Sjögren's syndrome, spasm, spina bifida, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, Still-Richardson-Olshewski syndrome, substantia nigra degeneration, Sturge-Weber syndrome, tardive dyskinesia, Tau disease, Tay-Sachs disease, tetanus, thoracic outlet syndrome, thyrotoxic myopathy, painful tic, Todd's paralysis, trigeminal neuralgia, tropical spastic paraplegia Troy syndrome, Onfrigh-Lundberg disease, vascular dementia, vertigo, von Eknomer disease, von Hippel-Lindau disease (VHL), von Recklinghausen disease, Wallenberg syndrome, Wernig-Hoffmann disease, Wernicke-Korsakov syndrome, West syndrome, Whipple's disease, Williams syndrome, Wilson's disease, Wollman's disease, X-linked spinal-bulbar muscular atrophy, Y-linked hearing loss, Zelweg syndrome, multiple sclerosis with brain atrophy, Lewis body dementia (LBD), or Anglemann syndrome.

[0114] Any aspect or implementation disclosed herein may be combined with any other aspect or implementation disclosed herein. Attached Figure Description

[0115] The patent or application document contains at least one color drawing. The Patent Office will provide a copy of the patent or patent application publication with the color drawing, as required and subject to payment of necessary fees.

[0116] Figure 1 A schematic diagram illustrating the in vivo study design in mice is provided.

[0117] Figure 2A The fragment analyzer data plots show uncapped H1N1 HA mRNA (left inset) and H1N1 HA protein expression (right inset). Figure 2B An exemplary LNP composition and its characteristics are shown.

[0118] Figures 3A to 3C The titer analysis of anti-H1N1 HA IgG / M antibody is shown. Figure 3A ELISA data of α-H1N1 HA IgG / M antibody levels for the composition shown are presented. Figure 3B ELISA data on α-H1N1 HA IgG / M antibody levels grouped by dose are shown. Figure 3C ELISA data on α-H1N1 HAIgG / M antibody levels administered at microdose levels (0.03 µg / dose and 0.1 µg / dose) are presented.

[0119] Figures 4A to 4CThe graph shows the T cell proliferation analysis on day 14. Figure 4A The proliferation analysis of all live cells is shown. Figure 4B The proliferation analysis of T helper cells is shown. Figure 4C The proliferation analysis of cytotoxic T cells is shown.

[0120] Figures 5A to 5C The graph shows the T cell proliferation analysis on day 28. Figure 5A The proliferation analysis of all live cells is shown. Figure 5B The proliferation analysis of T helper cells is shown. Figure 5C The proliferation analysis of cytotoxic T cells is shown.

[0121] Figures 6A to 6C The graph shows the T cell activation analysis on day 14. Figure 6A The activation analysis of all T cells is shown. Figure 6B The activation analysis of T helper cells is shown. Figure 6C The activation analysis of cytotoxic T cells is shown.

[0122] Figures 7A to 7C The graph shows the T cell activation analysis on day 28. Figure 7A The activation analysis of all T cells is shown. Figure 7B The activation analysis of T helper cells is shown. Figure 7C The activation analysis of cytotoxic T cells is shown.

[0123] Figures 8A to 8D A heatmap of cytokine expression in mouse serum after treatment with H1N1 HA mRNA LNP is shown. Figure 8A The cytokine heatmap analysis on day 14 is shown. Figure 8B The cytokine heatmap analysis on day 28 is shown. Figure 8C A heatmap analysis of cytokines was shown for micro-dose administration on day 14. Figure 8D A heatmap analysis of cytokines after micro-dose administration on day 28 is shown.

[0124] Figures 9A to 9B The titer analysis of anti-H1N1 HA IgG / M antibody is shown. Figure 9A ELISA data of α-H1N1 HA IgG / M antibody levels for the composition shown are presented. Figure 9B ELISA data on α-H1N1 HA IgG / M antibody levels grouped by dose (0.03 µg / dose, 1 µg / dose, and 10 µg / dose) are shown.

[0125] Figures 10A to 10B A heatmap of cytokine expression in cultured spleen cells after treatment with H1N1 HA mRNA LNP is shown. Figure 10A The cytokine heatmap analysis for day 7 is shown. Figure 10B The cytokine heatmap analysis on day 28 is shown.

[0126] Figures 11A to 11B A heatmap of cytokine expression in cultured lymph nodes after treatment with H1N1 HA mRNA LNP is shown. Figure 11A The cytokine heatmap analysis for day 7 is shown. Figure 11B The cytokine heatmap analysis on day 28 is shown.

[0127] Figures 12A to 12C Antibody and cytokine analyses of mice treated with RSV mRNA LNPs are shown. Figure 12A An ELISA graph showing the α-RSV antibody levels of the composition presented is shown. Figure 12A A heatmap of cytokine expression in cultured spleen cells after treatment with H1N1 HA mRNALNP is shown. Figure 12B The following is a heatmap analysis of cytokines in cultured spleen cells on days 7 and 28 after treatment with RSV mRNA LNP. Figure 12C The following is a cytokine heatmap analysis of cultured lymph nodes on days 7 and 28 after treatment with RSV mRNA LNP.

[0128] Figure 13 (Top) Shows the number of differentially expressed genes (DEGs) in innate cells. Figure 13 (Bottom) shows the number of differentially expressed genes (DEGs) in adaptive immune cells.

[0129] Figures 14A to 14K A volcano diagram of various immune cells is shown on day 1. Figure 14A A volcano diagram of initial CD8 T cells is shown. Figure 14B A volcano diagram of nascent CD4 T cells is shown. Figure 14C A volcano diagram of B cells is shown. Figure 14D A volcano diagram of T-reg cells is shown. Figure 14E A volcano diagram of natural killer (NK) cells is shown. Figure 14F A volcano diagram of monocyte-derived macrophages is shown. Figure 14G A volcano diagram of pDC1 cells is shown. Figure 14H A volcano diagram of cDC1 cells is shown. Figure 14I A volcano diagram of mDC cells is shown. Figure 14J A volcano diagram of effector CD4 T cells is shown. Figure 14K A volcano diagram of effector CD8 T cells is shown. Figure 14L A volcano diagram of cDC2 is shown.

[0130] Figures 15A to 15G The results show a comparison of single-cell RNA sequences administered to lymph node cells via vaccine injection, presented by uniform manifold approximation and projection (UMAP). Figure 15A A comparison of composition 2H and buffer solution is shown. Figure 15B A comparison of composition 2B and buffer solution is shown. Figure 15C A comparison of composition X / pH4 and buffer solution is shown. Figure 15D A comparison of composition X / pH6.5 and buffer solution is shown. Figure 15E A comparison of composition X / pH4, composition X / pH6.5, and buffer solution is shown. Figure 15F A comparison of composition 2B, composition 2H, and buffer solution is shown. Figure 15G A comparison of composition X / pH4, composition X / pH6.5, composition 2H, and buffer solution is shown.

[0131] Figures 16A to 16B Conditional survival rates of mice challenged with 100 PFU of H1N1 PR8 at 28 days are shown. Figure 16A The results show a comparison of conditional survival rates in mice that received composition 2B, composition X, or composition 2H. Figure 16B Conditional survival rates of mice administered with composition 2B (top), composition X (middle), or composition 2H (bottom) are shown.

[0132] Figures 17A to 17B Conditional survival rates of mice challenged with 60 PFU of H1N1 PR8 at 84 days are shown. Figure 17A The results show a comparison of conditional survival rates in mice that received composition 2B, composition X, or composition 2H. Figure 17B Conditional survival rates of mice administered with composition 2B (top), composition X (middle), or composition 2H (bottom) are shown.

[0133] Figures 18A to 18C Conditional survival rates of mice challenged with 60 PFU of H1N1 PR8 at 84 days are shown. Figure 18A Conditional survival rates of mice administered with composition V, composition 2B, composition X, or composition 2H are shown. Figure 18B The results show a comparison of conditional survival rates in mice that were administered composition V, composition 2B, or compositions X through 2H. Figure 18C The results show a comparison of conditional survival rates in mice administered with composition V, composition 2B, composition X, or composition 2H.

[0134] Figures 19A to 19CThe changes in body weight of mice challenged with 60 PFU of H1N1 PR8 at 84 days are shown. Figure 19A The changes in body weight of mice administered with composition V, composition 2B, composition X, or composition 2H are shown. Figure 19B The results show a comparison of weight changes in mice that received composition V, composition 2B, or compositions X through 2H. Figure 19C The results show a comparison of weight changes in mice that received composition V, composition 2B, composition X, or composition 2H.

[0135] Figure 20 An exemplary LNP composition and its characterization are shown.

[0136] Figures 21A to 21F In vivo brain imaging (IVIS) of a patient receiving the LNP composition is shown. ® ). Figure 21A In vivo imaging (IVIS) of mice treated with composition A before and after whole-brain MAV removal is shown. ® ), and in vivo imaging of brain slices. Figure 21B In vivo imaging (IVIS) of mice treated with composition W before and after whole-brain MAV removal is shown. ® ), and in vivo imaging of brain slices. Figure 21C In vivo imaging (IVIS) of mice treated with composition B before and after whole-brain MAV removal is shown. ® ), and in vivo imaging of brain slices. Figure 21D In vivo imaging (IVIS) of mice treated with composition G before and after whole-brain MAV removal is shown. ® ), and in vivo imaging of brain slices. Figure 21E In vivo imaging (IVIS) of mice treated with composition I before and after whole-brain MAV removal is shown. ® ), and in vivo imaging of brain slices. Figure 21F In vivo imaging (IVIS) is shown ® ) quantitative results.

[0137] Figures 22A to 22C In vivo imaging (IVIS) of the patient receiving the LNP composition was shown. ® ). Figure 22A Whole-body in vivo imaging (IVIS) of mice administered various LNP compositions is shown. ® ). Figure 22B In vivo organ imaging (IVIS) of mice administered various LNP compositions is shown. ® ). Figure 22C In vivo imaging (IVIS) is shown ®) quantitative results.

[0138] Figure 23 Immunofluorescence staining of the brain of tdTomato mice administered with composition A is shown.

[0139] Figures 24A to 24B Immunofluorescence staining of the brain of tdTomato mice administered with composition A is shown. Figure 24A Endothelial staining of tdTomato mice is shown (Paxinos & Franklin, Fig. 15). Figure 24B Endothelial staining of tdTomato mice is shown (Paxinos & Franklin, Fig. 18).

[0140] Figures 25A to 25B Immunofluorescence staining of the brain of tdTomato mice administered with composition A is shown. Figure 25A Neuronal staining (NeuN) in tdTomato mice is shown (Paxinos & Franklin, Fig. 59-59). Figure 25B Neuronal staining (NeuN) in tdTomato mice is shown (Paxinos & Franklin, Fig. 59-59).

[0141] Figure 26 Astrocyte staining (GFAP) of tdTomato mice treated with composition A is shown (Paxinos & Franklin). Figure 1 (Sniffing bulb).

[0142] Figure 27 Neuronal staining (NeuN) in tdTomato mice treated with composition A is shown (Paxinos & Franklin Fig. 43, cortex / thalamus / hypothalamus; top) and astrocyte staining (GFAP) in tdTomato mice treated with composition A is shown (Paxinos & Franklin Fig. 67, cerebellum / rhomboids; bottom).

[0143] Figure 28 An exemplary LNP composition and its characterization are shown.

[0144] Figures 29A to 29C Immunofluorescence staining of the brains of tdTomato mice administered with composition 2F, composition 2I, or composition 2B is shown. Figure 29A The CD31 staining of tdTomato mice that received composition 2I is shown (Paxinos & Franklin Fig. 58). Figure 29BThe CD31 staining of tdTomato mice that received composition 2B is shown (Paxinos & Franklin, Fig. 43). Figure 29C CD31 staining of tdTomato mice that received composition 2F, composition 2I or composition 2B is shown (Paxinos & Franklin Fig. 43 or 58).

[0145] Figures 30A to 30C Immunofluorescence staining of the brains of tdTomato mice administered with composition 2F, composition 2I, or composition 2B is shown. Figure 30A PDGFRβ staining of tdTomato mice that received composition 2I is shown (Paxinos & Franklin Fig. 58). Figure 30B PDGFRβ staining of tdTomato mice that received composition 2B is shown (Paxinos & Franklin Fig. 43). Figure 30C PDGFRβ staining of tdTomato mice administered with composition 2F, composition 2I or composition 2B is shown (Paxinos & Franklin Fig. 43 or 58).

[0146] Figures 31A to 31C Immunofluorescence staining of the brains of tdTomato mice administered with composition 2F, composition 2I, or composition 2B is shown. Figure 31A SMA and PDGFRβ staining of tdTomato mice that received composition 2I are shown (Paxinos & Franklin Fig. 58). Figure 31B SMA and PDGFRβ staining of tdTomato mice that received composition 2B are shown (Paxinos & Franklin Fig. 43). Figure 31C SMA staining of tdTomato mice that received composition 2F, composition 2I or composition 2B is shown (Paxinos & Franklin Fig. 43 or 58). Detailed Implementation

[0147] This document provides lipid nanoparticle (LNP) compositions that contain antigens (such as pathogen-associated antigens) or polynucleotides encoding antigens (such as pathogen-associated antigens). In some embodiments, the LNP compositions contain antigens from pathogens involved in infectious diseases. This disclosure also provides pharmaceutical compositions comprising LNP compositions and vaccines comprising LNP compositions. Methods of using LNP compositions, pharmaceutical compositions, and vaccines comprising LNP compositions are also provided herein.

[0148] This document also provides lipid nanoparticle (LNP) compositions comprising cancer antigens or polynucleotides encoding cancer antigens. In some embodiments, the LNP compositions comprise cancer antigens. This disclosure also provides pharmaceutical compositions comprising LNP compositions and vaccines comprising LNP compositions. Methods of using LNP compositions, pharmaceutical compositions, and vaccines comprising LNP compositions are also provided herein.

[0149] In some aspects, this disclosure relates to the surprising discovery by the inventors that the LNP compositions described herein produce in vivo an adaptive immune response at least as strong or stronger than that of a reference LNP composition (e.g., an LNP composition using SM-102 as an ionizable cationic lipid and / or lacking SORT lipids) for delivering the same polynucleotide encoding the same antigen. In some aspects, this disclosure relates to the surprising discovery by the inventors that the LNP compositions described herein produce an immune response with the same or lower cytokine response compared to a reference LNP composition. In some aspects, this disclosure relates to the surprising discovery by the inventors that the LNP compositions described herein produce an immune response with the same or higher cytokine response compared to a reference LNP composition.

[0150] This document also provides lipid nanoparticle (LNP) compositions comprising: a polypeptide or a polynucleotide encoding a polypeptide, such as a gene associated with a CNS disease; or a polynucleotide encoding a gene editor for editing genes associated with a CNS disease; or a polynucleotide encoding a polypeptide, such as a gene encoding a CNS disease; or a polynucleotide encoding a gene editor for editing genes associated with a CNS disease. This disclosure also provides pharmaceutical compositions comprising LNP compositions. Methods of using the LNP compositions and pharmaceutical compositions are also provided herein.

[0151] In some aspects, this disclosure relates to the surprising discovery by the inventors that the LNP compositions described herein generate in vivo an adaptive immune response at least as strong or stronger than that of a reference LNP composition (e.g., an LNP composition employing SM-102 as an ionizable cationic lipid and / or lacking SORT lipids) for delivering the same polynucleotide encoding the same polypeptide. In some aspects, this disclosure relates to the surprising discovery by the inventors that the LNP compositions described herein generate an immune response with the same or lower cytokine response compared to the reference LNP compositions. In some aspects, this disclosure relates to the surprising discovery by the inventors that the LNP compositions described herein generate an immune response with the same or higher cytokine response compared to the reference LNP compositions. Further advantages of the disclosed embodiments will become apparent from the following description of the embodiments.

[0152] Other advantages of the disclosed implementation scheme will become apparent from the following description of the implementation scheme.

[0153] I. Definition

[0154] Before describing embodiments of this disclosure, it should be understood that such embodiments are provided by way of example only, and various alternatives to the embodiments of this disclosure described herein may be employed in the practice of this invention. Numerous changes, modifications, and substitutions will be possible for those skilled in the art without departing from this invention.

[0155] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Various scientific dictionaries including those containing the terms included herein are well-known and available to those skilled in the art. While any methods and materials similar to or equivalent to those described herein may be used to practice or test this disclosure, preferred methods and materials are described herein. Therefore, a more complete description of the terms defined immediately thereafter can be obtained by referring to the entire specification.

[0156] The singular forms “a,” “a,” and “the” are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0157] As used herein, the term “about” means a range of values ​​that would reasonably be considered by one of ordinary skill in the art to be similar to the specified value. For example, “about” means within the standard deviation using measurement methods generally acceptable in the art. For example, “about” means a range extended to + / - 10%, + / - 5%, + / - 3%, or + / - 1% of the specified value.

[0158] The term "at least" as used in this article is followed by a number to indicate the beginning of a range from which that number begins (which can be a range with or without an upper limit, depending on the defined variable). For example, "at least 1" means 1 or greater than 1.

[0159] As used herein, the term "at most" followed by a number indicates the end of a range, which may be a range with a lower limit of 1 or 0, or a range without a lower limit, depending on the defined variable. For example, "at most 4" means 4 or less, and "at most 40%" means 40% or less. In this specification, when a range is given as "(first number) to (second number)" or "(first number) - (second number)", this means a range whose lower limit is the first number and whose upper limit is the second number. For example, 25mm to 100mm means a range whose lower limit is 25mm and whose upper limit is 100mm.

[0160] As used herein, the term "lipid nanoparticle" or "LNP" refers to a carrier or mediator formed from one or more lipid components for the delivery of payloads (e.g., nucleic acids, proteins, peptides, polypeptides, polynucleotides, or oligonucleotides) in the context of drug development. Lipid nanoparticles may have one or more lipids with at least one nanometer dimension (e.g., 1 nm to 1000 nm). Typically, lipid nanoparticle compositions for delivery consist of one or more lipids, such as, but not limited to, synthetic ionizable or cationic lipids, phospholipids, structural lipids, and polyethylene glycol (PEG) lipids. These compositions may also include other lipids. In some embodiments, at least one therapeutic agent (e.g., mRNA) may be trapped in the lipid portion of the lipid nanoparticle or in an aqueous space encapsulated by some or all of the lipid portions of the lipid nanoparticle, thereby protecting the at least one therapeutic agent from enzymatic degradation by other undesirable effects (e.g., adverse immune responses) induced by biological mechanisms of the target subject, tissue, and / or cells. In some embodiments, the lipid nanoparticles comprise at least one therapeutic agent (e.g., mRNA) organized within inverted lipid micelles and encapsulated within a lipid monolayer membrane, or embedded between adjacent lipid bilayers. In some embodiments, the morphology of the lipid nanoparticles differs from that of conventional liposomes, which are characterized by a lipid bilayer surrounding an aqueous core. In some embodiments, the lipid nanoparticles are substantially non-toxic. In some embodiments, the therapeutic agent (e.g., mRNA) is resistant to intracellular or intercellular enzymatic degradation in aqueous solutions.

[0161] As used herein, the term "SORT lipid" refers to lipids that, when included in a lipid nanoparticle (LNP) composition, enable the LNP to selectively and predictably target an organ, cell type, or tissue (e.g., as described in the following literature: Cheng et al., Nature 15:313-320 (2020); Wang et al., Nat. Protoc.18(1):265-291; and U.S. Patent Publications US 2022 / 0071916 A1 and US 2021 / 0259980 A1, the entire contents of which are incorporated herein by reference). For example, adding specific SORT lipids to an LNP can redirect (e.g., retarget) the LNP from the liver to the lungs. SORT lipids include, but are not limited to, permanently cationic lipids, anionic lipids, zwitterionic lipids, and ionizable cationic lipids. Unbound by theory, anionic SORT lipids generally favor delivery to the spleen, at least when administered intravenously; ionizable cationic SORT lipids generally favor delivery to the liver; permanent cationic SORT lipids generally favor delivery to the lungs; and zwitterionic SORT lipids favor delivery to the spleen.

[0162] As used herein, the term "ionizable cationic lipid" refers to lipids and lipid-like molecules having at least one pKa in the range of about 4.5–8, such that, without being bound by theory, they can promote the release of LNP payloads upon uptake into the endosome compartment of the cell. Ionizable cationic lipids can carry a neutral charge at pH above their pKa, but become positively charged at pH below their pKa to promote membrane fusion and subsequent cytoplasmic release. Exemplary ionizable cationic lipids have one or more nitrogen atoms, such as tertiary amine groups, in the pKa range of about 4.5–8.

[0163] As used herein, the term “permanent cationic lipid” refers to a lipid or lipid-like molecule that is positively charged in physiologically relevant solutions, regardless of pH (positively charged in the absence of pKa or in the absence of pKa greater than 8). Illustrative permanent cationic lipids may include quaternary ammonium groups and lack negatively charged phosphate groups. Without being bound by theory, permanent cationic lipids may act as SORT lipids by increasing the apparent pKa of LNP, as exemplified by, for example, Dilliard et al., PNAS USA. 118(52):e2109256118 (2021), the entire contents of which are incorporated herein by reference.

[0164] As used herein, the term "anionic lipid" refers to lipids that carry a negative charge at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoylphosphatidylglycerol (POPG), ethylphosphocholine, and other anionic modifying groups linked to neutral lipids.

[0165] As used herein, the term "phospholipid" refers to a lipid containing a phosphate group. The lipid component of a lipid nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. Typically, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.

[0166] As used herein, the term "sterol" refers to a subgroup of steroids having a hydroxyl group at the 3-position of ring A in a gonane ring system. "Cholesterol" is a sterol with a four-ring fused hydrocarbon structure (gonane ring system), these rings having a polar hydroxyl group at one end and an eight-carbon branched aliphatic tail at the other. Unbound by theory, the tetracyclic structure of cholesterol contributes to cell membrane fluidity because the molecule is in a trans conformation, making all parts of cholesterol, except the side chains, rigid and planar. Cholesterol affects the fluidity, thickness, compressibility, water permeability, and intrinsic curvature of the lipid bilayer, for example, in LNPs. For example, "sterol" can be cholesterol or sitosterol.

[0167] As used herein, the term "PEG lipid" refers to a lipid modified with polyethylene glycol units. In some embodiments, the PEG lipid comprises dimyristoylglycerol (DMG). In some embodiments, the PEG lipid comprises 1,2-distearate-sn-glycerol-3-phosphoethanolamine (DSPE).

[0168] As used herein, the phrase “N / P ratio” refers to the molar ratio of nitrogen in a lipid composition to phosphate in a polynucleotide payload.

[0169] As used herein, the term "apparent pKa" refers to the total dissociation constant of all titratable groups in a lipid nanoparticle. Apparent pKa is an experimentally determined value for a molecule or nanoparticle. Apparent pKa can be expressed as the pH at which the number of ionized (protonated) and deionized groups in the system are equal. The surface charge and ionic interactions of the nanomaterial assembled in the nanoparticle can be estimated from the apparent pKa. The apparent pKa of a nanoparticle can be the result of the average ratio of all ionized groups to deionized groups in the nanoparticle. Therefore, apparent pKa is not the intrinsic pKa value of any single molecule. The apparent pKa of nanoparticles can be measured using various techniques. For example, acid-base titration of 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) fluorescence method is widely used to determine the apparent pKa of blank nanoparticles.

[0170] As used herein, the phrase "lipid:RNA ratio" refers to the lipid mass (e.g., nanograms or milligrams) corresponding to one unit (i.e., nanograms or milligrams) of mRNA active pharmaceutical ingredient. Without being bound by theory, the lipid:RNA ratio may affect the encapsulation efficiency of lipid nanoparticles.

[0171] The term "molar percentage" is the number of moles of one component in a given mixture divided by the total number of moles in the given mixture, and then multiplied by 100%.

[0172] As used herein, the term "encapsulation" refers to the process of confining a payload within the LNP described herein. For example, "encapsulation" refers to confining an mRNA molecule within the LNP described herein. The term "encapsulation efficiency" refers to the proportion of payload encapsulated within or otherwise coupled to the lipid nanoparticle composition when an LNP is formed. Encapsulation efficiency can be determined by comparing the amount of input payload to the amount of payload in the LNP sample, or by comparing the amount of payload in the LNP to the free excess payload in the sample. For example, fluorescence detection analysis (e.g., RiboGreen) ™ This is used to determine encapsulation efficiency by measuring free RNA in a sample with intact LNPs and comparing it with total RNA in a sample treated to destroy LNPs.

[0173] The term "payload" refers to one or more bioactive molecules, such as small molecules, biomolecules, nucleic acids (e.g., DNA, RNA, siRNA, and shRNA), proteins, or peptides, contained in an LNP composition. For example, the payload may be covalently or nonvalently bound to, encapsulated in, coupled to, or form a complex with the LNP within the LNP composition.

[0174] The terms “antigen” or “immunogen” are used in their common literal sense, referring to a molecule, compound, or composition capable of inducing an immune response (cellular or humoral), such as a cytotoxic T lymphocyte (CTL) response, a B cell response (e.g., the production of antibodies that specifically bind to the antigenic epitope), an NK cell response, or any combination thereof, when administered to or expressed in an immunocompetent subject. Antigens may include peptides or polypeptides (including glycoproteins). For example, an antigen may include a peptide, polypeptide, or polypeptide complex that elicits an immune response. For example, an antigen may include one or more immunogenic epitopes associated with a viral pathogen or a cancer antigen. An antigen may be a full-length naturally occurring protein or a fragment of a naturally occurring protein. An antigen may be a full-length modified protein or a fragment of a modified protein, wherein the modified protein differs from the naturally occurring protein by one or more amino acids, one or more associated compounds (e.g., glycosylation, acetylation, methylation, or phosphorylation), etc. As used herein, the term antigen is not limited to the portion of a polypeptide or polypeptide complex containing an antigenic epitope.

[0175] As used herein, the term "pathogen-associated antigen" is an antigen that is derived from, co-derived from, or similar to antigens present in or on a pathogenic organism (e.g., an infectious microorganism). Like other antigens, pathogen-associated antigens elicit an immune response in subjects who come into contact with them. As used herein, pathogen-associated antigens may be identical to antigens associated with naturally occurring (i.e., wild-type) pathogens; alternatively, pathogen-associated antigens may be engineered or modified to differ from the corresponding antigens associated with naturally occurring pathogens.

[0176] As used herein, the term "cancer antigen" or "tumor-specific antigen" is an antigen derived from, or similar to, an antigen present in or on cancer cells, or in or on a tumor containing cancer cells. Like other antigens, cancer antigens elicit an immune response in a subject upon contact with them. As used herein, cancer antigens may be identical to antigens associated with naturally acquired (e.g., biopsy) cancer cells, cancer cells present in a subject, or cultured cancer cells; in some embodiments, cancer antigens are engineered or modified to differ from corresponding antigens associated with natural cancer cells. In this disclosure, the terms "cancer antigen," "tumor-specific antigen," etc., are synonymous.

[0177] An epitope is a part of an antigen that is recognized by the immune system (especially by antibodies or T-cell receptors).

[0178] As used herein, the term “immunogenicity” refers to, for example, the ability to stimulate an immune response via T cells, B cells, or both.

[0179] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment,” and “polynucleotide” are used interchangeably and are intended to refer to, but are not limited to, polymeric forms of nucleotides covalently linked together, which may have various lengths, be deoxyribonucleotides or ribonucleotides, or analogs, derivatives, or modifications thereof. Different polynucleotides may have different three-dimensional structures and may perform a variety of known or unknown functions. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, intergenic DNA (including but not limited to heterochromatin DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched-chain polynucleotides, plasmids, vectors, sequence-isolated DNA, sequence-isolated RNA, nucleic acid probes, and primers. Polynucleotides that can be used in the methods of this disclosure may comprise natural nucleic acid sequences and their variants, artificial nucleic acid sequences, or combinations of such sequences. When a nucleotide or nucleoside is chemically and / or structurally modified, the nucleotide or nucleoside may be referred to as a “modified nucleotide” or “modified nucleoside.” Polynucleotides containing modified nucleotides are called "modified polynucleotides," and polynucleotides containing modified nucleosides are called "modified polynucleotides." Modified nucleotides or modified nucleosides can reduce the innate immune response compared to unmodified nucleotides and unmodified nucleosides. See, for example, Karikó K, Buckstein M, Ni H, Weissman D, “Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity.” 2005 Aug;23(2):165-75, and scientific reports and patent documents published by Dr. Karikó and Dr. Weissman, including, for example, US20110143397 and WO2007024708, the entire contents of which are incorporated herein by reference. See also WO2016131052, the entire contents of which are incorporated herein by reference.

[0180] The terms “identity,” “same,” and “sequence identity” refer to the degree to which two best-aligned polynucleotide or polypeptide sequences remain unchanged across the entire alignment window of their components (e.g., nucleotides or amino acids). “Identity” can be readily calculated by known methods, including but not limited to those described in Needleman and Wunsch, J. Mol. Biol. 48:443 (1970). Thus, a polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. The terms “sequence identity percentage,” “identity percentage,” or “same as…” refer to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) compared to the test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are best aligned. In some embodiments, “identity percentage” may refer to the percentage of identical amino acids in an amino acid sequence. For sequence comparison, a sequence is used as a reference sequence and compared to the test sequence. The term “reference sequence” refers to the molecule compared to the test sequence. Sequence alignment methods used to compare and determine the percentage of sequence identity are well known in the art. The best sequence alignment for comparison can be performed, for example, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol.Biol.48:443.

[0181] As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA may be purified from natural sources, produced using recombinant expression systems, and optionally purified, chemically synthesized, etc. Where appropriate, such as in the case of chemically synthesized molecules, mRNA may contain nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. Unless otherwise stated, mRNA sequences are presented in a 5' to 3' orientation.

[0182] As used in this article, the term "shRNA" or "short hairpin RNA" refers to a short sequence of RNA that can form a tight hairpin bend and can be used to silence gene expression.

[0183] As used in this article, the term “microRNA” refers to a non-coding RNA consisting of approximately 22 ribonucleotides that silences messenger RNA by pairing with complementary sequence bases of target mRNA, thereby regulating gene expression in the post-transcriptional stage.

[0184] As used herein, the term "gene editing system" refers to a DNA or RNA editing system comprising one or more guide RNA elements and one or more RNA-guided endonuclease elements. The guide RNA element comprises target RNA or nucleic acid containing a nucleotide sequence substantially complementary to a nucleotide sequence at one or more target genomic regions, and the nucleic acid containing a nucleotide sequence encoding the target RNA. The RNA-guided endonuclease element comprises an endonuclease guided or carried to the target genomic region by the guide RNA element or a nucleic acid containing a nucleotide sequence encoding such an endonuclease.

[0185] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to a polymer of amino acid residues that optionally has one or more post-translational modifications (e.g., glycosylation, acetylation, methylation, and phosphorylation) and / or other modifications.

[0186] When the term "isolated" is applied to polynucleotides or peptides, it means that the polynucleotide or peptide is essentially free of other cellular components associated with it in its native state. It can be, for example, in a homogeneous state and can be in a dry state or in an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The major polynucleotide or peptide present as a key component in a formulation is essentially purified.

[0187] The term "variant" refers to a polypeptide or polynucleotide that has one or more insertions, deletions, or amino acid substitutions relative to a reference polypeptide or polynucleotide.

[0188] The term "subject" refers to a living organism to whom any composition as described herein may be administered. A subject may have a disease or condition that can be treated by administration of the nebulized pharmaceutical composition provided herein, or may be at risk of having such a disease or condition. Non-limiting examples of subjects include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, dairy cows, deer, and other non-mammals. In some embodiments, the subject is a human.

[0189] As used herein, the term "therapeutic effective amount" refers to an amount of therapeutic agent sufficient to treat a disease, condition, or symptom. For example, regarding the use of an LNP with an mRNA payload for the treatment of, for example, cystic fibrosis (CF) or primary ciliary dyskinesia (PCD), a therapeutic effective amount is a dose or concentration of mRNA (e.g., CFTR or PCD mRNA) capable of eradicating, inhibiting, preventing, or slowing the progression of all or part of, respiratory symptoms of, such as CF or PCD, or some combination thereof. Similarly, regarding the use of an LNP with an mRNA payload for the prevention of cancer; for the treatment of cancer or for reducing the severity of cancer; and / or for the inhibition of the growth, proliferation, progression, and / or metastasis of cancer cells, a therapeutic effective amount is a dose or concentration of mRNA (e.g., mRNA encoding a cancer antigen) capable of eradicating, inhibiting, preventing, or slowing the growth, proliferation, progression, and / or metastasis of cancer, or some combination thereof. For a given parameter, the therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as an increase or decrease in a "fold." For example, the therapeutically effective amount may have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or more relative to the control. "Therapeutically effective amount" can vary based on, for example, but not limited to, the compound, the disease or symptom and / or its symptoms, the severity of the disease or symptom and / or its symptoms, the age, weight, and / or health status of the subject to be treated, and the judgment of the prescribing physician. As used herein, the term "therapeuticly effective amount" can also refer to the amount of pathogen-associated antigens, cancer antigens, or polynucleotides encoding such antigens (such as pathogen-associated antigens) sufficient to immunize a subject. In any given case, the appropriate amount can be determined by those skilled in the art or can be determined by routine experiments.

[0190] As used herein, the term “delivery” means the delivery of a payload (e.g., a polynucleotide) from the subject’s site of administration to a target organ, tissue, or cell by means of the chemical or biophysical properties of a composition (e.g., an LNP composition). As used herein, the term “selective delivery” means delivery to a target organ, tissue, or cell at a greater rate or in greater quantity than to a reference non-target organ, tissue, or cell, or that the composition delivers a higher proportion of the total amount administered to the subject to the target organ, tissue, or cell compared to a reference composition. For example, selective delivery may mean delivering at least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%) of the total amount administered to the target organ, tissue, or cell. The term “selective delivery” is defined by comparing the proportion of an LNP composition or payload containing a selected lipid (e.g., SORT lipid) delivered to a target organ (e.g., the lung) with the proportion of a reference LNP composition in which the selected lipid is replaced by a control lipid delivered to the target organ.

[0191] "Prevention" or "avoidance" refers to suppressing the onset of disease in a subject or patient who may be at risk and / or susceptible to disease but has not yet experienced or exhibited any or all of the pathology or symptoms of disease, and / or slowing the onset of disease pathology or symptoms in a subject or patient who may be at risk and / or susceptible to disease but has not yet experienced or exhibited any or all of the pathology or symptoms of disease. Prevention can be complete (without detectable symptoms) or partial, thereby resulting in fewer observed symptoms than would be present in the absence of treatment. As is known in the art, immunization and vaccination, as described herein, help prevent disease (e.g., pathogen infection and / or disease or condition caused by pathogens), or cancerous disease or condition.

[0192] The term "administration" means providing the composition to a subject in a manner that allows the composition to have its intended effect. Administerment for vaccination or post-exposure prophylaxis may be performed via intramuscular injection, intravenous injection, intraperitoneal injection, or any other suitable route.

[0193] "Co-administration" means administering the composition described herein simultaneously, immediately before, or immediately after the administration of one or more other therapies. The compositions provided herein may be administered to a subject alone or in combination. Co-administration means administering compounds (more than one compound) simultaneously or sequentially, alone or in combination. Therefore, the formulation may also be combined with other active substances when needed (e.g., to reduce metabolic degradation).

[0194] The terms "pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that facilitate administration of an active agent to a subject and absorption by the subject, and can be included in the compositions of this disclosure without causing significant adverse toxicological effects on the subject or patient. They can also refer to excipients approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals (and more specifically, in humans). Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline solutions, lactated Ringer's solution, plain sucrose, plain glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose, amylose, or starch), fatty acid esters, carboxymethyl cellulose, polyvinylpyrrolidone, and pigments, etc. Such formulations can be sterilized and, if necessary, can be mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring substances, and / or aromatic substances, etc., without causing adverse reactions with the compounds disclosed herein. Those skilled in the art will recognize that other pharmaceutical excipients are useful in this disclosure.

[0195] As used herein, the term "pharmaceutical composition" refers to a mixture of substances suitable for administration to an individual, including pharmaceutical agents. For example, a pharmaceutical composition may comprise modified oligonucleotides and sterile aqueous solutions.

[0196] The term "expression" encompasses any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for protein detection, such as ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.

[0197] As used herein (and as fully understood in the art), “treatment” or “manipulation” also broadly includes any method that achieves a beneficial or desired outcome (including clinical outcomes) for a subject’s condition. Beneficial or desired clinical outcomes may include, but are not limited to, relief or improvement of one or more symptoms or conditions, reduction of the severity of the disease, stabilization of the disease state (i.e., non-deterioration), prevention of the spread or diffusion of the disease, delay or slowing the progression of the disease, improvement or relief of the disease state, reduction of disease recurrence, and remission, whether partial or complete, and whether detectable or undetectable. In other words, “treatment” as used herein includes any cure, improvement, or prevention of a disease (e.g., cancer). Treatment may prevent the onset of the disease; inhibit the spread of the disease, such as the metastasis of cancer cells or the expansion of a tumor; relieve the symptoms of the disease; completely or partially remove the underlying cause of the disease; shorten the duration of the disease; or a combination of these actions.

[0198] As used herein, the term "vaccine" refers to a composition that typically provides active acquired immunity against a pathogen or cancer cells or cancer. Vaccines generally contain one or more agents that can induce an immune response against a pathogen (i.e., a target pathogen or disease) in a subject. The immunogenic agent stimulates the body's immune system to recognize the agent as a threat or indication of the presence of the target pathogen or disease, thereby inducing immune memory so that the immune system can more easily recognize and destroy any subsequently exposed pathogen. Vaccines can be preventative (e.g., to prevent or mitigate the effects of future pathogen infection) or therapeutic (e.g., to treat pathogen infection). The administration of a vaccine is referred to as vaccination. In some examples, the vaccine composition may provide the subject with nucleic acids, such as mRNA encoding an antigenic polypeptide (e.g., a pathogen-associated antigenic polypeptide). The nucleic acid delivered to the subject by the vaccine composition may be expressed as an antigen (e.g., an antigenic protein) and confer immunity against the antigenic molecule (e.g., a pathogen-associated antigen molecule). In the context of vaccination against an infectious disease, the vaccine composition may provide mRNA encoding an antigenic protein associated with a specific pathogen (e.g., a pathogen-associated antigen), such as one or more peptides known to be expressed in pathogens (e.g., pathogenic bacteria or viruses).

[0199] As used in this article, the term "pathogen" refers to bacteria, viruses, fungi, protozoa, or any organism or infectious agent capable of causing disease.

[0200] The term “virus” or “viral particle” is used according to its ordinary literal meaning in virology and refers to a virion that contains a viral genome (e.g., DNA, RNA, single-stranded, double-stranded), a viral capsid, and associated proteins; and in the case of enveloped viruses (e.g., herpesviruses), it refers to an envelope containing lipids and optional host cell membrane components and / or viral proteins.

[0201] The term "adjuvant" refers to a pharmaceutically acceptable substance that enhances the immune response to an antigen (e.g., a pathogen-associated antigen) when co-administered with or before, during, or after administration of the antigen to a subject. In some cases, LNP polynucleotides (e.g., mRNA) can act as adjuvants.

[0202] The term "protective immune response" refers to an immune response that prevents and / or reduces the severity of pathogen infection when a subject is reinfected with the pathogen, or reduces the severity of cancer cell growth, or refers to an immune response that produces a level of protection-associated immune response. For example, vaccination can produce a protective immune response if it produces neutralizing antibodies in the plasma or serum of a subject (e.g., a human, pet, or agricultural animal) that protect the subject from subsequent infection or cancer cell or tumor proliferation, and / or if the amount of these antibodies is sufficient to provide protection for the subject receiving the trial.

[0203] The terms “immunization” and “immunizing” refer to administering the composition to a subject in an amount sufficient to elicit the desired immune response after one or more administration steps. Immunization may include one to ten or more administrations of the composition (e.g., injection), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more administrations. The first administration may not elicit a detectable immune response because, typically, each subsequent administration reinforces the immune response generated by the previous administration. As used herein, the term “immunizing” includes post-exposure prophylaxis.

[0204] As used herein, “cytokine” is a general term for proteins released by a population of cells that act as intercellular mediators to another cell. For example, as described in Penichet et al., J Immunol Methods 248:91-101 (2001). Cytokines include, but are not limited to, IFNγ, IL-10, IL-12p70, IL-1β, IL-2, IL-4, IL-5, IL-6, KC / GRO, TNF-α, IL-16, IL-17A, IL-17C, IL-21, IL-22, IL-23, IL-17E / IL-25, IL-15, IL-17F, IL-27P28 / IL-30, IL-31, IL-33, IL-17A / F, IL-9, OP-10, MCP-1, MIP-1α, MIP-2, and MIP-3α. Cytokine expression can be detected by methods known in the art, such as cytokine-specific enzyme-linked immunosorbent assay (ELISA).

[0205] II. Lipid Nanoparticle Composition

[0206] This disclosure covers LNP compositions that can be used to deliver a payload (e.g., an antigen, such as a pathogen-associated antigen) or a polynucleotide encoding an antigen to host cells. The LNPs are formulated to target different host cells in vitro or in vivo, and the payload is subsequently released into the host cells.

[0207] Payload

[0208] This disclosure covers a variety of payloads delivered via LNP compositions that can directly function as antigens (e.g., pathogen-associated antigens or cancer antigens) or indirectly function as antigens by encoding antigens. For example, the payload may be a pathogen-associated antigen or an antigenic fragment thereof, or it may be a polynucleotide encoding such an antigen or antigenic fragment. The antigen may also be a tumor-specific antigen or an antigenic fragment thereof, or it may be a polynucleotide encoding such an antigen or antigenic fragment.

[0209] In some implementations, the payload comprises a therapeutic peptide or a polynucleotide encoding a peptide. For example, the payload may be a polynucleotide encoding a gene associated with CNS disease, or a polynucleotide encoding a gene editor for editing genes associated with CNS disease.

[0210] A. Antigen

[0211] Exemplary antigens (e.g., pathogen-associated antigens or cancer antigens) include, but are not limited to, synthetic antigens, recombinant antigens, exogenous antigens, or homologous antigens, and antigenic substances may include, but are not limited to, proteins, peptides, polypeptides, lipids, glycolipids, carbohydrates, RNA, and DNA.

[0212] In some embodiments, the antigens in the LNP composition or the antigens expressed by the nucleic acids in the LNP composition include pathogen-associated antigens, namely microbial antigens or viral antigens. Pathogens include, but are not limited to, infectious viruses, infectious bacteria, infectious parasites, infectious fungi, and infectious protozoa.

[0213] Infectious diseases

[0214] Virus

[0215] Influenza virus. Influenza viruses are enveloped RNA viruses. Common influenza outbreaks lead to increased morbidity for pneumonia and lower respiratory tract infections. In the United States, influenza is estimated to cause 9 to 41 million infections, 140,000 to 710,000 hospitalizations, and 12,000 to 52,000 deaths annually. There are three types of influenza viruses: A, B, and C. Type A and B influenza viruses are further subtyped based on the viral surface proteins hemagglutinin (HA) and neuraminidase (NA). Currently, 18 HA and 11 NA subtypes have been identified.

[0216] In some implementation schemes, the influenza virus antigen comes from influenza A virus, influenza B virus, or influenza C virus.

[0217] In some embodiments, the influenza A virus may be selected from influenza A viruses characterized by hemagglutinin (HA) selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18. In some embodiments, the influenza A virus may be selected from influenza A viruses characterized by neuraminidase (NA) selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11.

[0218] The influenza virus strain can be any influenza virus strain. Non-limiting examples of influenza virus strains used for the purposes provided herein include A / California / 07 / 2009 (H1N1), A / Michigan / 45 / 2015 (H1N1), A / Netherlands / 602 / 2009 (H1N1), A / Hong Kong / 4801 / 2014 (H3N2), A / Vietnam / 1203 / 2004 (H5N1), and A / Vietnam / 1194 / 2004 (H5N1).

[0219] In some implementations, the influenza virus antigen is an antigen that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with HA protein, NA protein, NP (nuclear protein) protein, M1 (matrix protein 1) protein, M2 (matrix protein 2) protein, non-structural protein 1 (NS1), non-structural protein 2 (NS2), extranuclear transfer protein (NEP), polymerase acid protein (PA), polymerase basic protein PB1, PB1-F2, or polymerase basic protein 2 (PB2), or an antigen fragment that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the antigen fragment of the aforementioned proteins.

[0220] SARS-CoV. SARS-CoV-2 is a single-stranded sense RNA virus that can cause severe respiratory illness in humans. The SARS-CoV-2 viral spike (S) protein binds to angiotensin-converting enzyme 2 (ACE2), the entry receptor utilized by SARS-CoV-2. The spike (S) protein of coronaviruses is the major surface protein and a target for neutralizing antibodies in infected subjects or patients. Therefore, it is considered a potential protective antigen for vaccine design. The SARS-CoV N protein contains two distinct RNA-binding domains (N-terminal domain [NTD] and C-terminal domain [CTD]), which are linked by a poorly linked region (LKR) containing a serine / arginine-rich (SR-rich) domain (SRD). Due to the presence of positively charged amino acids, the SARS-CoV N-NTD and N-CTD have been reported to bind to the viral RNA genome. The LKR can promote oligomerization.

[0221] In some implementations, the SARS-CoV-2 antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the SARS-CoV-2 S protein, or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with its antigenic fragment. A fully characterized antigenic fragment of the S protein is its receptor-binding domain (RBD).

[0222] Respiratory syncytial virus (RSV). RSV is an antisense single-stranded RNA virus and is the most common cause of bronchiolitis. The virus is present in at least two antigenic subgroups (group A and group B). The proteins encoded by RSV are shown in Table 1.

[0223] Table 1. Exemplary proteins encoded by RSV

[0224]

[0225] Two RSV surface glycoproteins (i.e., attachment glycoprotein (G) and fusion (F) glycoprotein) mediate attachment and adhesion to respiratory epithelial cells. The G protein anchors and stabilizes viral particles on the surface of bronchial epithelial cells, while the F protein interacts with cellular glycosaminoglycans to mediate the fusion of RSV virions with host cells and delivers their contents into the host cells. In some embodiments, the RSV antigen includes the F protein, G protein, SH protein, L protein, P protein, N protein, M2 protein, M protein, NS1 protein, and NS2 protein.

[0226] In some embodiments, the RSV antigen is derived from an antigen having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the RSV F protein, or from an antigen fragment having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the RSV F protein.

[0227] Human metapneumovirus (hMPV) is an antisense single-stranded RNA virus that causes upper and lower respiratory tract infections. hMPV shares high homology with respiratory syncytial virus (RSV) in its surface glycoproteins. The F protein exists as a trimer and is a type I glycoprotein. In addition to the F protein, hMPV also encodes a glycoprotein (G), a matrix protein (M), a phosphoprotein (P), a nucleoprotein (N), and a SH protein. In some embodiments, the hMPV antigen includes the F, G, SH, P, N, and M proteins.

[0228] This disclosure is not limited to any particular hMPV strain. Exemplary hMPV strains include, but are not limited to, A1, A2, B1, B2, CAN98-75, CAN97-83, hMPV isolate TN / 92-4, hMPV isolate NL / 1 / 99, or hMPV isolate PER / CFO0497 / 2010 / B.

[0229] In some embodiments, the hMPV antigen comprises an hMPV antigenic polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the hMPV F protein, or having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with its antigenic fragment. In some embodiments, the hMPV antigen comprises an hMPV antigenic polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the hMPV G protein, or having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with its antigenic fragment.

[0230] Human parainfluenza virus type 3 (PIV3). PIV is an antisense single-stranded RNA virus that causes a variety of respiratory illnesses. It is a major cause of acute respiratory infections prevalent in infancy and early childhood.

[0231] The PIV3 genome encodes two envelope glycoproteins (hemagglutinin-neuraminidase (HN) and a fusion protein (F)), a matrix protein (M), a nucleocapsid protein (N), and several non-structural proteins, including a viral replicase (L). The PIV3 HN protein is essential for attachment and cell entry, while the PIV3 F protein promotes viral fusion and cell entry. In some embodiments, the PIV3 antigen comprises polynucleotides encoding the F, HN, L, N, and M proteins.

[0232] This disclosure is not limited to any particular PIV3 strain. The PIV3 strain can be any PIV3 strain. A non-limiting example of a PIV3 used for the purposes provided herein includes HPIV3 / Homo sapiens / PER / FLA4815 / 2008.

[0233] In some embodiments, the PIV3 antigen comprises a PIV3 antigenic polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the PIV3 F protein, or having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with its antigenic fragment. In some embodiments, the PIV3 antigen comprises a PIV3 antigenic polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the PIV3 HN protein, or having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with its antigenic fragment.

[0234] Middle East Respiratory Syndrome Coronavirus (MERS-CoV). MERS-CoV is a sense single-stranded RNS virus. It exhibits strong tropism for non-ciliated bronchial epithelial cells, evades the innate immune response, and antagonizes the production of interferon (IFN) in infected cells. Dipeptidyl peptidase 4 (DDP4, also known as CD26) has been identified as the functional cellular receptor for the spike protein of MERS-CoV. Proteins encoded by MERS-CoV are shown in Table 2.

[0235] Table 2. Exemplary proteins encoded by MERS-CoV

[0236]

[0237] In some implementations, the MERS-CoV antigen includes the MERS-CoV S protein, S1 subunit protein, S2 subunit protein, E protein, N protein, and M protein.

[0238] This disclosure is not limited to any specific strain of MERS-CoV. MERS-CoV strains can be any MERS-CoV strain. Non-limiting examples of MERS-CoV strains used for the purposes provided herein include Riyadh_14_2013, 2cEMC / 2012, and Hasa_1_2013.

[0239] Measles virus (MeV). Measles virus is a single-stranded RNA virus. The fusion protein (F) undergoes membrane fusion after the hemagglutinin (HA) protein binds to the receptor. In some embodiments, the MeV antigen includes the MeV F protein, HA protein, P protein, V protein, and C protein.

[0240] In some embodiments, the MeV antigen comprises a MeV antigenic polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the MeV F protein, or with its antigenic fragment. In some embodiments, the MeV antigen comprises a MeV antigenic polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the MeV HA protein, or with its antigenic fragment.

[0241] This disclosure is not limited to any particular MeV strain. Any MeV strain may be used. Non-limiting examples of MeV strains used for the purposes provided herein include B3 / B3.1, C2, D4, D6, D7, D8, G3, H1, Moraten, Rubovax, Mvi / New Jersey.USA / 45.05, MVi / Texas.USA / 4.07, AIK-C, MVi / New York.USA / 26.09, MVi / California.USA / 8.04, and MVi / Pennsylvania.USA / 20.09.

[0242] bacteria

[0243] Streptococci. Streptococci are Gram-positive bacteria that cause a variety of diseases and invasive infections. Streptococci are divided into three groups: Group A, Group B, and Group C. Group B streptococcal infections can lead to pneumonia, meningitis, and sepsis.

[0244] In some implementations, the streptococcal antigen comprises group A carbohydrates or modified group A carbohydrates.

[0245] Tuberculosis. Tuberculosis is an infectious disease usually caused by Mycobacterium tuberculosis, which can cause infection of the lungs or other tissues. The BCG vaccine is currently the most widely used vaccine in some countries. However, it does not prevent either primary infection or recurrence of latent lung infection. Therefore, the impact of BCG vaccination on TB transmission is limited. In some implementations, the tuberculosis antigen comprises an antigen listed in Table 5, or a polypeptide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an antigen listed in Table 5, or having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with its antigen fragment.

[0246] In some embodiments, the antigen comprises an H1N1 polypeptide sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NO: 1. In some embodiments, the antigen comprises an influenza virus antigen polypeptide sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NO: 2-26. In some embodiments, the antigen comprises a SARS-CoV-2 antigen polypeptide sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NO: 27-29. In some embodiments, the antigen comprises an RSV antigen polypeptide sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NO: 30-33. In some embodiments, the antigen comprises a PIV antigen polypeptide sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NO: 34-35. In some embodiments, the antigen comprises a MERS-CoV antigen polypeptide sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NO: 36-39. In some embodiments, the antigen comprises a measles virus antigen polypeptide sequence that is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to the polypeptide sequence encoded by SEQ ID NO: 40-43. In some embodiments, the antigen comprises a tuberculosis antigen polypeptide sequence that is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to the polypeptide sequence encoded by SEQ ID NO: 44-56. In some embodiments, the antigen comprises a polypeptide sequence that is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to the polypeptide sequence encoded by SEQ ID NO: 57-96.

[0247] Exemplary viral antigen polynucleotide sequences are shown in Table 3. It should be understood that T in the DNA polynucleotide sequence is T, and T in the RNA polynucleotide sequence is U.

[0248] Table 3 shows a list of viral antigens.

[0249] Table 3. Viral Antigens

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282] Table 4. Non-restrictive list of viral antigen polypeptide sequences

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293] Table 5. Bacterial Antigens

[0294]

[0295]

[0296]

[0297]

[0298]

[0299] lung cancer

[0300] Non-small cell lung cancer (NSCLC) is grouped together because their prognosis and management are largely the same. There are three main subtypes: squamous cell lung cancer, adenocarcinoma, and large cell lung cancer. Six genes were selected to stimulate the immune system to allow for the treatment of lung cancer, especially non-small cell lung cancer (NSCLC).

[0301] 5T4

[0302] 5T4 is a trophoblast glycoprotein that is highly expressed in the placenta and various human cancers, including colorectal, gastric, renal, and ovarian cancers, but rarely in normal tissues. It plays a role in multiple biological and pathological processes, including extensive cell migration during embryogenesis, implantation-associated cell invasion, and tumor metastasis during tumorigenesis and progression.

[0303] In some implementations, the 5T4 antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 101.

[0304] Survival proteins

[0305] Survival protein (containing protein 5 in the baculovirus IAP repeat sequence) is highly expressed in most human cancer cells of epithelial and hematopoietic origin, and its overexpression is associated with cancer progression, poor prognosis, drug resistance, and short patient survival. It has been shown to inhibit apoptosis, promote cell proliferation, and enhance angiogenesis. Consistent with its role in these processes, it also plays a role in cancer progression. Due to the significant differences in survival protein expression between normal and malignant tissues, and its causal role in cancer progression, survival protein is currently under investigation as a potential tumor marker. Cytotoxic T cells recognizing survival protein epitopes can be induced in vitro and through vaccination of patients with leukemia, breast cancer, and melanoma. Immunization of mice with dendritic cells transfected with survival protein-RNA resulted in long-term resistance to lymphomas expressing survival protein, demonstrating the potential of survival protein as a cancer cell antigen.

[0306] In some embodiments, the survival protein antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 102. In some embodiments, the 5T4 antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 103.

[0307] NY-ESO-1

[0308] NY-ESO-1 is reportedly a cancer / testis antigen expressed as mRNA in a variety of human tumors, including melanoma, ovarian cancer, breast cancer, thyroid cancer, prostate cancer, bladder cancer, colon cancer, Burkitt lymphoma, glioma, and hepatocellular carcinoma. Vaccination has induced NY-ESO-1-specific antibody responses and / or specific CD8 and CD4 T cell responses against a broad range of NY-ESO-1 epitopes. Furthermore, extensive NY-ESO-1-specific immune responses, including antibody and CD4 and CD8 T cell responses, have been observed after co-immunization with resected NY-ESO-1-expressing melanoma in patients with NY-ESO-1-expressing melanoma.

[0309] In some implementations, the NY-ESO-1 antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 104.

[0310] MAGE-C1 and MAGE-C2

[0311] MAGE-C1 (melanoma antigen family-C1) and MAGE-C2 (melanoma antigen family-C2) are not expressed in a group of normal tissues tested, excluding the testes. In tumor samples, they are frequently expressed in seminoma, melanoma, and bladder cancer. They are also expressed in a significant proportion of head and neck cancers, breast cancer, non-small cell lung cancer, prostate adenocarcinoma, and sarcoma.

[0312] In some embodiments, the MAGE-C1 antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 105. In some embodiments, the MAGE-C2 antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 106.

[0313] Prostate cancer

[0314] Prostate-specific antigen (PSA)

[0315] Prostate-specific antigen (PSA) is an androgen-regulated kallikrein-like serine protease produced only by epithelial cells of all types of prostate tissue (benign and malignant). PSA is currently the most widely used tumor marker for screening, diagnosing, and monitoring prostate cancer. Specifically, several immunoassays for detecting serum PSA are widely used in clinical practice.

[0316] In some implementations, the PSA antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 107.

[0317] Prostate-specific membrane antigen (PSMA)

[0318] Prostate-specific membrane antigen (PSMA), or folate hydrolase 1 (FOLH1), is a type II transmembrane glycoprotein. PSMA expression is primarily confined to prostate tissue, but detectable levels of PSMA mRNA have also been observed in brain, salivary gland, small intestine, and renal cell carcinomas. PSMA is highly expressed in most primary and metastatic prostate cancers. Specifically, PSMA is highly expressed in angiogenesis in prostate cancer cells and non-prostate solid tumors and is a target for anticancer imaging and therapeutic agents. PSMA acts as a glutamate carboxypeptidase (GCPII) on small molecule substrates, including folic acid, the anticancer drug methotrexate, and the neuropeptide N-acetyl-L-aspartyl-L-glutamate. In prostate cancer, PSMA expression has been confirmed to be associated with disease progression, with the highest expression levels in hormone-refractory and metastatic disease. PSMA is considered a biomarker for prostate cancer and is under further investigation as an imaging and therapeutic target.

[0319] In some implementations, the PSMA antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 108.

[0320] Prostate stem cell antigen (PSCA)

[0321] Prostate stem cell antigen (PSCA) is widely overexpressed at all stages of prostate cancer, including high-grade prostatic intraepithelial neoplasia (PIN), androgen-dependent prostate cancer, and androgen-independent prostate cancer. The PSCA gene shows 30% homology with stem cell antigen-2, a member of the Thy-1 / Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA mRNA expression is highly upregulated in both androgen-dependent androgen-independent prostate cancer xenografts. In situ mRNA analysis localizes PSCA expression to the basal cell epithelium, the putative stem cell compartment of the prostate. PSCA can be used as a prostate cancer marker to differentiate between malignant prostate cancer, normal prostate, and non-malignant prostate cancer.

[0322] In some implementations, the PSCA antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 109.

[0323] Six-transmembrane prostatic epithelial antigen (STEAP)

[0324] Six-transmembrane prostatic epithelial antigen (STEAP) is a novel cell surface protein primarily expressed in human prostate tissue and other common malignancies, including prostate cancer, bladder cancer, colon cancer, and ovarian tumors, as well as Ewing's sarcoma, suggesting its potential function as a near-universal tumor antigen. In particular, STEAP is highly expressed in primary prostate cancer but its expression is limited in normal tissues.

[0325] In some implementations, the STEAP antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 110.

[0326] Prostatic acid phosphatase (PAP)

[0327] Prostatic acid phosphatase (PAP) is an enzyme secreted by the epithelial cells of the prostate gland that catalyzes the conversion of orthophosphate monoesters into alcohols and orthophosphates. PAP is strongly expressed in >95% of normal adult prostate tissue samples (including normal tissue near tumors) and >95% of primary adenocarcinomas. PAP expression can be detected in some normal human tissues other than the prostate (e.g., kidneys, lungs, testes, colon, and pancreas), but at levels approximately one to two orders of magnitude lower, and PAP is generally considered a tissue-specific prostate antigen, highly expressed in both normal and malignant prostate cells. Furthermore, PAP has been shown to be strongly expressed in bone metastases of prostate cancer and may play a causal role in the osteoblastic phase of this disease. PAP has been shown to induce long-term CD4+ and CD8+ T cell responses, including CTL responses, in patients.

[0328] In some implementations, the PAP antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 111.

[0329] Mucin 1 (MUC1)

[0330] Mucin 1 (MUC1) is a large mucin glycoprotein normally expressed on the luminal surface of glandular epithelium. Its function in normal epithelium is to lubricate and protect epithelial cells. MUC1 expression is typically increased, no longer confined to the luminal surface, and is characterized by aberrant glycosylation in many human malignancies, including prostate cancer. MUC1 is expressed in approximately 60% of primary prostate cancers and 90% of lymph node metastases. Furthermore, 86% of MUC1-positive primary prostate tumors have a Gleason score ≥7, suggesting an association with more aggressive disease. Both overexpression and underexpression of MUC1 have been found to increase the risk of prostate cancer progression. MUC1 has been shown to be immunogenic and has been described as inducing specific immune responses in patients, including CD8+ CTLs and IgM antibodies. In a phase II trial in patients with advanced non-small cell lung cancer, MUC1 administration using different vaccination methods trended toward clinical benefit and appeared to be well-tolerated. Vaccination of prostate cancer patients with the same vaccine targeting MUC1 was associated with prolonged PSA doubling time in some patients.

[0331] In some implementations, the MUC1 antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 112.

[0332] Furthermore, the antigen, antigenic protein, or antigenic peptide encoded by at least one mRNA of the composition according to the invention may comprise fragments or variants of those sequences. Such fragments or variants typically comprise sequences that have at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% or even 97% sequence homology relative to the entire wild-type sequence, whether at the nucleic acid level or the amino acid level.

[0333] Table 6 shows exemplary polypeptide sequences of cancer antigens.

[0334] Table 6. Polypeptide sequences of antigens

[0335]

[0336]

[0337]

[0338] neoantigen

[0339] Neoantigens are a class of tumor-specific antigens produced by tumor-specific mutations that alter the amino acid sequence of proteins encoded by the genome. The peptides resulting from these mutations play a crucial role in the immune response against cancer cells by labeling them as foreign substances on T cells.

[0340] In some embodiments, the antigens in the LNP composition include tumor-specific antigens, i.e., neoantigens. Tumors include, but are not limited to, adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), invasive breast cancer (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), colonic adenocarcinoma (COAD), chronic lymphocytic leukemia (CLL), colorectal cancer (CRC), diffuse large B-cell lymphoma (DLBCL), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), chromophobe renal cell carcinoma (KICH), clear cell renal cell carcinoma (KIRC), and papillary renal cell carcinoma (K). IRP, acute myeloid leukemia (LAML), hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), multiple myeloma (MM), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), rectal adenocarcinoma (READ), cutaneous melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thyroid adenocarcinoma (THCA), endometrioid carcinoma of the uterine corpus (UCEC) or uterine carcinosarcoma (UCS).

[0341] In some implementations, the cancer or cancer antigen (e.g., conventional cancer antigen) is one of the following antigens: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5, AGS-16, angiopoietin 2, B2M, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinoembryonic antigen. Antigen, CTLA4, Cripto, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EGFR (e.g., EGFR-L858R), EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, ICOS, IGF1R, integrin av, integrin anb, FAG-3, Fewis Y, mesothelin, c-MET, MN, carbonic anhydrase IX, MUC1, MUC16, Nectin-4, NKGD2, NOTCH, OX40, OX40F, PD-1, PDF1, PSCA, PSMA, RANKE, ROR1, ROR2, SFC44A4, multiligand proteoglycan-1, TACI, TAG-72, tendinin, TIM3, TRAIFR1, TRAIFR2, VEGFR-1, VEGFR-2, VEGFR-3 and / or their variants.

[0342] In some implementations, the neoantigen peptide includes at least one peptide listed in Table 7.

[0343] Tumor-specific mutations include those listed in Table 7.

[0344] Table 7. Exemplary sequences of neoantigens

[0345]

[0346]

[0347]

[0348] In some implementations, the length of the antigen (e.g., pathogen-associated antigen or cancer antigen) is greater than 9 amino acids, greater than 10 amino acids, greater than 11 amino acids, greater than 12 amino acids, greater than 13 amino acids, greater than 14 amino acids, greater than 15 amino acids, greater than 16 amino acids, greater than 17 amino acids, greater than 18 amino acids, greater than 19 amino acids, greater than 20 amino acids, greater than 25 amino acids, greater than 30 amino acids, greater than 35 amino acids, greater than 40 amino acids, greater than 45 amino acids, greater than 50 amino acids, greater than 100 amino acids, greater than 200 amino acids, greater than 300 amino acids, greater than 400 amino acids, greater than 500 amino acids, greater than 600 amino acids, greater than 700 amino acids, greater than 800 amino acids, greater than 900 amino acids, greater than 1000 amino acids, greater than 1500 amino acids, or greater than 2000 amino acids.

[0349] B. Polynucleotides

[0350] In some embodiments, the nucleic acid or polynucleotide encoding an antigen (e.g., a pathogen-associated antigen) includes, but is not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threonucleic acid (TNA), diol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a β-D-ribose configuration, α-LNA having an α-L-ribose configuration (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization and 2'-amino-α-LNA with 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or hybrids or combinations thereof.

[0351] In some implementations, the polynucleotide comprises messenger RNA (mRNA), short hairpin RNA (shRNA), or microRNA.

[0352] In some embodiments, the polynucleotide comprises one or more modifications selected from the group consisting of: pyridine-4-ketoribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurate methyluridine, 1-taurate methyl-2-thiouridine, 1-taurate methyl-4-thiouridine, 5-methyluridine, 1-methyl -Pseudouridine, 4-Thio-1-methyl-pseudouridine, 2-Thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-Thio-1-methyl-1-denitro-pseudouridine, dihydrouridine, dihydropseudouridine, 2-Thio-dihydrouridine, 2-Thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine Pyrrolo-cytidine, pyrrolo-pseudo-cytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudo-cytidine, 4-thio-1-methyl-pseudo-cytidine, 4-thio-1-methyl-1-deazo-pseudo-cytidine, 1-methyl-1-deazo-pseudo-cytidine, zablaline, 5-aza-zablaline, 5-methyl-zablaline, 5-aza-2-thio-zablaline, 2-thio-zablaline, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, 2-aminopurine, 2,6-di Aminopurine, 7-denitro-adenine, 7-denitro-8-aza-adenine, 7-denitro-2-aminopurine, 7-denitro-8-aza-2-aminopurine, 7-denitro-2,6-diaminopurine, 7-denitro-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenosine, 2-methylthio-adenosine and 2-methoxy-adenosine, inosine, 1-methyl-inosine, woyoside, woyoside, 7-deazo-guanosine, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof.

[0353] In some implementations, the polynucleotide may encode at least one antigen (e.g., a pathogen-associated antigen or a cancer antigen) or a fragment thereof. The antigen may be a peptide, polypeptide, or protein.

[0354] In some implementations, the length of the polynucleotide is greater than 30 nucleotides, greater than 50 nucleotides, greater than 100 nucleotides, greater than 200 nucleotides, greater than 300 nucleotides, greater than 400 nucleotides, greater than 500 nucleotides, greater than 600 nucleotides, greater than 700 nucleotides, greater than 800 nucleotides, greater than 900 nucleotides, greater than 1000 nucleotides, greater than 1500 nucleotides, greater than 2000 nucleotides, greater than 2500 nucleotides, greater than 3000 nucleotides, greater than 3500 nucleotides, greater than 4000 nucleotides, greater than 4500 nucleotides, or greater than 5000 nucleotides.

[0355] In some embodiments, the polynucleotide comprises about 50 to about 100,000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 5,000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 2,500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 1,000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 300 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 200 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 100 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 100,000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 5,000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 2,500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 1,000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 300 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 200 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 100,000 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 5,000 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 2,500 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 1,000 nucleotides. In some embodiments, the polynucleotide comprises about 1,000 to about 100,000 nucleotides. In some embodiments, the polynucleotide comprises about 1,000 to about 5,000 nucleotides. In some embodiments, the polynucleotide comprises about 1,000 to about 2,500 nucleotides. In some embodiments, the polynucleotide comprises about 1,000 to about 2,000 nucleotides.

[0356] In some embodiments, the mRNA molecule is about 50 nucleotides long. In some embodiments, the mRNA molecule is about 100 nucleotides long. In some embodiments, the mRNA molecule is about 200 nucleotides long. In some embodiments, the mRNA molecule is about 300 nucleotides long. In some embodiments, the mRNA molecule is about 400 nucleotides long. In some embodiments, the mRNA molecule is about 500 nucleotides long. In some embodiments, the mRNA molecule is about 600 nucleotides long. In some embodiments, the mRNA molecule is about 700 nucleotides long. In some embodiments, the mRNA molecule is about 800 nucleotides long. In some embodiments, the mRNA molecule is about 900 nucleotides long. In some embodiments, the mRNA molecule is about 1000 nucleotides long. In some embodiments, the mRNA molecule is about 2000 nucleotides long. In some embodiments, the mRNA molecule is about 3000 nucleotides long. In some embodiments, the mRNA molecule is about 4000 nucleotides long. In some embodiments, the mRNA molecule is about 5000 nucleotides long.

[0357] In some embodiments, the mRNA comprises the viral polynucleotides shown in Table 3. In some embodiments, the mRNA comprises the bacterial polynucleotides shown in Table 5. In some embodiments, the mRNA encodes the viral antigens shown in Table 4.

[0358] In some implementations, the mRNA encoding H1N1 contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1.

[0359] In some implementations, the mRNA encoding the influenza virus antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2-26.

[0360] In some implementations, the mRNA encoding the SARS-CoV-2 antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 27-29.

[0361] In some embodiments, the mRNA encoding the RSV antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 30-33.

[0362] In some embodiments, the mRNA encoding the PIV antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 34-35.

[0363] In some implementations, the mRNA encoding the MERS-CoV antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 36-39.

[0364] In some embodiments, the mRNA encoding the measles virus antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 40-43.

[0365] In some embodiments, the mRNA encoding the tuberculosis antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 44-56.

[0366] In some implementations, the mRNA encodes a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 57-96.

[0367] In some embodiments, the mRNA contains the lung cancer polynucleotides shown in Table 8. In some embodiments, the mRNA contains the prostate cancer polynucleotides shown in Table 9. In some embodiments, the mRNA encodes a neoantigen shown in Table 10.

[0368] In some embodiments, the mRNA encoding the 5T4 antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 113-116.

[0369] In some embodiments, the mRNA encoding the survival protein antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 117-120.

[0370] In some embodiments, the mRNA encoding the NY ESO-1 antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 121-124.

[0371] In some embodiments, the mRNA encoding the MAGE-C1 antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 125-129.

[0372] In some embodiments, the mRNA encoding the MAGE-C2 antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 130-133.

[0373] In some embodiments, the mRNA encoding the PSA (prostate-specific antigen) antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:136-138.

[0374] In some embodiments, the mRNA encoding the PSMA (prostate-specific membrane antigen) antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 139-143.

[0375] In some implementations, the mRNA encoding the PSCA (prostate stem cell antigen) antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:144-148.

[0376] In some embodiments, the mRNA encoding the six-transmembrane prostatic epithelial antigen (STEAP) contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 149-153.

[0377] In some implementations, the mRNA encoding the PAP (prostate acid phosphatase) antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:154-157.

[0378] In some embodiments, the mRNA encoding the MUC1 (mucin 1) antigen contains a polynucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 158-161.

[0379] Table 8. Polynucleotide Sequences

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407] In some embodiments, the polynucleotide encodes one or more, two or more, three or more polynucleotides that encode one or more, two or more, three or more antigens (e.g., pathogen-associated antigens or cancer antigens). In some embodiments, the polynucleotide encodes one or more, two or more, three or more polynucleotides that encode antigens derived from different pathogens.

[0408] Examples of pathogens include, but are not limited to, infectious fungi that infect mammals (more specifically, humans).

[0409] C. Central Nervous System (CNS) Diseases

[0410] Central nervous system disorders, also known as central nervous system ailments, are neurological disorders that affect the structure or function of the brain or spinal cord, which form the central nervous system (CNS). In some implementations, CNS disorders include acid lipase disorders, acid maltase deficiency, acid storage disorders, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Addie pupils, Addie syndrome, adrenoleukodystrophy, agnosia, Eckhardt syndrome, Eckhardt-Gutier syndrome, Alexander disease, Alpert disease, and alternating hemiplegia. Hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, hemangioma, hypoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, aromatic L-amino acid decarboxylase deficiency (AADC deficiency), aspartate glucosamineuria, Asperger's syndrome, ataxia, ataxia-telangiectasia (Lewis-Barr syndrome), ataxia and cerebellar or spinocerebellar degeneration, attention deficit hyperactivity disorder (ADHD). Hyperactivity disorder, autism, autonomic dysfunction, Bass syndrome, Barten disease, Becker's muscle rigidity, Behçet's disease, Bell's palsy, Bernhardt-Rose syndrome, Binswanger disease, Bloch-Sulzberger syndrome, Bradbury-Egelston syndrome, Brown-Seckal syndrome, medullary spinal muscular atrophy, CADASIL, Canavan disease, burning neuralgia, cavernous malformation, cavernous hemangioma, central cervical spinal cord syndrome, central spinal cord syndrome, central pontine myelinolysis, ceramide deficiency Cerebellar degeneration, cerebellar hypoplasia, cerebral beriberi, cerebral gigantism, cerebral palsy, brain-ocular-facial-skeletal syndrome (COFS), cholesterol ester storage disease, chorea, chorea-acanthosis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, cavitary brain, congenital myasthenia gravis, corticobasal degeneration, cranial arteritis, Klebsiella encephalitis, Creutzfeldt-Jakob disease, Cushing's syndrome, cystinosis, giant cell inclusion body disease. Inclusion body disease), oculoclonus-myoclonus syndrome, Dandy-Walker syndrome, Danon disease, Dawson's disease, Demosier syndrome, Degerina-Klempke palsy, dementia, dentate nucleus-cerebellar ataxia, dentate nucleus-red nucleus atrophy, dermatomyositis, developmental motor disorders, Dweck syndrome, diffuse sclerosis, autonomic dysfunction, writing disorders, reading disorders, dysphagia, motor disorders, myoclonic cerebellar ataxia, progressive cerebellar ataxia.Epilepsy (e.g., Amish infantile epilepsy syndrome [AIES], benign familial infantile epilepsy [BFIS], benign familial neonatal epilepsy [BFNS], childhood absence epilepsy [CAE], childhood-onset epileptic encephalopathy [COEE], Dravet syndrome [DS], early infantile epileptic encephalopathy [EIEE], familial adult myoclonic epilepsy [FAME], familial febrile seizures [FFS], familial focal epilepsy with variable lesions [FFEVF], familial infantile myoclonic epilepsy [FIME], familial temporal lobe epilepsy [FTLE], focal epilepsy and speech disorders with or without intellectual disability [FESD], generalized epilepsy with paroxysmal motor disorders [GEPD], generalized epilepsy with febrile seizures plus [GEFS+], idiopathic generalized epilepsy [IGE], juvenile absence epilepsy [JA] [E], Juvenile myoclonic epilepsy [JME], Myoclonic-atonic epilepsy [MAE], Nocturnal frontal lobe epilepsy [NFLE], Progressive myoclonic epilepsy [PME], 5'-pyridoxine oxidase deficiency [PNPOD], Pyridoxine-dependent epilepsy [EPD], and severe myoclonic epilepsy in infancy [SMEI]), Fabry disease, Fall syndrome, Familial autonomic dysfunction, Familial hemangioma, Familial idiopathic basal ganglia calcification, Familial periodic paralysis, Familial spastic paralysis, Fabry disease, fibromuscular dysplasia, Fisher syndrome, Infantile relaxation syndrome, Friedreich ataxia, Frontotemporal dementia, Fucoidoside storage disease, Galactosylsialic acid storage disease, Gaucher disease, Systemic ganglioside storage disease, Göstahl syndrome, GSS disease, Giant axonal neuropathy, Giant cell arteritis, Giant cell inclusion body disease (giant Cellinclusion disease, globular leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, GM1 ganglioside storage disease, GM2 ganglioside storage disease (Tai-Sachs disease), Guillain-Barré syndrome, Hallewarden-Scholes disease, persistent unilateral headache, alternating hemiplegia (hemiplegia) Alterans syndrome, hereditary spastic paraplegia, hereditary ataxic polyneuritis, Holmes-Addie syndrome, holoencephaly, Hughes syndrome, Huntington's disease, hydrocephalus, hydrocephalus, Cushing's syndrome, immune-mediated encephalomyelitis, inclusion body myositis, incontinence pigmentosa, infantile hypotonia, infantile axonal dystrophy, occipital cleft with exposed head, Isaac syndrome, Jubert syndrome, Karns-Sell syndrome, Kennedy disease, Kingsburn syndrome, Klein-Levin syndrome, Krippel-Fair syndrome, Krippel-Trenone syndrome (KTS), Klüfer-Busy syndrome, Korsakov amnesia syndrome, Crabbe's disease, Kugelberg-Weyland disease, Lambert-Eton myasthenia gravis syndrome, Landau-Cleven syndrome, lateral femoral cutaneous nerve entrapment syndrome, lateral medullary syndrome, Lewy's disease.Lennox-Gasstor syndrome, Lesch-Niehan syndrome, Levin-Critchley syndrome, Lewy body dementia, lipoprotein storage disorders, lissencephaly, locked-in syndrome, Luger's disease, lupus, Lyme disease, Machado-Joseph disease, megalencephaly, alpha-mannoside storage disorders, beta-mannoside storage disorders, Méridov-Robb syndrome, Menkes disease, lateral femoral cutaneous neuralgia, metachromatic leukodystrophy, microcephaly, Miller-Fischer syndrome, Möbius syndrome, mucopolysaccharidosis type IH (Heller syndrome), mucopolysaccharidosis type IH / S (Heller-Shee syndrome), mucopolysaccharidosis type IS (Shee syndrome), mucopolysaccharidosis type II (Hunter syndrome), mucopolysaccharidosis type III-A (Samfilipo syndrome type A), mucopolysaccharidosis type III Mucopolysaccharidosis type B (Samfelipo syndrome), Mucopolysaccharidosis type III-C (Samfelipo syndrome type C), Mucopolysaccharidosis type III-D (Samfelipo syndrome type D), Mucopolysaccharidosis type IV-B (Mochio syndrome type B), Mucopolysaccharidosis type VI (Maroto-Rami syndrome), Mucopolysaccharidosis type VII (Slay syndrome), Mucopolysaccharidosis type IX (Natovich syndrome), Multiple sclerosis, Muscular dystrophy, Myasthenia gravis, Myelinolytic diffuse sclerosis, Narcolepsy, Neuroacanthocytosis, Neurofibromatosis, Neuroleptic malignant syndrome, Sarcoidosis, Niemann-Pick disease, Otahara syndrome, Olivary corpus pontocerebellar atrophy Oculoclonus myoclonus, O'Sullivan-McLeod syndrome, pantothenic kinase-related neurodegenerative diseases, paraneoplastic syndromes, paresthesia, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal unilateral headache, Paroxysmal-Robber syndrome, Peyre's disease, Pena-Shokel syndrome type II, periventricular leukomalacia, phytanoic acid storage disease, Pick's disease, piriformis syndrome, polymyositis, Pompe disease, post-poliomyelitis syndrome, posterior cortical atrophy, primary dentate nucleus atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive hemifacial atrophy, progressive motor ataxia, progressive multifocal leukoencephalopathy, progressive sclerotic gray matter dystrophy, progressive supranuclear leukoencephalopathy. Paralysis, prosopagnosia, Ramsey Hunt syndrome type I, Ramsey Hunt syndrome type II, Rasmussen encephalitis, Reifsum disease, Rett syndrome, Reye's syndrome, Riley-Day syndrome, Sandhof disease, Hildberg disease, Setterberg disease, Shay-Dreg syndrome, Sjögren's syndrome, spasticity, spina bifida, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olshefsky syndrome, striatal substantia nigra degeneration, Sturge-Weber syndrome, tardive dyskinesia, Tau proteinosis, Ty-Sachs disease, thoracic outlet syndrome, thyrotoxic myopathy, painful tic, Todd's paralysis, trigeminal neuralgia, tropical spastic paraplegia.Troy syndrome, vascular dementia, von Eknomer disease, von Hippel-Lindau disease (VHL), von Recklinghausen disease, Wallenberg syndrome, Wernig-Hoffmann disease, Wernicke-Korsakov syndrome, West syndrome, Whipple's disease, Williams syndrome, Wilson's disease, Wollman's disease, X-linked spinal-bulbar muscular atrophy, Zelweg syndrome, multiple sclerosis with brain atrophy, Lewis body dementia (LBD), or Anglemann syndrome.

[0411] Alzheimer's disease

[0412] Alzheimer's disease is the most common type of dementia. It is a progressive disease that initially presents as mild memory loss and can lead to a loss of the ability to converse and respond to the environment. It involves parts of the brain that control thinking, memory, and language.

[0413] Amyotrophic Lateral Sclerosis (ALS)

[0414] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a neurological disorder that affects motor neurons. ALS causes loss of muscle control.

[0415] Huntington's disease

[0416] Huntington's disease is a hereditary disorder that causes progressive degeneration of nerve cells in the brain. It leads to changes in the central regions of the brain, affecting motor, mood, and cognitive skills.

[0417] Parkinson's disease

[0418] Parkinson's disease is a brain disorder that causes involuntary or uncontrolled movements, such as tremors, rigidity, and difficulty with balance and coordination. Lewy bodies, abnormal clumps of alpha-synuclein, have been observed in the brain cells of patients.

[0419] Spinal muscular atrophy (SMA)

[0420] Spinal muscular atrophy (SMA) is a motor neuron disease involving the loss of motor neurons in the spinal cord. SMA is a genetic disorder that affects the central nervous system, peripheral nervous system, and voluntary muscle movement (skeletal muscle).

[0421] In some implementations, genes involved in CNS diseases include, but are not limited to, 3R tau, 4R tau, AARS, ABCD1, ACOX1, ADGRV1, ADRA2B, AGA, AGER, ALDH7A1, ALG13, ALS2, ANG, ANXA11, APP, ARHGEF9, ARSA, ARSB, ARV1, ASAH1, ASPA, ATN1, ATP10A, ATP13A2, ATXN1, ATXN2, ATXN3, BAX, BCL-2, BDNF, BICD2, C9orf72, CACNA1A, CACNA1H, CACNB4, CASR, CCNF, CDKL5, CERS1, CFAP410, CHCHD10. CHD2, CHMP2B, CHRNA2, CHRNA4, CHRNA7, CHRNB2, CLCN2a, CLN1, CLN2, CLN3, CLN5, CLN6, CLN8, CNTN2, CPA6, CSTB, CTNS, CTSA, CTSD, DAO, DCTN1, DE PDC5, DMD, DNAJB2, DNM1, DOCK7, DRD2, DYNC1H1, EEF1A2, EFHC1, EGLN1, EPHA4, EPM2A, ERBB4, FGF12, FIG4, FRRS1L, FTL, FUCA1, FUS, FXN, GAA, GAB RA1, GABRB1, GABRB3, GABRD, GABRG2, GAL, GALC, GALNS, GBA, GFAP, GLA, GLB1, GLE1, GLT8D1, GNAO1, GNS, GOSR2, GPR98, GRIA1, GRIA2, GRIK1, GRIN 1. GRIN2A, GRIN2B, GRIN2D, GSTM1, GUF1, GUSB, HCN1, HGSNAT, HNRNPA1, HTT, HYAL1, IDS, IDUA, IGHMBP2, IL-1, IT15, ITPA, JPH3, KCNA2, KCNB1, KC NC1, KCNMA1, KCNQ2, KCNQ3, KCNT1, KCTD7, LAL, LAMP2, LGI1, LMNB2, LRRK2, MAN2B1, MAN2B2, MAN2C1, MANBA, MATR3, MBD5, MFSD8, NAGA, NAGLU, NEC AP1, NEFH, NEK1, NEU1, NHLRC1, NPC1, NPC2, NR4A2, NTRK2, OCA2, OPTN, PARK2, PARK7, PCDH19, PEX1, PEX2, PEX3, PEX5, PEX6, PEX10, PEX11B, PEX12,PEX13, PEX14, PEX16, PEX19, PEX26, PFN1, PINK1, PLCB1, PNPO, PON1, PON2, PON3, PPARGC1A, PRDM8, PRICKLE1, PRKN, PRNP, PRPH, PRRT2, PSAP, S 106β, SCARB2, SCN1A, SCN1B, SCN2A, SCN8A, SCN9A, SCN9Ab, SETX, SGSH, SIGMAR1, SIK1, SKP1, SLC1A1, SLC1A2, SLC2A1, SLC6A1, SLC9A6, SLC12A 5. SLC13A5, SLC25A12, SLC25A22, SLCA17A5, SMN1, SMPD1, SNCA, SNRPN, SOD1, SPG11, SPTAN1, SQSTM1, ST3GAL3, ST3GAL5, STX1B, STXBP1, SYP, S YT1, SZT2, TAF15, TARDBP, TBC1D24, TBCE, TBK1, TBP, TITF-1, TREM2, UBA5, UBE1, UBE3A, UBQLN2, UCH-L1, UNC13A, VAPB, VCP, VPS35, WWOX, and XBP1. ,

[0422] In some embodiments, the payload comprises a polynucleotide, optionally mRNA, shRNA, or microRNA. In some embodiments, the mRNA encodes a polypeptide or protein selected from the groups shown in Tables 9, 10, and / or SEQ ID NO: 201-207. In some embodiments, the mRNA encodes a gene editing system or a component thereof. In some embodiments, the payload comprises a polypeptide or protein.

[0423] In some implementations, the polypeptide includes a peptide or protein capable of restoring the function of a defective protein in the subject's body.

[0424] Table 9. Illustrative sequences of genes associated with CNS diseases

[0425]

[0426] In some embodiments, the SOD1 polypeptide comprises a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to that of SEQ ID NO: 201. In some embodiments, the SMN-1 polypeptide comprises a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to that of SEQ ID NO: 202. In some embodiments, the conjugate polypeptide comprises a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to that of SEQ ID NO: 203. In some embodiments, the conjugate polypeptide comprises a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to that of SEQ ID NO: 204. In some embodiments, the conjugate polypeptide comprises a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 205. In some embodiments, the GAD-65 polypeptide comprises a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 206. In some embodiments, the GAD-67 polypeptide comprises a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 207.

[0427] Polynucleotides

[0428] In some embodiments, the lipid compositions described herein comprise one or more polynucleotides. In some embodiments, the polynucleotides encode one or more polypeptides described herein.

[0429] Exemplary nucleic acids or polynucleotides of the present invention include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threonucleic acid (TNA), diol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a β-D-ribose configuration, α-LNA having an α-L-ribose configuration (diastereomers of LNA), 2'-amino-LNA having 2'-amino functionalization and 2'-amino-α-LNA having 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or hybrids or combinations thereof.

[0430] Furthermore, it should be understood that this disclosure is not limited to the specific polynucleotides disclosed herein. However, the scope of this disclosure is not limited to any particular source, sequence, or type of polynucleotide, as those skilled in the art can readily identify related homologs of polynucleotides from a wide variety of other sources, including those from non-human species (e.g., mice, rats, rabbits, dogs, monkeys, gibbons, chimpanzees, apes, baboons, cattle, pigs, horses, sheep, cats, and other species). It is contemplated that polynucleotides used in this disclosure may comprise sequences based on naturally occurring sequences. Taking into account the degeneracy of the genetic code, the sequence has at least about 50%, typically at least about 60%, more typically about 70%, most typically about 80%, preferably at least about 90%, and most preferably about 95% of the nucleotide sequence identical to that of the naturally occurring sequence. In some embodiments, the polynucleotide is a sequence complementary to the naturally occurring sequence, or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and 100% complementary to the naturally occurring sequence. This article considers longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or longer.

[0431] In some embodiments, the polynucleotides used herein may be derived from genomic DNA, i.e., directly cloned from the genome of a specific organism. In some embodiments, the polynucleotides comprise complementary DNA (cDNA). cDNA plus natural introns or introns derived from another gene is also considered; such engineered molecules are sometimes referred to as “small genes.” The term “cDNA” is intended to refer to DNA prepared using messenger RNA (mRNA) as a template. The advantage of using cDNA, as opposed to genomic DNA or DNA polymerized from genomic, unprocessed, or partially processed RNA templates, is that cDNA primarily contains the coding sequence of the corresponding protein. Sometimes, a complete or partial genomic sequence may be preferred, such as where non-coding regions are optimally required for expression, or where non-coding regions such as introns will be targeted in an antisense strategy.

[0432] In some embodiments, the polynucleotide comprises one or more segments comprising small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), primary microRNA (pri-miRNA), long non-coding RNA (lncRNA), messenger RNA (mRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), antisense RNA (RNA), guide RNA, DNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), or double-stranded RNA (dsRNA). In some embodiments, the polynucleotide encodes at least one of the therapeutic (or prophylactic) agents described herein.

[0433] In some implementations, the length of the polynucleotide is greater than 30 nucleotides, greater than 50 nucleotides, greater than 100 nucleotides, greater than 200 nucleotides, greater than 300 nucleotides, greater than 400 nucleotides, greater than 500 nucleotides, greater than 600 nucleotides, greater than 700 nucleotides, greater than 800 nucleotides, greater than 900 nucleotides, greater than 1000 nucleotides, greater than 1500 nucleotides, greater than 2000 nucleotides, greater than 2500 nucleotides, greater than 3000 nucleotides, greater than 3500 nucleotides, greater than 4000 nucleotides, greater than 4500 nucleotides, or greater than 5000 nucleotides.

[0434] In some embodiments, the mRNA is about 50 nucleotides long. In some embodiments, the mRNA molecule is about 100 nucleotides long. In some embodiments, the mRNA molecule is about 200 nucleotides long. In some embodiments, the mRNA molecule is about 300 nucleotides long. In some embodiments, the mRNA molecule is about 400 nucleotides long. In some embodiments, the mRNA molecule is about 500 nucleotides long. In some embodiments, the mRNA molecule is about 600 nucleotides long. In some embodiments, the mRNA molecule is about 700 nucleotides long. In some embodiments, the mRNA molecule is about 800 nucleotides long. In some embodiments, the mRNA molecule is about 900 nucleotides long. In some embodiments, the mRNA molecule is about 1000 nucleotides long. In some embodiments, the mRNA molecule is about 2000 nucleotides long. In some embodiments, the mRNA molecule is about 3000 nucleotides long. In some embodiments, the mRNA molecule is about 4000 nucleotides long. In some embodiments, the mRNA molecule is about 5000 nucleotides long.

[0435] In some embodiments, the polynucleotide comprises about 50 to about 100,000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 5,000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 2,500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 1,000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 300 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 200 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 100 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 100,000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 5,000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 2,500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 1,000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 300 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 200 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 100,000 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 5,000 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 2,500 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 1,000 nucleotides. In some embodiments, the polynucleotide comprises about 1,000 to about 100,000 nucleotides. In some embodiments, the polynucleotide comprises about 1,000 to about 5,000 nucleotides. In some embodiments, the polynucleotide comprises about 1,000 to about 2,500 nucleotides. In some embodiments, the polynucleotide comprises about 1,000 to about 2,000 nucleotides.

[0436] In some embodiments, the LNP composition comprises mRNA with a lipid:mRNA (weight / weight) ratio between 5:1 and 40:1. In some implementations, the LNP comprises mRNA with a lipid:mRNA ratio between 10:1 and 40:1, 15:1 and 40:1, 20:1 and 40:1, 25:1 and 40:1, 30:1 and 40:1, 35:1 and 40:1, 20:1 and 35:1, 25:1 and 35:1, 30:1 and 35:1, 20:1 and 30:1, 25:1 and 30:1, 20:1 and 25:1, 25:1 and 30:1, 25:1 and 35:1, 20:1 and 36:1, 25:1 and 36:1, 5:1 and 45:1, 20:1 and 40:1, 25:1 and 40:1, 35:1 and 40:1, or 30:1 and 40:1. In some embodiments, the LNP contains mRNA with a lipid:mRNA ratio of 30:1. In some embodiments, the LNP contains mRNA with a lipid:mRNA ratio of 40:1.

[0437] In some implementations, the mRNA encodes a gene or part of a gene associated with the CNS diseases shown in Table 10.

[0438] Table 10. Exemplary genes associated with CNS

[0439]

[0440] In some implementations, the CNS disease is Alzheimer's disease, and at least one transgene contains an mRNA containing cDNA of a gene selected from or consisting of the following groups: 3R tau, 4R tau, AGER, APP, BAX, BCL-2, CHRNA7, DRD2, GFAP, GRIA1, GRIA2, GRIK1, GRIN1, IL-1, SLC1A1, SYP, and SYT1.

[0441] In some implementations, the CNS disease is amyotrophic lateral sclerosis (ALS), and at least one transgene contains an mRNA comprising cDNA of a gene selected from or consisting of the following groups: ALS2, ANG, ANXA11, ATXN2, C9orf72, CHMP2B, CFAP410, CHCHD10, CCNF, DAO, DCTN1, EPHA4, ERBB4, FIG4, FUS, GLE, GLT8D1, HNRNPA1, MATR3, NEFH, NEK1, OPTN, PFN1, PON1, PON2, PON3, PPARGC1A, PRPH, SETX, SIGMAR1, SMN1, SOD1, SPG11, SQSTM1, TAF15, TARDBP, TBK1, TREM2, UBQLN2, UNC13A, VAPB, and VCP.

[0442] In some implementations, the CNS disease is Huntington's disease, and at least one transgene contains an mRNA comprising cDNA of a gene selected from or consisting of the following groups: ATN1, ATXN1, ATXN2, ATXN3, FTL, HTT, IT15, JPH3, PRNP, SLC2A3, TBP, TITF-1, and XBP1.

[0443] In some implementations, the CNS disease is Parkinson's disease, and at least one transgene contains an mRNA comprising cDNA of a gene selected from or consisting of the following groups: ATP13A2, BDNF, EGLN1, GBA, GSTM1, LRRK2, NR4A2, NTRK2, PARK2, PARK7, PINK1, PRKN, S106β, SKP1, SNCA, VPS35, and UCH-L1.

[0444] Modified polynucleotides

[0445] In some embodiments, the polynucleotide comprises one or more modifications selected from the group consisting of: pyridine-4-ketoribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurate methyluridine, 1-taurate methyl-2-thiouridine, 1-taurate methyl-4-thiouridine, 5-methyluridine, 1-methyl -Pseudouridine, 4-Thio-1-methyl-pseudouridine, 2-Thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-Thio-1-methyl-1-denitro-pseudouridine, dihydrouridine, dihydropseudouridine, 2-Thio-dihydrouridine, 2-Thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine Pyrrolo-cytidine, pyrrolo-pseudo-cytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudo-cytidine, 4-thio-1-methyl-pseudo-cytidine, 4-thio-1-methyl-1-deazo-pseudo-cytidine, 1-methyl-1-deazo-pseudo-cytidine, zablaline, 5-aza-zablaline, 5-methyl-zablaline, 5-aza-2-thio-zablaline, 2-thio-zablaline, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, 2-aminopurine, 2,6-di Aminopurine, 7-denitro-adenine, 7-denitro-8-aza-adenine, 7-denitro-2-aminopurine, 7-denitro-8-aza-2-aminopurine, 7-denitro-2,6-diaminopurine, 7-denitro-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenosine, 2-methylthio-adenosine and 2-methoxy-adenosine, inosine, 1-methyl-inosine, woyoside, woyoside, 7-deazo-guanosine, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof.

[0446] In some embodiments, the polynucleotide of this disclosure comprises a modified pyrimidine, such as a modified uridine. In some cases, the uridine analogue is selected from pseudouridine (Ψ), 1-methylpseudouridine (m). 1 P), 2-thiouridine (s) 2 U), 5-methyluridine (m 5 U), 5-methoxyuridine (mo) 5 U), 4-thiouridine (s) 4 U), 5-bromouridine (Br) 5 U), 2'O-methyluridine (U2'm), 2'-amino-2'-deoxyuridine (U2'NH2), 2'-azido-2'-deoxyuridine (U2'N3) and 2'-fluoro-2'-deoxyuridine (U2'F).

[0447] Modifications in non-translated areas

[0448] In some embodiments, the polynucleotides of this disclosure (such as nucleic acid constructs, vectors, or polynucleotides) may contain one or more untranslated regions. The untranslated region may contain any number of modified or unmodified nucleotides. The untranslated region (UTR) of a gene is transcribed but not translated into a polypeptide. In some cases, the untranslated sequence may increase the stability of the polynucleotide and improve translation efficiency. Regulatory features of the UTR may be incorporated into the modified mRNA molecules of this disclosure, for example, to increase molecular stability. Specific features may also be incorporated to ensure controlled downregulation of transcripts, thereby preventing them from being misdirected to unwanted organ sites. Some 5' UTRs play a role in translation initiation. The 5' UTR may contain a Kozak sequence, which is involved in the ribosome-initiated translation of many genes. The Kozak sequence may have a concordant sequence GCC(R)CCAUGG, where R is a purine (adenine or guanine) located three bases upstream of the start codon (AUG). The 5' UTR may form secondary structures that are involved in the binding of translation elongation factors. In some cases, the stability of the polynucleotide molecules disclosed herein and protein production can be increased by engineering features found in genes typically expressed in large quantities in specific target organs. For example, the introduction of 5' UTRs of liver-expressed mRNAs, such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII, can be used to increase the expression of polynucleotides in the liver. Similarly, the use of 5' UTRs from myoproteins (MyoD, myosin, myoglobin, myopoietin, forcein) for endothelial cells (Tie-1, CD36), bone marrow cells (C / EBP, AML1, G-CSF, GM-CSF, CD1 lb, MSR, Fr-1, i-NOS), leukocytes (CD45, CD18), adipose tissue (CD36, GLUT4, ACRP30, liponein), and lung epithelial cells (SP-A / B / C / D) can be used to increase the expression of polynucleotides in desired cells or tissues.

[0449] Other non-UTR sequences may be incorporated into the 5' (or 3' UTR) UTR of the polynucleotides disclosed herein. The 5' and / or 3' UTRs can provide stability and / or translation efficiency for the polynucleotides. For example, introns or portions of intron sequences may be incorporated into the flanking regions of the polynucleotide. Incorporation of intron sequences can also increase the translation rate of the polynucleotide.

[0450] In some implementations, the 3' UTR may contain segments of adenosine and uridine embedded therein. These AU-rich tags are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be classified into the following categories: Class I AREs, which contain several scattered copies of the AUUUA motif within a U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs, which have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of ARE include GM-CSF and TF-α. Class III AREs, which are less well-defined. These U-rich regions do not contain the AUUUA motif; c-Jun and myopoietin are two well-studied examples of this type of element. Proteins that bind to AREs may destabilize messenger RNA (mRNA), while members of the ELAV family (such as HuR) may increase mRNA stability. HuR can bind AREs of all three classes. Engineering a HuR-specific binding site into the 3' UTR of a polynucleotide molecule allows HuR to bind, thereby stabilizing the signal in vivo. Engineering of AU-rich elements (AREs) in the 3' UTR can be used to modulate polynucleotide stability. One or more copies of an ARE can be engineered into a polynucleotide to regulate its stability. AREs can be identified, removed, or mutated to increase intracellular stability, thereby increasing the translation and production of the resulting protein. Transfection assays can be performed using polynucleotides in relevant cell lines, and protein production can be measured at different time points post-transfection. For example, cells can be transfected with different ARE-engineered molecules, and ELISA kits targeting the relevant proteins can be used to measure protein production at 6, 12, 24, 48, and 7 days post-transfection.

[0451] In some embodiments, polynucleotides such as the nucleic acid constructs, vectors, polynucleotides, or compositions disclosed herein may include an engineered 5' cap structure, or the 5' cap may be added to the polynucleotide intracellularly. The 5' cap structure of mRNA can participate in binding to mRNA cap-binding proteins (CBPs), which are responsible for mRNA stability and translational capacity in cells by associating with poly(A)-binding proteins to form mature pseudocircular mRNA material. The 5' cap structure can also participate in extranuclear transfer, increased mRNA stability, and assist in the removal of 5' proximal introns during mRNA splicing.

[0452] In some implementations, the polynucleotide, such as a nucleic acid construct, vector, or polynucleotide, may be 5' capped, thereby creating a 5'-Gppp N-3'-triphosphate bond between the terminal guanosine cap residue and the sense nucleotide transcribed at the 5' end of the mRNA molecule. The cap structure may contain a modified or unmodified 7-methylguanosine linked to the first nucleotide via a 5'-5' triphosphate bridge. This 5'-guanosine cap can then be methylated to produce an N7-methyl-guanosine residue (Cap-0 structure). The ribose of the terminal and / or anteterminal transcribed nucleotides at the 5' end of the mRNA may also optionally be 2'-O-methylated (Cap-1 structure). Decapping of the 5'-guanosine cap structure can be targeted to polynucleotide molecules, such as mRNA molecules, for degradation by hydrolysis and cleavage. In some cases, the cap may contain further modifications, including methylation of the 2' hydroxyl groups of the first two riboses at the 5' end of the mRNA. For example, eukaryotic cap-1 has a methylated 2'-hydroxyl group on the first ribose, while cap-2 has methylated 2'-hydroxyl groups on the first two riboses. The 5' cap can be chemically analogous to the 3' end of an RNA molecule (binding the 5' carbon of the capped ribose, and having a free 3'-hydroxyl group at both the 5' and 3' ends of the capped transcript). This dual modification can provide significant resistance to 5' exonucleases. Non-limiting examples of 5' cap structures that can be used with polynucleotides include, but are not limited to, m 7 G(5')ppp(5')N(Cap-0)m 7 G(5')ppp(5')N1mpNp(Cap-1)and m 7 G(5')-ppp(5')N1mpN2mp(Cap-2).

[0453] Modification of the modified mRNA disclosed herein can produce a non-hydrolyzable cap structure, which prevents decapping and thus increases the mRNA half-life, while promoting efficient translation. Because cap structure hydrolysis requires cleavage of the 5'-ppp-5' triphosphate bond, modified nucleotides can be used during the capping reaction. For example, the vaccinia capping enzyme from New England Biolabs (Ipswich, MA) can be used with guanosine α-thiophosphate nucleotides according to the manufacturer's instructions to generate thiophosphate bonds in the 5'-ppp-5' cap. Other modified guanosine nucleotides, such as α-methylphosphonates and selenium phosphate nucleotides, can be used. Additional modifications include, but are not limited to, 2'-O-methylation of the ribose of the 5' terminal and / or 5' preterminal nucleotides of the mRNA at the 2'-hydroxyl group of the sugar ring. A variety of different 5' cap structures can be used to generate 5' caps for polynucleotides.

[0454] Modified mRNA can be capped post-transcriptionally. According to this disclosure, the 5' cap may contain an endogenous cap or a cap analogue. According to this disclosure, the 5' cap may contain a guanine analogue. Useful guanine analogues include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-dezo-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0455] In some implementations, the untranslated region may contain any number of nucleotides. The length of the untranslated region may include about 1 to about 10 bases or base pairs, about 10 to about 20 bases or base pairs, about 20 to about 50 bases or base pairs, about 50 to about 100 bases or base pairs, about 100 to about 500 bases or base pairs, about 500 to about 1000 bases or base pairs, about 1000 to about 2000 bases or base pairs, or about 2000 to about 3000 bases or base pairs. The length of a base pair, approximately 3,000 to approximately 4,000 bases or base pairs, approximately 4,000 to approximately 5,000 bases or base pairs, approximately 5,000 to approximately 6,000 bases or base pairs, approximately 6,000 to approximately 7,000 bases or base pairs, approximately 7,000 to approximately 8,000 bases or base pairs, approximately 8,000 to approximately 9,000 bases or base pairs, or approximately 9,000 to approximately 10,000 bases or base pairs. The length of the untranslated region can include, for example, at least 1 base or base pair, 2 base or base pairs, 3 base or base pairs, 4 base or base pairs, 5 base or base pairs, 6 base or base pairs, 7 base or base pairs, 8 base or base pairs, 9 base or base pairs, 10 base or base pairs, 20 base or base pairs, 30 base or base pairs, 40 base or base pairs, 50 base or base pairs, 60 base or base pairs, 70 base or base pairs, 80 base or base pairs, 90 base or base pairs, 100 base or base pairs, 200 base or base pairs. The length of a base pair, 300 bases or base pairs, 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 700 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 1000 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs, 6000 bases or base pairs, 7000 bases or base pairs, 8000 bases or base pairs, 9000 bases or base pairs, or 10000 bases or base pairs.

[0456] In some embodiments, the polynucleotides of this disclosure may comprise a polyA sequence. The polyA sequence (e.g., a polyA tail) may comprise any number of nucleotides. The length of the polyA sequence may include about 1 to about 10 bases or base pairs, about 10 to about 20 bases or base pairs, about 20 to about 50 bases or base pairs, about 50 to about 100 bases or base pairs, about 100 to about 500 bases or base pairs, about 500 to about 1000 bases or base pairs, about 1000 to about 2000 bases or base pairs, about 2000 to about 3000 bases or base pairs. The length of the polyA sequence is approximately 3,000 to 4,000 bases or base pairs, approximately 4,000 to 5,000 bases or base pairs, approximately 5,000 to 6,000 bases or base pairs, approximately 6,000 to 7,000 bases or base pairs, approximately 7,000 to 8,000 bases or base pairs, approximately 8,000 to 9,000 bases or base pairs, or approximately 9,000 to 10,000 bases or base pairs. In some embodiments, the polyA sequence is at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides. The length of the polyA sequence can include, for example, at least 1 base or base pair, 2 bases or base pairs, 3 bases or base pairs, 4 bases or base pairs, 5 bases or base pairs, 6 bases or base pairs, 7 bases or base pairs, 8 bases or base pairs, 9 bases or base pairs, 10 bases or base pairs, 20 bases or base pairs, 30 bases or base pairs, 40 bases or base pairs, 50 bases or base pairs, 60 bases or base pairs, 70 bases or base pairs, 80 bases or base pairs, 90 bases or base pairs, 100 bases or base pairs, or 200 bases. The length of a base pair, or 300 bases or base pairs, 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 700 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 1000 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs, 6000 bases or base pairs, 7000 bases or base pairs, 8000 bases or base pairs, 9000 bases or base pairs, or 10000 bases or base pairs. The polyA sequence may include a length of up to 100 bases or base pairs, 90 bases or base pairs, 80 bases or base pairs, 70 bases or base pairs, 60 bases or base pairs, 50 bases or base pairs, 40 bases or base pairs, 30 bases or base pairs, 20 bases or base pairs, 10 bases or base pairs, or 5 bases or base pairs.

[0457] Gene editing payload

[0458] The LNP disclosed herein may contain one or more components for gene editing, such as, but not limited to, guide RNA, tracr RNA, sgRNA, mRNA encoding gene or base editing proteins, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly spaced short palindromic repeat (CRISPR) nucleases (e.g., Cas9), a DNA template for gene editing, or combinations thereof. In some embodiments, the LNP payload may be suitable for genome editing technologies. In some embodiments, the genome editing technology may be CRISPR or TALEN. In some embodiments, the LNP may contain one or more mRNAs capable of encoding gene editing or base editing proteins. In some embodiments, the LNP may simultaneously contain mRNA encoding gene or base editing proteins and one or more guide RNAs. In some embodiments, the LNP may contain at least one nucleic acid suitable for genome editing technologies, such as CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA (gRNA), and a DNA repair template. In some implementations, the activity of the CRISPR nuclease may be altered; for example, the nuclease may be modified to be a nicking enzyme instead of performing double-strand cuts, or it may bind to a sequence specified by the guide RNA but without enzymatic activity. In some implementations, the base-editing protein may be a fusion protein comprising a deaminase domain and a sequence-specific DNA-binding domain, such as an inactive CRISPR nuclease.

[0459] Gene editing methods

[0460] The LNP or pharmaceutical composition described herein may contain a payload of any conventional gene editing method. In some embodiments, the gene-editing component may be selectively delivered to cells of a target organ. In some embodiments, the target organ may be the lung. In some embodiments, the cells of the target organ may be lung cells. In some embodiments, the cells may be ciliated cells, goblet cells, secretory cells, rod cells, basal cells, or ionized cells.

[0461] In some implementations, gene editing can be targeted editing. Targeted editing can be achieved through nuclease-independent or nuclease-dependent methods.

[0462] Non-nuclease-dependent targeted editing, such as base editing and / or leader editing, can involve precise modifications to DNA sequences without producing double-strand breaks. Homologous recombination can be guided by homologous sequences flanked by exogenous polynucleotides in endogenous sequences to be introduced via enzymatic mechanisms in target organ cells.

[0463] Base editing allows the conversion of one DNA base pair to another at a specific target site. In some embodiments, the nuclease may be a fusion of a deaminase with a modified Cas9 protein (dCas9) or other engineered Cas variants. In some embodiments, base editing can change C (cytosine) to T (thymine) or A (adenine) to G (guanine) in endogenous DNA. Guide RNA can be programmed to target specific genomic locations of interest in target organ cells.

[0464] Leader editing allows for more complex and precise DNA modifications, including insertions, deletions, and all 12 possible base-to-base conversions (A, C, G, T) without double-strand breaks. Leader editing guide RNAs can be designed, consisting of a guide sequence and a template for the desired editing. A leader editing protein (PE2), capable of combining reverse transcriptase and a Cas9 variant, can be guided to the target site via the leader editing guide RNA. The Cas9 variant can create a single-strand break (nick) in the DNA. The reverse transcriptase can then use the template sequence of the leader editing guide RNA to copy the desired change into the nicked strand of the DNA. Subsequently, the cellular repair mechanisms of the target organ cells can repair the nick via homology-directed repair (HDR), thereby incorporating the edited sequence.

[0465] Nuclease-dependent methods can achieve higher-frequency targeted editing by specifically introducing double-strand breaks (DSBs) using rare cleaving nucleases (e.g., endonucleases). This nuclease-dependent targeted editing can also utilize DNA repair mechanisms, such as non-homologous end joining (NHEJ), which can occur in response to DSBs. In some embodiments, DNA repair mediated by NHEJ may result in a small number of random insertions or deletions of endogenous nucleotides (insertions / deletions). In contrast to NHEJ-mediated repair, repair can also occur via homology-directed repair (HDR). When a donor template containing foreign genetic material flanked by a pair of homologous arms is present, the foreign genetic material can be introduced into the genome via HDR, potentially leading to targeted integration of the foreign genetic material. In some embodiments, nuclease-dependent targeted editing nucleases may include, but are not limited to, CRISPR-Cas9, CRISPR-Cas12 (Cpf1), CRISPR-Cas13, C2c2, C2c6, NgAgo, and / or TALEN.

[0466] The use of CRISPR-Cas gene editing technology to create genomic deletions in cells (e.g., knocking out genes in cells) is a well-known technique. See, for example, Bauer et al., J Vis Exp. 95:e52118 (2015). Available endonucleases capable of introducing specific target DSBs include, but are not limited to, ZFN, TALEN, and CRISPR / Cas9.

[0467] In some implementations, targeted gene editing can be achieved using a dual integrase box exchange (DICE) system that utilizes phiC31 and Bxb1 integrases.

[0468] CRISPR-Cas9 gene editing system

[0469] The CRISPR-Cas9 system is a naturally occurring biological defense mechanism in prokaryotic cells that has been repurposed as an RNA-guided DNA-targeting platform for gene editing. It relies on the DNA nuclease Cas9 and two non-coding RNAs, crriprRNA (crRNA) and trans-activating RNA (tracrRNA), to target and cleave DNA. CRISPR is a family of DNA sequences found in the genomes of bacteria and archaea containing DNA fragments (spacer regions) that are similar to foreign DNA previously exposed to the cell (e.g., by viruses that infect or attack prokaryotes). Prokaryotes can use these DNA fragments to detect and destroy similar reintroduced foreign DNA, such as when encountering similar viruses during a subsequent attack. Transcription of CRISPR loci can form RNA molecules containing spacer sequences that can bind to and target Cas (CRISPR-associated) proteins capable of recognizing and cleaving foreign DNA. Many types and classes of CRISPR / Cas systems have been described, for example, in Koonin et al., Curr Opin Microbiol 37:67-78 (2017).

[0470] crRNA drives the sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing (typically pairing with about 20 nucleotides in the target DNA). Changing the sequence of the 5' last 20 nucleotides of crRNA allows the CRISPR-Cas9 complex to target specific loci. If the target sequence is immediately followed by a specific short DNA motif (with the sequence NGG) called the prespacer adjacent motif (PAM), the CRISPR-Cas9 complex can only bind to DNA sequences containing a sequence that matches the first 20 nucleotides of the crRNA.

[0471] tracrRNA can hybridize with the 3' end of crRNA to form an RNA-double-stranded structure, which can bind to the Cas9 endonuclease to form a catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.

[0472] Once the CRISPR-Cas9 complex binds to DNA at the target site, the two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, producing a double-strand break (DSB), in which both strands of DNA terminate at a base pair (flat end).

[0473] After the CRISPR-Cas9 complex binds to DNA at a specific target site and forms a site-specific DSB, cells can repair the DSB using two main DNA repair pathways: non-homologous end joining (NHEJ) and homologous directed repair (HDR). NHEJ is a highly active repair mechanism in most cell types, including non-dividing cells. NHEJ can be error-prone and often results in the removal or addition of one to several hundred nucleotides at the DSB site, although such modifications are usually less than 20 nucleotides. The resulting insertions and deletions (insertions and deletions) can disrupt either the coding or non-coding regions of a gene. Alternatively, HDR can repair the DSB with high fidelity using long segments of homologous donor DNA, whether endogenous or exogenous. HDR is active only in dividing cells and occurs at a relatively low frequency in most cell types.

[0474] CRISPR endonucleases: In some embodiments, the Cas9 endonuclease can be used in the CRISPR method for the genetic engineering of target organ cells of the LNP described herein. In some embodiments, the Cas9 enzyme can be derived from *Streptococcus pyogenes*, although other Cas9 homologs may also be used. In some embodiments, the Cas9 enzyme can be wild-type Cas9. In some embodiments, the Cas9 enzyme can be a modified form of Cas9 (e.g., an evolved form of Cas9, or a Cas9 ortholog or variant). In some embodiments, Cas9 can be replaced by another RNA-guided endonuclease, such as Cpf1 (a class II CRISPR / Cas system).

[0475] In some implementations, the CRISPR / Cas system may contain components derived from type I, type II, or type III systems. In some implementations, the CRISPR / Cas system may contain components derived from type 1 and type 2 CRISPR / Cas systems, having types I through V, or types II, V, and VI, respectively (Makarova et al., Nat Rev Microbiol 13(11):722-36(2015); Shmakov et al., Mol Cell 60:385-397 (2015)).

[0476] Class 2 CRISPR / Cas systems can have a single protein effector. Type II, V, and VI Cas proteins can be single-protein RNA-guided endonucleases, referred to herein as class 2 Cas nucleases. Class 2 Cas nucleases can include, for example, but not limited to, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. The Cpf1 nuclease is homologous to Cas9 and contains a RuvC-like nuclease domain.

[0477] In some embodiments, the Cas nuclease may be derived from a type II CRISPR / Cas system (e.g., the Cas9 protein from the CRISPR / Cas9 system). In some embodiments, the Cas nuclease may be derived from two types of CRISPR / Cas systems (single-protein Cas nucleases, such as the Cas9 protein or the Cpf1 protein). The Cas9 and Cpf1 protein families are enzymes with DNA endonuclease activity and can be directed to cleave desired nucleic acid targets by designing appropriate guide RNAs, which will be explained further below.

[0478] In some embodiments, the Cas nuclease may contain more than one nuclease domain. In some embodiments, the Cas9 nuclease may contain at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease may introduce a DSB into the target sequence. In some embodiments, the Cas9 nuclease may be modified to contain only one functional nuclease domain. For example, the Cas9 nuclease may be modified such that one of the nuclease domains is mutated or completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease may be modified to lack a functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease may be modified to lack a functional HNH-like nuclease domain. In some embodiments where only one nuclease domain is functional, the Cas9 nuclease may be a cleavage enzyme that can introduce single-strand breaks (nicks) into the target sequence. In some embodiments, conserved amino acids within the Cas9 nuclease domain may be substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease nickase may contain amino acid substitutions in the RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC-like nuclease domain may include D10A (based on *Streptococcus pyogenes* Cas9 nuclease). In some embodiments, the nickase may contain amino acid substitutions in the HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH-like nuclease domain may include, but are not limited to, E762A, H840A, N863A, H983A, and D986A (based on *Streptococcus pyogenes* Cas9 nuclease).

[0479] In some embodiments, the Cas nuclease may be derived from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a type I CRISPR / Cas system. For example, the Cas nuclease may be a Cas3 nuclease. In some embodiments, the Cas nuclease may be derived from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease may be derived from a type IV CRISPR / Cas system. In some embodiments, the Cas nuclease may be derived from a type V CRISPR / Cas system. In some embodiments, the Cas nuclease may be derived from a type VI CRISPR / Cas system.

[0480] Type I CRISPR / Cas systems utilize a large effector complex called Cascade (a CRISPR-associated complex for antiviral defense) for targeted binding and interference. The Cascade complex can contain multiple Cas proteins, including Cas3, which is responsible for disrupting the target DNA. Type II CRISPR / Cas systems, particularly the CRISPR-Cas9 system, utilize a single Cas9 protein guided by synthetic guide RNA (sgRNA) to introduce double-strand breaks in the target DNA for subsequent repair or modification. Type III CRISPR / Cas systems utilize Csm (CRISPR-Cas isoform multiprotein) or Cmr (CRISPR-Cas isoform ribonucleoprotein) complexes for interference. Type III CRISPR / Cas systems can target RNA molecules in addition to DNA. Type V CRISPR / Cas systems, including Cpf1 (also known as Cas12) and C2c2 (also known as Cas13), utilize single effector proteins for interference. Type VI CRISPR / Cas systems can utilize a single Cas protein, such as C2c2 (also known as Cas13), to target and cleave RNA molecules, making the system usable for RNA editing and manipulation.

[0481] Guide RNA (gRNA): CRISPR technology may involve using a nucleic acid targeting the genome that can guide one or more endonucleases to a specific target sequence within the target gene for gene editing at that specific target sequence. The nucleic acid targeting the genome can be RNA. The RNA targeting the genome is referred to herein as "guide RNA" or "gRNA". The guide RNA may contain at least one spacer sequence and a CRISPR repeat sequence, the at least one spacer sequence being capable of hybridizing with a target nucleic acid sequence within the target gene for editing.

[0482] In type II gRNAs, the gRNA may also contain a second RNA sequence called tracrRNA. In type II gRNAs, the CRISPR repeat sequence and the tracrRNA sequence can hybridize to form a double strand. In type V gRNAs, the crRNA can form a double strand. In both systems, the double strand can bind a site-directing peptide, allowing the guide RNA and the site-directing peptide to form a complex. In some implementations, the genomic nucleic acid can provide target specificity to the complex through its association with the site-directing peptide. Therefore, the genomic nucleic acid can guide the activity of the site-directing peptide.

[0483] As will be understood by those skilled in the art, each guide RNA can be designed to include a spacer sequence complementary to its genomic target sequence. See Jinek et al., Science 337:816-821 (2012); Deltcheva et al., Nature 471:602-607 (2011).

[0484] In some implementations, the nucleic acid targeting the genome (e.g., gRNA) can be a double-stranded guide RNA comprising two RNA molecular strands. The first strand may contain an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence in the 5' to 3' direction. The second strand may contain a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0485] In some implementations, the nucleic acid targeting the genome (e.g., gRNA) can be a single-molecule guide RNA (sgRNA). In type II systems, the sgRNA may include, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single-molecule guide adapter, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension may include elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide adapter may connect the minimal CRISPR repeat sequence and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension may include one or more hairpins. In type V systems, the single-molecule guide RNA may include a minimal CRISPR repeat sequence and a spacer sequence in the 5' to 3' direction.

[0486] The spacer sequence in the gRNA is a sequence (e.g., a 20-nucleotide sequence) that can define a target sequence (e.g., a DNA target sequence, such as a genomic target sequence) of interest (e.g., DNAI1 or CFTR). In some embodiments, the spacer sequence can be in the range of 15 to 30 nucleotides. For example, the spacer sequence can contain 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the spacer sequence can contain 20 nucleotides.

[0487] The target sequence is located within a target gene (e.g., DNAI1 or CFTR), and may be adjacent to a PAM sequence and may be a sequence to be modified by an RNA-guided nuclease (e.g., Cas9). The target sequence is located on the PAM strand of a target nucleic acid, which is a double-stranded molecule containing both a PAM strand and a complementary non-PAM strand. Those skilled in the art will recognize that the gRNA spacer region sequence can hybridize with a complementary sequence located in the non-PAM strand of the target nucleic acid of interest. Therefore, the gRNA spacer region sequence can be an RNA equivalent of the target sequence. The gRNA spacer region can interact with the target nucleic acid of interest in a sequence-specific manner through hybridization (i.e., base pairing). Therefore, the nucleotide sequence of the spacer region can vary depending on the target sequence of the target nucleic acid of interest.

[0488] In the CRISPR / Cas system, the spacer region sequence can be programmed to hybridize with the 5' end of the target nucleic acid in a PAM that is recognized by the Cas9 enzyme used in the system. The spacer region can be a perfect match to the target sequence or it can have a mismatch. Each Cas9 enzyme is capable of recognizing a specific PAM sequence in the target DNA. For example, *Streptococcus pyogenes* recognizes a PAM containing the 5'-NRG-3' sequence in the target nucleic acid, where R can contain A or G, N can be any nucleotide, and N can be immediately adjacent to the 3' end of the target nucleic acid sequence targeted by the spacer region sequence.

[0489] In some embodiments, the target nucleic acid sequence may have a length of about 20 nucleotides. In some embodiments, the target nucleic acid may have a length of less than about 20 nucleotides. In some embodiments, the target nucleic acid may have a length of more than about 20 nucleotides. In some embodiments, the target nucleic acid may have a length of at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid may have a length of up to 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid sequence may have 20 bases immediately adjacent to the 5' end of the first nucleotide of PAM. For example, in a sequence containing 5'-NNNNNNNNNNNNNNNNNNNNNRG-3', the target nucleic acid may be a sequence corresponding to N, where N can be any nucleotide, and the underlined NRG sequence may be Streptococcus pyogenes PAM.

[0490] Guide RNA can target any sequence of interest via the spacer region sequence in crRNA. In some embodiments, the complementarity between the spacer region sequence of the guide RNA and the target sequence in the target gene can be approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the spacer region sequence of the guide RNA can be 100% complementary to the target sequence in the target gene. In other embodiments, the spacer region sequence of the guide RNA and the target sequence in the target gene can contain up to 10 mismatches, for example, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 mismatch.

[0491] The length of the spacer sequence in the gRNA can depend on the CRISPR / Cas9 system and the components used to edit any target gene (e.g., DNAI1 or CFTR). For example, different Cas9 proteins from different bacterial species may have different optimal spacer sequence lengths. Therefore, the spacer sequence length can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more 50 nucleotides. In some embodiments, the spacer sequence length can be 18 to 24 nucleotides. In some embodiments, the target sequence length can be 19 to 21 nucleotides. In some embodiments, the spacer sequence length can contain 20 nucleotides.

[0492] In some embodiments, the gRNA may be sgRNA, which may include a 20-nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may include a spacer sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may include a spacer sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may include a spacer sequence of variable length having about 17 to 30 nucleotides at the 5' end of the sgRNA sequence.

[0493] In some embodiments, the gRNA may comprise unmodified ribonucleic acid. In some embodiments, the gRNA may comprise modified ribonucleic acid. Various types of RNA modifications can be introduced during or after the chemical synthesis and / or enzymatic production of RNA, such as modifications that enhance stability, reduce the likelihood or extent of an innate immune response, and / or enhance other properties, as described in the art. In some embodiments, non-naturally modified nucleobases can be introduced into any gRNA during or after synthesis. In some embodiments, the modification can be located at a nucleoside internucleotide bond, a purine or pyrimidine base, or a sugar. In some embodiments, the modification can be introduced at the end of the gRNA by chemical synthesis or by a polymerase.

[0494] In some implementations, more than one guide RNA may be used with the CRISPR / Cas nuclease system. Each guide RNA may contain a different target sequence, allowing the CRISPR / Cas system to cleave more than one target nucleic acid. In some implementations, one or more guide RNAs may have the same or different properties, such as activity or stability within the Cas9 RNP complex. Where more than one guide RNA can be used, each guide RNA may be encoded on the same or different vectors. The promoters used to drive the expression of more than one guide RNA may be the same or different.

[0495] In some implementations, enzymatic or chemical linking methods can be used to conjugate polynucleotides or regions thereof with different functional parts, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc.

[0496] In some embodiments, the CRISPR / Cas nuclease system may contain multiple gRNAs, for example, 2, 3, or 4 gRNAs. These multiple gRNAs can target different sites within the same target gene. Alternatively, the multiple gRNAs can target different genes. In some embodiments, the guide RNA and the Cas protein can form a ribonucleoprotein (RNP), such as a CRISPR / Cas complex. The guide RNA guides the Cas protein to a target sequence on one or more target genes (e.g., DNAI1 and CFTR), whereby the Cas protein cleaves the target gene at the target site. In some embodiments, the CRISPR / Cas complex may be a Cpf1 / guide RNA complex. In some embodiments, the CRISPR complex may be a type II CRISPR / Cas9 complex. In some embodiments, the Cas protein may be the Cas9 protein. In some embodiments, the CRISPR / Cas9 complex may be a Cas9 / guide RNA complex.

[0497] In some implementations, the insertion / deletion frequency (editing frequency) of a specific CRISPR / Cas nuclease system containing one or more specific gRNAs can be determined using TIDE analysis, which can be used to identify highly efficient gRNA molecules for editing target genes. In some implementations, highly efficient gRNAs can produce gene editing frequencies higher than 80%. For example, a gRNA can be considered highly efficient if it produces a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0498] Other gene editing methods

[0499] In addition to the CRISPR system disclosed herein, other gene editing systems known in the art can also be used as payloads for the LNPs described herein. In some embodiments, other gene editing systems may include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), restriction endonucleases, broad-spectrum nucleases, homing endonucleases, etc.

[0500] ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA-binding domain (ZFBD). This ZFBD can be a polypeptide domain that can bind DNA in a sequence-specific manner via one or more zinc fingers. The zinc finger can be a domain of approximately 30 amino acids within the ZFBD, and its structure can be stabilized by coordination with zinc ions. Examples of zinc fingers include, but are not limited to, C2H2, C3H, and C4 zinc fingers. The designed ZFBD can be a domain not found in nature, and its design / composition is primarily generated by reasonable criteria, such as applying substitution rules and computerized algorithms to process information in databases storing existing ZFP design information and binding data. The selected ZFBD can be a domain not found in nature, and its generation can be primarily achieved through empirical processes such as phage display, interaction trapping, or heterozygous selection. In some embodiments, the ZFN can be a fusion of a FokI nuclease and a zinc finger DNA-binding domain.

[0501] TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA-binding domain. A “transcription activator-like effector DNA-binding domain,” “TAL effector DNA-binding domain,” or “TALE DNA-binding domain” is a polypeptide domain of a TAL effector protein responsible for binding the TAL effector protein to DNA. TAL effector proteins can be secreted by plant pathogens of the genus *Xanthomonas* during infection. These proteins can enter the nucleus of plant cells, bind effector-specific DNA sequences via their DNA-binding domains, and activate gene transcription of these sequences via their transactivation domains. The specificity of the TAL effector DNA-binding domain can depend on an incomplete 34-amino acid repeat sequence of variable number of effector residues, which may exhibit polymorphism at selected repeat positions, known as variable repetition double residues (RVD). In some embodiments, TALEN can be a fusion polypeptide of a FokI nuclease and a TAL effector DNA-binding domain.

[0502] Other examples of suitable targeted nucleases may include, but are not limited to, Bxb1, phiC31, PhiBT1, and Wβ / SPBc / TP901-1, whether used alone or in combination. Bxb1 nuclease, also known as Bxb1 integrase, is a site-specific recombinase derived from mycobacteriophase Bxb1. Bxb1 integrase catalyzes site-specific recombination between two specific DNA sequences, known as attachment (att) sites. Bxb1 integrase recognizes a specific 48-base pair sequence within the attachment site. phiC31 nuclease (also known as phiC31 integrase) is derived from the bacterial bacteriophage phiC31. phiC31 nuclease catalyzes site-specific recombination between two specific DNA sequences, known as attB (attachment site in bacterial bacteriophages) and attP (attachment site in bacteriophages). phiC31 nuclease can facilitate the integration of DNA fragments flanked by attB and attP into the genome of target organ cells. phiBT1 nuclease can be integrated into attachment sites different from those of phiC31. Wβ / SPBc / TP901-1 nuclease, also known as bacterial phage P2 Bxb1 Cre nuclease, is a site-specific recombinase derived from the temperate bacterial phage P2.

[0503] D. Lipids

[0504] This disclosure covers LNP compositions that contain an antigen (e.g., a pathogen-associated antigen or a cancer antigen) or a polynucleotide encoding an antigen as described herein, and a mixture of lipids for delivery to host cells. Exemplary lipids contemplated for use in the LNP compositions described herein include ionizable cationic lipids, selective organ-targeting lipids, helper lipids, structural lipids, and polyethylene glycol-conjugated lipids (PEG lipids).

[0505] In one aspect, this disclosure provides a lipid nanoparticle composition comprising an antigen (e.g., a pathogen-associated antigen or a cancer antigen) or a polynucleotide encoding an antigen, and selective organ-targeting (SORT) lipids; and ionizable cationic lipids, auxiliary lipids, sterols and / or polyethylene glycol-conjugated lipids (PEG lipids).

[0506] In some embodiments, the LNP composition comprises one or more, two or more, three or more polynucleotides encoding one or more, two or more, three or more antigens (e.g., pathogen-associated antigens). In some embodiments, the LNP composition comprises one or more, two or more, three or more polynucleotides encoding antigens from different pathogens.

[0507] In some embodiments, the LNP composition comprises one or more, two or more, three or more polynucleotides encoding one or more, two or more, three or more antigens (e.g., cancer antigens). In some embodiments, the LNP composition comprises one or more, two or more, three or more polynucleotides encoding antigens derived from different cancer cell types.

[0508] In some embodiments, the LNP composition delivers one or more, two or more, three or more polynucleotides that encode one or more, two or more, three or more antigens (e.g., pathogen-associated antigens). In some embodiments, the LNP composition delivers one or more, two or more, three or more polynucleotides that encode antigens from different pathogens.

[0509] In some embodiments, the LNP composition delivers one or more, two or more, three or more polynucleotides that encode one or more, two or more, three or more antigens (e.g., cancer antigens). In some embodiments, the LNP composition delivers one or more, two or more, three or more polynucleotides that encode antigens from different cancer cell types.

[0510] In some embodiments, lipid nanoparticles deliver one or more, two or more, three or more antigens (e.g., pathogen-associated antigens) to host cells. In some embodiments, lipid nanoparticles deliver one or more, two or more, three or more antigens from different pathogens.

[0511] In some embodiments, lipid nanoparticles deliver one or more, two or more, three or more antigens (e.g., cancer antigens) to host cells. In some embodiments, lipid nanoparticles deliver one or more, two or more, three or more antigens from different cancer cell types.

[0512] In some embodiments, the LNP composition comprises mRNA with a lipid:mRNA (weight / weight) ratio between 5:1 and 40:1. In some embodiments, the LNP composition comprises mRNA with a lipid:mRNA ratio between 10:1 and 40:1, 15:1 and 40:1, 20:1 and 40:1, 25:1 and 40:1, 30:1 and 40:1, 35:1 and 40:1, 20:1 and 35:1, 25:1 and 35:1, 30:1 and 35:1, 20:1 and 30:1, 25:1 and 30:1, 20:1 and 25:1, 25:1 and 30:1, 25:1 and 35:1, 20:1 and 36:1, 25:1 and 36:1, or 5:1 and 45:1.

[0513] Ionizable cationic lipids

[0514] In some embodiments, the lipid composition comprises an ionizable cationic lipid. In some embodiments, the ionizable cationic lipid contains one or more groups that are protonated at physiological pH but may deprotonate and become uncharged at pH values ​​above the lipid's pKa. The ionizable group may contain one or more protonable amines capable of forming cationic groups at physiological pH. The ionizable cationic lipid compound may further comprise one or more lipid components, such as two or more C6-C6 lipids. 24 Fatty acids with alkyl or alkenyl carbon groups. These lipid groups can be linked by ester bonds or further added to sulfur atoms via Michael addition. In some embodiments, these compounds can be dendritic polymers, dendrites, polymers, or combinations thereof.

[0515] The lipid nanoparticle composition may contain one or more ionizable (e.g., ionizable amino) lipids (e.g., lipids that may be positively charged or partially positively charged at physiological pH). Ionizable cationic lipids may be selected from the non-restricted group consisting of: 3-(eicosylamino)-N1,N1,4-tridodecyl-1-piperazine ethylamine (KL10), N1-[2-(eicosylamino)ethyl]N1,N4,N4-tridodecyl-1,4-piperazine diethylamine (KL22), 14,25-tetradecyl-15,18,21,24-tetraaza-octacosane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), and heptadecyl-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLi). n-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3(3)-cholest-5-en-3-yloxy]octyl}oxy)N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA) (2R) and (2S)-2-({8-[(3(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propyl-1-amine (octyl-CLinDMA (2S)), 4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate (ALC-0315) or heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102). In addition to these, ionizable cationic lipids can also be lipids containing cyclic amine groups.

[0516] Ionizable cationic lipids can also be compounds disclosed in International Publication No. WO2017075531, the entire contents of which are incorporated herein by reference. Ionizable cationic lipids can also be compounds disclosed in International Publication No. WO2015199952, the entire contents of which are incorporated herein by reference. In one embodiment, the ionizable cationic lipid may be selected from, but is not limited to, international publications WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2008103276, WO2013086373, WO2015199952, WO Ionizable cationic lipids described in U.S. Patent Nos. 2015074085 and WO2013086354, U.S. Patent Nos. 7,893,302, 7,404,969, 8,283,333 and 8,466,122, and U.S. Patent Publications US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541 and US20130225836; the entire contents of each of these documents are incorporated herein by reference.

[0517] In some embodiments, the ionizable cationic lipid comprises 2 to 6 hydrophobic tails, typically alkyl or alkenyl, such as C6-C. 24 It may have an alkyl or alkenyl group, but may also have at least one, at least two, at least three, at least four, at least five or more than six tails.

[0518] dendritic polymers

[0519] In some embodiments, the ionizable cationic lipid is a dendritic polymer. A dendritic polymer is a polymer exhibiting regular dendritic branching, formed by adding branching layers sequentially or generationally to or from a core, and characterized by a core, at least one internal branching layer, and a surface branching layer. (See Petar R. Dvornic and Donald A. Tomalia in Chem. in Britain, 641-645, August 1994.) In other embodiments, as used herein, the term "dendritic polymer" is intended to include, but is not limited to, a molecular structure having an internal core, an inner layer (or "generation") regularly connected to the initiator core consisting of repeating units, and an outer surface connected to the outermost generation consisting of terminal groups. A "dendritic body" is a class of dendritic polymers having branches emanating from a focal point, which is or can be directly or via a connecting portion connected to the core to form a larger dendritic polymer. In some embodiments, the dendritic polymer structure has repeating groups radiating outward from a central core, the number of each repeating unit doubling with each branch. In some embodiments, the dendritic polymers described herein may be described as small molecules, medium-sized molecules, lipids, or lipid-like materials. These terms can be used to describe compounds described herein that have a dendritic appearance (e.g., molecules radiating outward from a single focal point).

[0520] Although dendritic polymers are polymers, they may be superior to conventional polymers because they possess controllable structures, a single molecular weight, numerous and controllable surface functionalities, and typically exhibit a globular conformation after reaching a certain generation. Dendritic polymers can be prepared by sequential reactions of each repeating unit to produce monodisperse, dendritic, and / or generational polymeric structures. A single dendritic polymer consists of a central core molecule, with dendritic wedges attached to one or more functional sites on this central core. Depending on the assembly monomers used during preparation, the dendritic surface layer can have a variety of functional groups disposed thereon, including anionic, cationic, hydrophilic, or lipophilic groups.

[0521] The physical properties of dendritic polymers can be tuned by altering the functional groups and / or chemical properties of the core, repeating units, and surface or capping groups. Some properties that can be modified include, but are not limited to, solubility, toxicity, immunogenicity, and bioattachment ability. Dendritic polymers are typically described by the number of repeating units in their generations or branches. A dendritic polymer consisting only of a core molecule is called generation 0, while each consecutive repeating unit along all branches is generation 1, generation 2, and so on, up to the capping or surface groups. In some embodiments, a half-generation may be produced only by a first condensation reaction with an amine, rather than a second condensation reaction with a thiol.

[0522] The preparation of dendritic polymers requires a degree of synthetic control through a series of stepwise reactions, which involve building the dendritic polymer by each successive group. Dendritic polymer synthesis can be convergent or divergent. During divergent dendritic polymer synthesis, molecules assemble from the core to the periphery in a stepwise process involving linking one generation to the previous one and then changing functional groups for the next reaction stage. Functional group transformation is essential to prevent uncontrolled polymerization. Such polymerization will produce highly branched, non-monodispersive molecules, also known as hyperbranched polymers. Due to steric effects, the recurring dendritic polymer units continue to react, generating spherical or globular molecules until steric saturation becomes so large that it prevents complete reaction of a particular generation and destroys the monodispersity of the molecules. Therefore, in some embodiments, dendritic polymers of generations G1–G10 are particularly considered. In some embodiments, the dendritic polymer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range thereof. In some embodiments, the dendritic polymers used herein are G0, G1, G2, or G3. However, the possible generations (such as 11, 12, 13, 14, 15, 20, or 25) can be increased by reducing the number of spacer units in the branched polymer.

[0523] Furthermore, dendritic polymers possess two main chemical environments: one formed by specific surface groups on the terminal branches, and the other is the interior of the dendritic structure. Thanks to the higher-order structure of the dendritic structure, the interior is protected from the influence of the bulk medium and surface groups. It is precisely because of these different chemical environments that dendritic polymers exhibit many different potential applications, including therapeutic uses.

[0524] In some embodiments of the lipid composition, dendritic polymers are assembled using the different reactivity of acrylate and methacrylate groups with amines and thiols. The dendritic polymers may include secondary or tertiary amines formed by reacting acrylate groups with primary or secondary amines, and thioethers formed by reacting methacrylates with mercapto groups. Additionally, the repeating units of the dendritic polymer may contain groups that are biodegradable under physiological conditions. In some embodiments, these repeating units may contain one or more geminal diether, ester, amide, or disulfide groups. In some embodiments, the core molecule is a monoamine, which allows dendritic polymerization in only one direction. In other embodiments, the core molecule is a polyamine having multiple different dendritic branches, each dendritic branch containing one or more repeating units. The dendritic polymer can be formed by removing one or more hydrogen atoms from the core. In some embodiments, these hydrogen atoms are on heteroatoms (such as nitrogen atoms). In some embodiments, the capping groups are lipophilic groups, such as long-chain alkyl or alkenyl groups. In other embodiments, the capping groups are long-chain haloalkyl or haloalkenyl groups. In other embodiments, the capping group is an aliphatic or aromatic group containing an ionizable group such as an amine (−NH2) or a carboxylic acid (−CO2H). In still other embodiments, the capping group is an aliphatic or aromatic group containing one or more hydrogen bond donors (such as hydroxyl, amide, or ester).

[0525] Ionizable cationic lipids may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be separable in optically active or racemic form. Therefore, unless a specific stereochemical or isomeric form is specifically indicated, all chiral, diastereomeric, racemic, epimeric, and geometrical isomers of the chemical formula are contemplated. Ionizable cationic lipids may exist as racemic mixtures and mixtures of racemates, as single enantiomers, mixtures of diastereomers, and as individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral center of the ionizable cationic lipid may have an S or R configuration. Furthermore, one or more ionizable cationic lipids are expected to exist as structural isomers. In some embodiments, the compounds have the same formula but differ in the way the core nitrogen atom is linked. It is not desired to be bound by any theory; such ionizable cationic lipids are believed to exist because the starting monomer reacts first with a primary amine and then statistically with any secondary amine present. Therefore, structural isomers may present as mixtures of fully reacted primary amines followed by reacted secondary amines.

[0526] Chemical formulas used to represent ionizable cationic lipids typically show only one of several possible tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is described for a given formula, and whichever is the most general, it means all tautomers of the given formula.

[0527] The ionizable cationic lipids disclosed herein also have the following advantages: compared with compounds known in the prior art, they can be more effective, less toxic, have a longer duration of action, be more potent, produce fewer side effects, be more easily absorbed and / or have better pharmacokinetic characteristics (e.g., higher oral bioavailability and / or lower clearance), and / or have other useful pharmacological, physical or chemical properties, whether or not used for the indications described herein.

[0528] Furthermore, the atoms constituting ionizable cationic lipids are intended to include all isotopic forms of such atoms. As used herein, isotopes include atoms with the same atomic number but different mass numbers. By way of general example and not limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include... 13 C and 14 C.

[0529] It should be recognized that the specific anion or cation that forms part of any ionizable cationic lipid in any salt form presented herein is not a critical factor, provided that the salt is pharmacologically acceptable as a whole. Other examples of pharmaceutically acceptable salts and their preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), the entire contents of which are incorporated herein by reference.

[0530] In some embodiments of the lipid composition, the ionizable lipid is a dendritic polymer or dendrite. In some embodiments, the ionizable cationic lipid comprises an ammonium group that is positively charged at physiological pH and contains at least two hydrophobic groups. In some embodiments, the ammonium group is positively charged at a pH of about 6 to about 8. In some embodiments, the ionizable cationic lipid is a dendritic polymer or dendrite. In some embodiments, the ionizable cationic lipid comprises at least two C6-C... 24 Alkyl or alkenyl groups.

[0531] dendritic polymers of formula (I)

[0532] In some embodiments of the lipid composition, the ionizable cationic lipid comprises at least two C8-C atoms. 24Alkyl groups. In some embodiments, the ionizable cationic lipid is further defined as a dendritic polymer:

[0533] Core - (Repeating Unit) n -Terminal groups (DI)

[0534] One or more core hydrogen atoms are replaced by repeating units, and in which:

[0535] The core has the following formula:

[0536] (D-II)

[0537] in:

[0538] X1 is an amino group or C1-C. 12 Alkylamino, C1-C 12 Dialkylamino, C3-C 12 Heterocyclic alkyl, C5-C 12 heteroaryl groups or their substituted forms;

[0539] R1 represents amino, hydroxyl, mercapto, or C1-C. 12 Alkylamino or C1-C 12 Dialkylamino, or a substituted form of any of these groups; and

[0540] a is 1, 2, 3, 4, 5, or 6; or

[0541] The core has the following formula:

[0542] (D-III)

[0543] in:

[0544] X2 is N(R5) y ;

[0545] R5 is hydrogen, C1-C 18 Alkyl or substituted C1-C 18 Alkyl; and

[0546] y can be 0, 1, or 2, provided that the sum of y and z is 3;

[0547] R2 represents amino, hydroxyl, thiol, or C1-C groups. 12 Alkylamino or C1-C 12 Dialkylamino, or a substituted form of any of these groups;

[0548] b is 1, 2, 3, 4, 5, or 6; and

[0549] z is 1, 2, or 3; the prerequisite is that the sum of z and y is 3; or

[0550] The core has the following formula:

[0551] (D-IV)

[0552] in:

[0553] X3 is -NR6-, where R6 is hydrogen, C1-C8 alkyl or C1-C8 substituted alkyl, -O- or C1-C8 alkylamino diester, C1-C8 alkoxy diester, C6-C8 aromatic diester, C5-C8 heteroaromatic diester, C3-C8 heterocyclic alkane diester, or a substituted form of any of these groups;

[0554] R3 and R4 are each independently amino, hydroxyl, mercapto, or C1-C. 12 Alkylamino or C1-C 12 Dialkylamino, or a substituted form of any of these groups; or a group of the following formula: −N(R f ) f (CH2CH2N(R c )) e R d , , or ;

[0555] in:

[0556] e and f are each independently 1, 2, or 3; the prerequisite is that the sum of e and f is 3.

[0557] R c R d and R f Each is independently hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl;

[0558] c and d are each independently 1, 2, 3, 4, 5, or 6; or

[0559] The core is C1-C 18 Alkylamines, C1-C 36 Dialkylamines, C3-C 12 Heterocyclic alkanes, or substituted forms of any of these groups;

[0560] The repeating unit contains a degradable diacyl group, or a degradable diacyl group and a connector;

[0561] Degradable diacyl groups have the following formula:

[0562] (D-VII)

[0563] in:

[0564] A1 and A2 are each independently −O−, -S−, or −NR. a -, where:

[0565] R a It is hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl;

[0566] Y3 is C1-C 12 Alkyl, C1-C 12 alkendiyl, C6-C 12 Aromatic dimethyl groups, or substituted forms of any of these groups; or groups of the following formula:

[0567] or

[0568] in:

[0569] X3 and X4 are C1-C 12 Alkyl, C2-C 12 alkendiyl, C6-C 12 Aromatic dimethyl groups, or substituted forms of any of these groups;

[0570] Y5 is a covalent bond, C1-C 12 Alkyl, C1-C 12 alkendiyl, C6-C 12 Aromatic dimethyl groups, or substituted forms of any of these groups; and

[0571] R9 is a C1-C8 alkyl or a substituted C1-C8 alkyl;

[0572] The linker group has the following formula:

[0573] (D-VI)

[0574] in:

[0575] Y1 is C1-C 12 Alkyl, C1-C 12 alkendiyl, C6-C 12 Aromatic dimethyl groups, or substituted forms of any of these groups; and

[0576] Each of them Independently represents the connection point with another repeating unit or end group; and

[0577] The end-capping group has the following formula:

[0578] (D-VIII)

[0579] in:

[0580] Y4 is an alkane diene or C1-C. 18 Alkyl groups, where C1-C 18 One or more hydrogen atoms on the alkane diene have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, −OCH2CH3, -SCH3 or -OC(O)CH3;

[0581] R 10 For hydrogen, carboxyl, hydroxyl,

[0582] C6-C 12 Aryl, C1-C 12 Alkylamino, C1-C 12 Dialkylamino, C3-C 12 N-Heterocyclic alkyl groups, -C(O)N(R) 11 )-C1-C6 alkane dimethyl-C3-C 12 Heterocyclic alkyl, -C(O)-C1-C 12 Alkyl-amino, -C(O)-C1-C 12 Dialkylamino or −C(O)−C3-C 12 N-Heterocyclic alkyl groups, wherein:

[0583] R 11 It is hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl;

[0584] The last degradable diacyl group in the chain is attached to the end capping group;

[0585] n is 0, 1, 2, 3, 4, 5, or 6;

[0586] Or its pharmaceutically acceptable salt.

[0587] In some implementations, the end-capping group is further defined by the following formula:

[0588] (D-VIII)

[0589] in:

[0590] Y4 is C1-C 18 Alkyl groups; and

[0591] R 10 It is hydrogen. In some embodiments, A1 and A2 are each independently -O- or -NR. a -

[0592] In some embodiments of the dendritic polymer of formula (DI), the end-capping group is a structure selected from the structures in Table 6.

[0593] In some embodiments of the dendritic polymer of formula (DI), the core is further defined by the following formula:

[0594] (D-III)

[0595] in:

[0596] X2 is N(R5) y ;

[0597] R5 is hydrogen or a C1-C8 alkyl or a substituted C1-C8 alkyl group. 18 Alkyl; and

[0598] y can be 0, 1, or 2, provided that the sum of y and z is 3;

[0599] R2 is an amino, hydroxyl, or thiol group, or a C1-C group. 12 Alkylamino, C1-C 12 Dialkylamino, or a substituted form of any of these groups;

[0600] b is 1, 2, 3, 4, 5, or 6; and

[0601] z is 1, 2, or 3; the prerequisite is that the sum of z and y is 3.

[0602] In some embodiments of the dendritic polymer of formula (DI), the core is further defined by the following formula:

[0603] (D-IV)

[0604] in:

[0605] X3 is -NR6-, where R6 is hydrogen, C1-C8 alkyl or substituted C1-C8 alkyl, -O- or C1-C8 alkylaminodiyl, C1-C8 alkoxydiyl, C1-C8 aromaticdiyl, C1-C8 heteroaromaticdiyl, C1-C8 heterocyclic alkanediyl, or a substituted form of any of these groups;

[0606] R3 and R4 are each independently amino, hydroxyl, or thiol groups, or C1-C... 12 Alkylamino, dialkylamino, or substituted forms of any of these groups; or groups of the following formula: −N(R f ) f (CH2CH2N(R c )) e R d , , or ;

[0607] in:

[0608] e and f are each independently 1, 2, or 3; the prerequisite is that the sum of e and f is 3.

[0609] R c R d and R f Each is independently hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl;

[0610] c and d can each be 1, 2, 3, 4, 5 or 6 independently.

[0611] In some embodiments of the dendritic polymer of formula (I), the capping group is represented by the following formula:

[0612] (D-VIII),

[0613] in:

[0614] Y4 is an alkane dimethyl group. (C≤18) ;and

[0615] R 10 It is hydrogen.

[0616] In some embodiments of the dendritic polymer of formula (DI), the core of the structure of formula (D-IV) is:

[0617] , , , , , , , , , , , , , , , , , or , or their pharmaceutically acceptable salts.

[0618] In some embodiments of the dendritic polymer of formula (DI), the core comprises the structural formulas shown in Table 12 and their pharmaceutically acceptable salts, where * indicates the connection point between the core and the repeating unit (i.e., the hydrogen of the core is replaced by the repeating unit here).

[0619] In some embodiments of the dendritic polymer of formula (DI), the degradable diacyl group is further defined as:

[0620] .

[0621] In some embodiments of the dendritic polymer of formula (DI), the joint is further defined as (D-VI),

[0622] Where Y1 is a C1-C8 alkane dienoyl or substituted C1-C 12 Alkyl dimethyl

[0623] In some embodiments, R6 is H in the core of formula (D-IV). In some embodiments, R6 is a C1-C8 alkyl group in the core of formula (D-IV). In some embodiments, R6 is a substituted alkyl group (e.g., an alkyl group substituted with -NH2, an alkyl group substituted with -NHCH3, or an alkyl group substituted with -NHCH2CH3) in the core of formula (D-IV).

[0624] In some embodiments, one or two hydrogen atoms in the core are replaced by repeating units. In some embodiments, three or four hydrogen atoms in the core are replaced by repeating units. In some embodiments, five hydrogen atoms in the core are replaced by repeating units. In some embodiments, six hydrogen atoms in the core are replaced by repeating units.

[0625] In some implementations, the ionizable lipid is a compound having the formula DA:

[0626] (DA),

[0627] in:

[0628] R D1 It is a C1-C4 alkyl group;

[0629] z1 and z2 are each independently 1, 2, or 3; and

[0630] z3 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0631] In some implementations, in compounds of formula DA, R D1 It is a methyl group.

[0632] In some implementations, in compounds of formula DA, z1 and z2 are each 2.

[0633] In some embodiments, the core of formula D-II, D-III, or D-IV has the structure shown in Table 9, and the end-capping group of formula D-VII has the structure shown in Table 10. In each structure in Tables 3 and 4, Indicates the connection point with the following structures: The core in Table 8 and the end capping groups in Table 9 are connected at opposite ends of the structure.

[0634] In some embodiments of the dendritic polymer of formula (DI), the dendritic polymer is selected from the group consisting of:

[0635] , , , ,

[0636] and ;

[0637] And their pharmaceutically acceptable salts.

[0638] dendritic polymer of formula (X)

[0639] In some embodiments of the lipid composition, the ionizable cationic lipid is a dendritic polymer of the following formula: In some embodiments, the ionizable cationic lipid is a dendritic polymer of the following formula:

[0640] .

[0641] In some embodiments of the lipid composition, the ionizable cationic lipid is a (g) generation dendritic polymer having the following structural formula:

[0642] ,

[0643] Or its pharmaceutically acceptable salt, wherein:

[0644] (a) The core contains the following structural formula (X) 核心 ):

[0645] ,

[0646] in:

[0647] Q is independently a covalent bond, -O-, -S-, or -NR each time it appears. 2 -or-CR 3a R 3b -;

[0648] R 2 R is independent for each occurrence. 1g or -L 2 -NR 1e R 1f ;

[0649] R 3a and R 3b Each time it appears, it is independently hydrogen or optionally substituted (e.g., C1-C6, such as C1-C3) alkyl;

[0650] R 1a R 1b R 1c R 1d R 1e R 1f and R 1g (If present) each occurrence is independently of the junction with the branch, hydrogen, or optional substitution (e.g., C1-C). 12 )alkyl;

[0651] L 0 L 1 and L 2 Each occurrence is independently selected from covalent bonds, alkylene, heteroalkylene, [alkylene]-[heterocyclic alkyl]-[alkylene], [alkylene]-(aryl)-[alkylene], heterocyclic alkyl, and aryl; or,

[0652] Alternatively, L 1 Part of R 1c and R 1d One of them forms (e.g., C4-C6) a heterocyclic alkyl group (e.g., containing one or two nitrogen atoms and optionally other heteroatoms selected from oxygen and sulfur); and

[0653] x 1 It can be 0, 1, 2, 3, 4, 5, or 6; and

[0654] (b) Each of the multiple (N) branches independently contains the following structure (X 分支 ):

[0655] ,

[0656] in:

[0657] * indicates the connection point between the branch and the core;

[0658] g is 1, 2, 3, or 4;

[0659] Z=2 (g-1) ;

[0660] When g=1, G=0; or when g≠1, G= ;

[0661] (c) Each diacyl group independently contains the structural formula ,in:

[0662] * indicates a connection point near the end of the diacyl group;

[0663] ** indicates the connection point at the distal end of the diacyl group;

[0664] Y 3 Independently substituted for each occurrence (e.g., C1-C) 12 ); alkylene, optionally substituted (e.g., C1-C) 12 ) alkenyl or optionally substituted (e.g., C1-C) 12 ) Aromatic hydrocarbon group;

[0665] A 1 and A 2 Each occurrence is independently -O-, -S-, or -NR. 4 -,in:

[0666] R 4 It is hydrogen or optionally substituted (e.g., C1-C6) alkyl;

[0667] m 1 and m 2 Each occurrence is independently 1, 2, or 3; and

[0668] R 3c R 3d R 3e and R 3f Each of these components is independently hydrogen or optionally substituted (e.g., C1-C8) alkyl groups; and

[0669] (d) Each linker group independently contains the structural formula ,

[0670] in:

[0671] ** indicates the connection point between the connector and the proximal diacyl group;

[0672] *** indicates the connection point between the connector and the distal diacyl group; and

[0673] Y1 is independently optional substituted each time it appears (e.g., C1-C). 12 ) alkylene, optionally substituted (e.g., C1-C) 12 ) alkenyl or optionally substituted (e.g., C1-C) 12 ) aromatic hydrocarbon group; and

[0674] (e) Each capping group is independently selected from optionally substituted (e.g., C1-C) 18 Such as C4-C 18 ) alkyl thiols and optional substituted (e.g., C1-C) 18Such as C4-C 18 ) Alkenyl thiols.

[0675] In X 核心 In some implementations, Q is independently covalent, -O-, -S-, -NR each time it appears. 2 -or-CR 3a R 3b In X 核心 In some implementations, Q is a covalent bond independently each time it occurs. In X 核心 In some implementations, Q is independently -O- each time it occurs. In X 核心 In some implementations, Q is independently -S- each time it occurs. In X 核心 In some implementations, Q is independently -NR each time it occurs. 2 And R 2 R is independent for each occurrence. 1g or -L 2 -NR 1e R 1f In X 核心 In some implementations, Q is independently -CR each time it occurs. 3a R 3b R 3a And R 3a and R 3b Each time it appears, it is independently hydrogen or an optional substituted alkyl group (e.g., C1-C6, such as C1-C3).

[0676] In X 核心 In some implementation schemes, R 1a R 1b R 1c R 1d R 1e R 1f and R 1g (If present) each occurrence is independently of the branch junction, hydrogen, or optionally substituted alkyl group. In X 核心 In some implementation schemes, R 1a R 1b R 1c R 1d R 1e R 1f and R 1g (If present) each occurrence is independently a connection point with the branch, a hydrogen atom. In X 核心 In some implementation schemes, R 1a R 1b R 1c R 1d R 1e R1f and R 1g (If present) each occurrence is independently a junction point with a branch, optionally substituted alkyl group (e.g., C1-C). 12 ).

[0677] In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each time it appears, it is independently selected from covalent bonds, alkylene, heteroalkylene, [alkylene]-[heterocyclic alkyl]-[alkylene], [alkylene]-(aryl)-[alkylene], heterocyclic alkyl, and aryl; or, alternatively, L 1 Part of R 1c and R 1d One of them forms a heterocyclic alkyl group (e.g., C4-C6, and containing one or two nitrogen atoms and optionally other heteroatoms selected from oxygen and sulfur). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each instance can independently be a covalent bond. In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each time it appears, it can independently be hydrogen. In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each time it appears, it can independently be an alkylene group (e.g., C1-C). 12 (e.g., C1-C6 or C1-C3). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each occurrence can independently be a heteroalkylene group (e.g., C1-C). 12 (e.g., C1-C8 or C1-C6). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each instance can independently be a heteroalkylene group (e.g., C2-C8 epoxides, such as oligomers (ethylene oxides)). In X 核心 In some implementation schemes, L 0 L 1 and L 2Each occurrence can independently be [alkylene]-[heterocyclic alkyl]-[alkylene][(e.g., C1-C6)alkylene]-[(e.g., C4-C6)heterocyclic alkyl]-[(e.g., C1-C6)alkylene]. In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each occurrence can independently be [alkylene]-(arylene)-[alkylene], for example, [(e.g., C1-C6)alkylene]-(arylene)-[(e.g., C1-C6)alkylene]. In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each occurrence can independently be [alkylene]-(arylene)-[alkylene], for example, [(e.g., C1-C6)alkylene]-phenylene-[(e.g., C1-C6)alkylene]. In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each occurrence can independently be a heterocyclic alkyl group (e.g., C4-C6 heterocyclic alkyl group). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each time it appears, it can be independently arylene (e.g., phenylene). In X 核心 In some implementation schemes, L 1 Part of R 1c and R 1d One of them forms a heterocyclic alkyl group. In X 核心 In some implementation schemes, L 1 Part of R 1c and R 1d One of them forms a heterocyclic alkyl group (e.g., C4-C6 heterocyclic alkyl group), and the heterocyclic alkyl group may contain one or two nitrogen atoms and optional additional heteroatoms selected from oxygen and sulfur.

[0678] In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each time it appears, it is independently selected from covalent bonds, C1-C6 alkylene groups (e.g., C1-C3 alkylene groups), C2-C 12 (e.g., C2-C8) epoxides (e.g., oligomeric (ethylene oxide), such as -(CH2CH2O) 1-4-(CH2CH2)-), [(C1-C4)alkylene]-[(C4-C6)heterocyclic alkylene]-[(C1-C4)alkylene] (e.g., ) and [(C1-C4)alkylene]-phenylene-[(C1-C4)alkylene] (e.g., ). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each occurrence is independently selected from C1-C6 alkylene groups (e.g., C1-C3 alkylene groups) and -(C1-C3 alkylene-O). 1-4 -(C1-C3 alkylene), -(C1-C3 alkylene)-phenylene-(C1-C3 alkylene)- and -(C1-C3 alkylene)-piperazinyl-(C1-C3 alkylene)-. In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each instance is independently a C1-C6 alkylene group (e.g., C1-C3 alkylene groups). In some embodiments, L 0 L 1 and L 2 Each occurrence is independently C2-C. 12 (e.g., C2-C8) epoxides, such as -(C1-C3 alkylene-O) 1-4 -(C1-C3 alkylene). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each time it appears, it is independently selected from [(C1-C4)alkylene]-[(C4-C6)heterocyclic alkyl]-[(C1-C4)alkylene] (e.g., -(C1-C3alkylene)-phenylene-(C1-C3alkylene)-) and [(C1-C4)alkylene]-[(C4-C6)heterocyclic alkyl]-[(C1-C4)alkylene] (e.g., -(C1-C3alkylene)-piperazinyl-(C1-C3alkylene)-).

[0679] In X 核心 In some implementation schemes, x 1 It can be 0, 1, 2, 3, 4, 5, or 6. In X 核心 In some implementation schemes, x 1 It is 0. In X 核心 In some implementation schemes, x 1 The value is 1. In X 核心 In some implementation schemes, x 1 It is 2. In X 核心In some implementation schemes, x 1 It is 3. In X 核心 In some implementation schemes, x 1 It is 4. In X 核心 In some implementation schemes, x 1 It is 5. In X 核心 In some implementation schemes, x 1 It is 6.

[0680] In X 核心 In some implementation schemes, the core includes a structure: (For example, ). In X 核心 In some implementation schemes, the core includes a structure: In X 核心 In some implementation schemes, the core includes a structure: (For example, , , or ). In X 核心 In some implementation schemes, the core includes a structure: (For example, ). In X 核心 In some implementation schemes, the core includes a structure: In X 核心 In some implementation schemes, the core includes a structure: (For example, , , or ). In X 核心 In some implementation schemes, the core includes a structure: (For example, Such as or ). In X 核心 In some implementation schemes, the core includes a structure: Where Q' is -NR 2 -or-CR 3a R 3b -;q 1 and q 2 Each is independently 1 or 2. In X 核心 In some implementation schemes, the core includes a structure: or (For example, , , or ). In X 核心 In some implementation schemes, the core includes a structured or (For example, , , or ), where ring A is an optional aryl group or an optional substituted group (e.g., C3-C). 12 Such as C3-C5) heteroaryl groups. In X 核 In some implementation schemes, the core includes a structured .

[0681] In X 核心 In some implementations, the core comprises the structural formulas shown in Table 11 and their pharmaceutically acceptable salts, where * indicates the connection point of the core to one of the multiple branches.

[0682] In some implementations, the multiple (N) branches include at least 3 branches, at least 4 branches, and at least 5 branches. In some implementations, the multiple (N) branches include at least 3 branches. In some implementations, the multiple (N) branches include at least 4 branches. In some implementations, the multiple (N) branches include at least 5 branches.

[0683] In X 分支 In some implementations, g is 1, 2, 3, or 4. In X 分支 In some implementations, g is 1. In X 分支 In some implementations, g is 2. In X 分支 In some implementations, g is 3. In X 分支 In some implementations, g is 4.

[0684] In X 分支 In some implementation schemes, Z=2 (g-1) And when g=1, G=0. In X 分支 In some implementation schemes, Z=2 (g-1) And when g≠1, .

[0685] In X 分支 In some implementations, g=1, G=0, Z=1, and each of the multiple branches contains a structure. .

[0686] In X 分支 In some implementations, g=2, G=1, Z=2, and each of the multiple branches contains a structure.

[0687] .

[0688] In X 分支In some implementations, g=3, G=3, Z=4, and each of the multiple branches contains a structure.

[0689] .

[0690] In X 分支 In some implementations, g=4, G=7, Z=8, and each of the multiple branches contains a structure.

[0691] .

[0692] In some embodiments, the dendritic polymers with algebraic (g) = 1 described herein have the following structure: .

[0693] In some embodiments, the dendritic polymers with algebraic (g) = 1 described herein have the following structure: .

[0694] Exemplary formulations of the dendritic polymers described herein for generations 1-4 are shown in Table 11. The number of diacyl groups, linker groups, and capping groups can be calculated based on g.

[0695] Table 11. Formulations of dendritic polymer groups based on algebra (g)

[0696]

[0697] In some implementations, the diacyl group independently comprises the structural formula * indicates a connection point near the diacyl group, and ** indicates a connection point far from the diacyl group.

[0698] In X 分支 In some embodiments of the diacyl group, Y 3 Each time it appears, it is independently optionally substituted; alkylene, optionally substituted alkenyl, or optionally substituted aromatic. In X 分支 In some embodiments of the diacyl group, Y 3 Each time it appears, it is independently an optionally substituted alkylene group (e.g., C1-C). 12 ). In X 分支 In some embodiments of the diacyl group, Y 3 Each time it appears, it is independently an optional substituted alkenyl group (e.g., C1-C). 12 ). In X 分支 In some embodiments of the diacyl group, Y 3 Each time it appears, it is independently an optionally substituted aromatic group (e.g., C1-C). 12 ).

[0699] In X分支 In some embodiments of the diacyl group, A 1 and A 2 Each occurrence is independently -O-, -S-, or -NR. 4 -. In X 分支 In some embodiments of the diacyl group, A 1 and A 2 Each occurrence is independently -O-. In X 分支 In some embodiments of the diacyl group, A 1 and A 2 Each occurrence is independently -S-. In X 分支 In some embodiments of the diacyl group, A 1 and A 2 Each occurrence is independently -NR 4 - and R 4 It is hydrogen or an optionally substituted alkyl group (e.g., C1-C6). In X 分支 In some embodiments of the diacyl group, m 1 and m 2 Each occurrence is independently 1, 2, or 3. In X 分支 In some embodiments of the diacyl group, m 1 and m 2 Each occurrence is independently set to 1. In X 分支 In some embodiments of the diacyl group, m 1 and m 2 Each occurrence is independently 2. In X 分支 In some embodiments of the diacyl group, m 1 and m 2 Each occurrence is independently 3. In X 分支 In some embodiments of the diacyl group, R 3c R 3d R 3e and R 3f Each time it appears, it is independently hydrogen or optionally substituted alkyl. In X 分支 In some embodiments of the diacyl group, R 3c R 3d R 3e and R 3f Each time it appears, it is independently hydrogen. In X 分支 In some embodiments of the diacyl group, R 3c R 3d R 3e and R 3f Each of them is independently an optional substituted (e.g., C1-C8) alkyl group.

[0700] In some embodiments of the diacyl group, A 1 It is -O or -NH-. In some embodiments of the diacyl group, A 1 For -O-. In some embodiments of the diacyl group, A 2 It is -O or -NH-. In some embodiments of the diacyl group, A 2 For -O-. In some embodiments of the diacyl group, Y 3 For C1-C 12 (e.g., C1-C6, such as C1-C3) alkylene groups.

[0701] In some embodiments of the diacyl group, the diacyl group independently comprises the structural formula each time it appears. (For example, Such as ), and optional R 3c R 3d R 3e and R 3f Each of them is independently hydrogen or C1-C3 alkyl.

[0702] In some implementations, the linker group independently comprises the structural formula ** indicates the junction point between the connector and the proximal diacyl group, and *** indicates the junction point between the connector and the distal diacyl group.

[0703] In X 分支 In some embodiments of the connector group (if present), Y1 is independently, each time it appears, an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted aromatic group. In X 分支 In some embodiments, Y1 is independently, each time it appears, an optionally substituted alkylene group (e.g., C1-C1). 12 ). In X 分支 In some embodiments of the connector group (if present), Y1 is independently, each time it appears, an optionally substituted alkenyl group (e.g., C1-C1). 12 ). In X 分支 In some embodiments, Y1 is independently, each time it appears, a optionally substituted aromatic group (e.g., C1-C1). 12 ).

[0704] In X 分支 In some embodiments of the end-capping groups, each end-capping group is independently selected from optionally substituted alkyl thiols and optionally substituted alkenyl thiols. In X 分支In some embodiments of the end-capping groups, each end-capping group is an optionally substituted alkylthiol (e.g., C1-C1). 18 Such as C4-C 18 ). In X 分支 In some embodiments of the end-capping groups, each end-capping group is an optionally substituted alkenyl thiol (e.g., C1-C1). 18 Such as C4-C 18 ).

[0705] In X 分支 In some embodiments of the end-capping groups, each end-capping group is independently C1-C1. 18 alkenyl thiols or C1-C 18 Alkyl thiols, wherein the alkyl or alkenyl moiety is optionally substituted by one or more substituents, each independently selected from: halogens, C6-C... 12 Aryl, C1-C 12 Alkylamino, C4-C6 N-heterocyclic alkyl, -OH, -C(O)OH, −C(O)N(C1-C3 alkyl)−(C1-C6 alkylene)−(C1-C 12 alkyl-amino), −C(O)N(C1-C3 alkyl)−(C1-C6 alkylene)−(C4-C6 N-heterocyclic alkyl), −C(O)−(C1-C 12 Alkyl-amino) and −C(O)−(C4-C6 N-heterocyclic alkyl), and the C4-C6 N-heterocyclic alkyl portion of any of the aforementioned substituents may optionally be replaced by C1-C3 alkyl or C1-C3 hydroxyalkyl.

[0706] In X 分支 In some embodiments of the end-capping groups, each end-capping group is independently C1-C1. 18 (For example, C4-C) 18 ) alkenyl thiols or C1-C 18 (For example, C4-C) 18 Alkyl thiols, wherein the alkyl or alkenyl moiety is optionally substituted by one or more substituents, each independently selected from: halogens, C6-C... 12 Aryl (e.g., phenyl), C1-C 12 (e.g., C1-C8)alkylamino (e.g., C1-C6 monoalkylamino (such as -NHCH2CH2CH2CH3) or C1-C8 dialkylamino (such as...) , , , C4-C6 N-heterocyclic alkyl groups (e.g., N-pyrrolidinyl alkyl groups) ), N-piperidinyl ( ), N-azacycloheptanyl ( ), -OH, -C(O)OH, -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C1-C 12 Alkyl-amino (e.g., monoalkylamino or dialkylamino) (e.g., ), -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C4-C6 N-heterocyclic alkyl) (e.g., -C(O)-(C1-C) 12 Alkyl-amino (e.g., monoalkylamino or dialkylamino) and -C(O)-(C4-C6 N-heterocyclic alkyl) (e.g., ), wherein the C4-C6 N-heterocyclic alkyl portion of any of the aforementioned substituents may optionally be substituted with a C1-C3 alkyl or a C1-C3 hydroxyalkyl. In X 分支 In some embodiments of the end-capping groups, each end-capping group is independently C1-C1. 18 (e.g., C4-C) 18 Alkyl thiols, wherein the alkyl moiety is optionally substituted with a substituent -OH. In X 分支 In some embodiments of the end-capping groups, each end-capping group is independently C1-C1. 18 (For example, C4-C) 18 ) alkyl thiols, wherein the alkyl moiety is optionally substituted by a substituent selected from the following: C1-C 12 (e.g., C1-C8)alkylamino (e.g., C1-C6 monoalkylamino (such as -NHCH2CH2CH2CH3) or C1-C8 dialkylamino (such as...) , , , C4-C6N-heterocyclic alkyl groups (e.g., N-pyrrolidinyl alkyl groups) and C4-C6N-heterocyclic alkyl groups (e.g., N-pyrrolidinyl alkyl groups) ), N-piperidinyl ( ), N-azacycloheptanyl ( In X) 分支 In some embodiments of the end-capping groups, each end-capping group is independently C1-C1. 18 (For example, C4-C) 18 ) alkenyl thiols or C1-C 18 (For example, C4-C) 18 ) Alkyl thiols. In X 分支 In some embodiments of the end-capping groups, each end-capping group is independently C1-C1. 18 (For example, C4-C) 18 Alkyl thiols.

[0707] Table 12. Exemplary Core Structure

[0708]

[0709]

[0710]

[0711]

[0712] In X 核心 In some implementation schemes, the core includes a structure selected from the following groups: , , , , , , , , , , , , , , , , , , , , , and their pharmaceutically acceptable salts, where * indicates the connection point between the core and one of the multiple branches.

[0713] In X 分支 In some embodiments, each capping group is independently selected from the structures in Table 13. In some embodiments, the dendritic polymer described herein may comprise capping groups selected from Table 13 or pharmaceutically acceptable salts thereof.

[0714] Table 13. Exemplary capping groups / peripheral structures

[0715]

[0716]

[0717]

[0718] In some embodiments, the dendritic polymer of formula (X) is selected from those structures shown in Table 14 and their pharmaceutically acceptable salts.

[0719] Table 9. Exemplary ionizable cationic lipid dendritic polymers

[0720]

[0721]

[0722]

[0723]

[0724]

[0725]

[0726]

[0727]

[0728]

[0729]

[0730]

[0731]

[0732]

[0733]

[0734]

[0735]

[0736]

[0737]

[0738]

[0739]

[0740]

[0741]

[0742]

[0743] In some embodiments, the dendritic polymer is 2A2-SC14. In some embodiments, the dendritic polymer is 2A6-SC14. In some embodiments, the dendritic polymer is 2A9-SC14. In some embodiments, the dendritic polymer is 3A3-SC10. In some embodiments, the dendritic polymer is 3A3-SC14. In some embodiments, the dendritic polymer is 4A5-SC10. In some embodiments, the dendritic polymer is 3A5-SC14. In some embodiments, the dendritic polymer is 4A1-SC12. In some embodiments, the dendritic polymer is 4A3-SC12. In some embodiments, the dendritic polymer is 5A1-SC12. In some embodiments, the dendritic polymer is 5A1-SC8. In some embodiments, the dendritic polymer is 5A2-2-SC12. In some embodiments, the dendritic polymer is 5A3-1-SC12. In some embodiments, the dendritic polymer is 5A3-1-SC8. In some embodiments, the dendritic polymer is 5A4-1-SC12. In some embodiments, the dendritic polymer is 5A4-1-SC8. In some embodiments, the dendritic polymer is 5A5-SC8. In some embodiments, the dendritic polymer is 5A5-SC12. In some embodiments, the dendritic polymer is 5A2-4-SC12. In some embodiments, the dendritic polymer is 5A2-4-SC10. In some embodiments, the dendritic polymer is 5A3-2-SC8. In some embodiments, the dendritic polymer is 5A3-2-SC12. In some embodiments, the dendritic polymer is 5A4-2-SC8. In some embodiments, the dendritic polymer is 5A4-2-SC12. In some embodiments, the dendritic polymer is 6A4-SC8. In some embodiments, the dendritic polymer is 6A4-SC12. In some embodiments, the dendritic polymer is 2A2-g2-SC12. In some embodiments, the dendritic polymer is 2A2-g2-SC8. In some embodiments, the dendritic polymer is 2A11-g2-SC12. In some embodiments, the dendritic polymer is 2A11-g2-SC8. In some embodiments, the dendritic polymer is 3A3-g2-SC12. In some embodiments, the dendritic polymer is 3A3-g2-SC8. In some embodiments, the dendritic polymer is 3A5-g2-SC12. In some embodiments, the dendritic polymer is 2A11-g3-SC12. In some embodiments, the dendritic polymer is 2A11-g3-SC8. In some embodiments, the dendritic polymer is 1A2-g4-SC12. In some embodiments, the dendritic polymer is 4A1-g2-SC12. In some embodiments, the dendritic polymer is 1A2-g4-SC8.In some embodiments, the dendritic polymer is 4A1-g2-SC8. In some embodiments, the dendritic polymer is 4A3-g2-SC12. In some embodiments, the dendritic polymer is 4A3-g2-SC8. In some embodiments, the dendritic polymer is 1A2-g3-SC12. In some embodiments, the dendritic polymer is 1A2-g3-SC8. In some embodiments, the dendritic polymer is 2A2-g3-SC12. In some embodiments, the dendritic polymer is 2A2-g3-SC8. In some embodiments, the dendritic polymer is 5A2-4-SC8. In some embodiments, the dendritic polymer is 5A5-SC8. In some embodiments, the dendritic polymer is 5A2-6-SC8. In some embodiments, the dendritic polymer is 5A2-1-SC8. In some embodiments, the dendritic polymer is 5A2-2-SC8. In some embodiments, the dendritic polymer is 4A1-SC5. In some embodiments, the dendritic polymer is 4A1-SC8. In some embodiments, the dendritic polymer is 4A3-SC6. In some embodiments, the dendritic polymer is 4A3-SC7. In some embodiments, the dendritic polymer is 4A3-SC8. In some embodiments, the dendritic polymer is 5A4-2-SC5. In some embodiments, the dendritic polymer is 5A4-2-SC6. In some embodiments, the dendritic polymer is 5A2-4-SC8. In some embodiments, the dendritic polymer is 3A5-g2-SC8. In some embodiments, the dendritic polymer is 5A2-SC8.

[0744] Other ionizable cationic lipids

[0745] In some embodiments of the lipid composition, the ionizable cationic lipid comprises the structural formula (D-I'):

[0746] (D-I'),

[0747] in:

[0748] a is 1 and b is 2, 3 or 4; or alternatively, b is 1 and a is 2, 3 or 4;

[0749] m is 1 and n is 1; or, alternatively, m is 2 and n is 0; or, alternatively, m is 2 and n is 1; and

[0750] R 1 R 2 R 3 R 4 R 5 and R 6Each can be independently selected from the following groups: H, -CH2CH(OH)R 7 -CH(R) 7 )CH2OH、-CH2CH2C(=O)OR 7 -CH2CH2C(=O)NHR 7 and -CH2R 7 , where R 7 Independently selected from C3-C 18 Alkyl group, C3-C with one C=C double bond 18 Protecting groups of alkenyl and amino groups, -C(=NH)NH2, poly(ethylene glycol) chains, and acceptor ligands;

[0751] The prerequisite is R 1 To R 6 At least two parts of it are independently selected from -CH2CH(OH)R 7 -CH(R) 7 )CH2OH、-CH2CH2C(=O)OR 7 -CH2CH2C(=O)NHR 7 or -CH2R 7 , where R 7 Independently selected from C3-C 18 Alkyl groups or C3-C groups with one C=C double bond 18 alkenyl; and

[0752] One or more nitrogen atoms shown in formula (D-I') can be protonated to provide ionizable cationic lipids.

[0753] In some embodiments of the ionizable cationic lipid of formula (D-I'), a is 1. In some embodiments of the ionizable cationic lipid of formula (D-I'), b is 2. In some embodiments of the ionizable cationic lipid of formula (D-I'), m is 1. In some embodiments of the ionizable cationic lipid of formula (D-I'), n is 1. In some embodiments of the ionizable cationic lipid of formula (D-I'), R... 1 R 2 R 3 R 4 R 5 and R 6 Each is independently H or -CH2CH(OH)R 7 In some embodiments of the ionizable cationic lipid of formula (D-I'), R 1 R 2 R 3 R 4 R 5 and R 6Each independently is H or In some embodiments of the ionizable cationic lipid of formula (D-I'), R 1 R 2 R 3 R 4 R 5 and R 6 Each independently is H or In some embodiments of the ionizable cationic lipid of formula (D-I'), R 7 For C3-C 18 Alkyl (e.g., C6-C) 12 alkyl).

[0754] In some embodiments, the ionizable cationic lipid of formula (D-I') is 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentadecane-11,25-diol: .

[0755] In some embodiments, the ionizable cationic lipid of formula (D-I') is (11R,25R)-13,16,20-tris((R)-2-hydroxydodecyl)-13,16,20,23-tetraazapentadecane-11,25-diol: .

[0756] Other ionizable cationic lipids that may be used in the compositions and methods described herein include those described in International Patent Publications WO2010144740, WO2013149140, WO2016118725, WO2016118724, WO2013063468, WO2016205691, WO2015184256, WO2016004202, WO2015199952, WO2017004143, WO2017075531, WO2017117528, WO2017049245, WO2017173054, and WO2015095340, the entire contents of which are incorporated herein by reference for all purposes. Examples of such ionizable cationic lipids include, but are not limited to, those shown in Table 15.

[0757] Table 15. Exemplary ionizable cationic lipids

[0758]

[0759]

[0760]

[0761]

[0762]

[0763]

[0764]

[0765]

[0766]

[0767]

[0768]

[0769]

[0770] In some embodiments of the lipid composition, the ionizable cationic lipid is present in the composition at a molar percentage of about 10% to about 25%.

[0771] In some embodiments of the lipid composition, the ionizable cationic lipid is present in the composition in a molar percentage of about 5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%.

[0772] In some embodiments of the lipid composition, the ionizable cationic lipids are present in the composition in molar percentages of about 5% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 60%, about 15% to about 50%, about 15% to about 40%, about 15% to about 30%, about 15% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, or about 10% to about 25%.

[0773] In some embodiments of the lipid composition, the ionizable lipid is present at a molar percentage of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30%. In some embodiments of the lipid composition, the ionizable lipid is present at a molar percentage of at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, or at most about 30%.

[0774] Selective organ-targeting (SORT) lipids

[0775] The lipid composition may contain additional anionic lipids, ionizable cationic lipids, or permanent cationic lipids. In some embodiments of the lipid composition, the lipid (e.g., nanoparticle) composition is preferentially delivered to the target organ.

[0776] In some embodiments of the lipid composition, the additional lipid comprises a permanently positively charged portion (i.e., a permanently cationic lipid). The permanently positively charged portion can be positively charged at physiological pH, such that the additional lipid (e.g., SORT lipid) carries a positive charge when delivering polynucleotides to cells. In some embodiments, the positively charged portion is a quaternary ammonium or quaternary ammonium ion. In some embodiments, the additional lipid (e.g., SORT lipid) comprises an antiion, or otherwise co-exists with or interacts with an antiion.

[0777] In some embodiments of the lipid composition, the additional lipid is a permanently cationic lipid (i.e., comprising one or more hydrophobic components and a permanently cationic group). The permanently cationic lipid may contain groups that carry a positive charge regardless of pH. One type of permanently cationic group that can be used in the permanently cationic lipid is a quaternary ammonium group. The permanently cationic lipid may comprise the following structural formula: (SI), where:

[0778] Y1, Y2, or Y3 are each independently X1C(O)R1 or X2N + R3R4R5;

[0779] The prerequisite is that at least one of Y1, Y2, and Y3 is X2N. + R3R4R5;

[0780] R1 is C1-C 24 Alkyl, C1-C 24 Substituted alkyl groups, C1-C 24 alkenyl, C1-C 24 Substituted alkenyl groups;

[0781] X1 is either 0 or NR a , where R a It is hydrogen, C1-C4 alkyl, or C1-C4 substituted alkyl;

[0782] X2 is a C1-C6 alkane dimethyl or a C1-C6 substituted alkane dimethyl;

[0783] R3, R4, and R5 are each independently C1-C 24 Alkyl, C1-C 24 Substituted alkyl groups, C1-C24 alkenyl, C1-C 24 Substituted alkenyl groups; and

[0784] A1 is a compound containing X2N. + Anions with equal numbers of R3R4R5 groups and charges.

[0785] In some embodiments, additional permanent cationic lipids (e.g., SORT lipids) have the following structural formula: (S-II), where:

[0786] R6-R9 are each independently C1-C 24 Alkyl, C1-C 24 Substituted alkyl groups, C1-C 24 alkenyl, C1-C 24 Substituted alkenyl groups; provided that at least one of R6-R9 is a C8-C group. 24 ;and

[0787] A2 is a monovalent anion.

[0788] In some embodiments, the permanent cationic lipids are 1,2-dilauroyl-sn-glycerol-3-ethylphosphocholine (12:0 EPC), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (16:0 EPC), 1,2-distearatel-sn-glycerol-3-ethylphosphocholine (18:0 EPC), 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (18:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-ethylphosphocholine (16:0-18:0 EPC), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:1 EPC), and dimethyl dioctadecylammonium (18:0 EPC). DDAB), 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), 1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP), 1,2-dioleoyl-3-trimethylammonium-propane (18:1 TAP, DOTAP), or 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA).

[0789] In some embodiments of the lipid composition, the SORT (alternative) lipid is an ionizable cationic lipid (e.g., comprising one or more hydrophobic components and an ionizable group, such as a tertiary amine group). The ionizable, positively charged portion can be positively charged at physiological pH. One ionizable group that can be used in the ionizable cationic lipid is a tertiary amine group. In some embodiments of the lipid compositions disclosed herein, the additional lipid (e.g., the additional lipid (e.g., the SORT lipid)) has the following structural formula: (S-I'a), where:

[0790] R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 Alkenyl or any other substituted form; and

[0791] R3 and R3' are each independently C1-C6 alkyl or substituted C1-C6 alkyl.

[0792] In some implementations of formula (S-I'a), R1 and R2 are each independently C8-C 24 Alkenyl (e.g., hexadecene, heptadecanene, or octadecene). In some embodiments of formula (S-I'a), R3 and R3' are each independently C1-C6 alkyl (e.g., methyl or ethyl). In some embodiments of formula (S-I'a), R1 and R2 are each independently C8-C6 alkyl. 24 The alkenyl group (e.g., hexadecane, heptadecane, or octadecene) and R3 and R3' are each independently C1-C6 alkyl (e.g., methyl or ethyl).

[0793] In some embodiments, the ionizable cationic lipid is 1,2-distearyl-3-dimethylammonium-propane (18:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP), 1,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP), 1,2-dioleoyl-3-dimethylammonium-propane (18:1 DAP, DODAP), or 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA).

[0794] In some embodiments of the lipid composition, the additional ionizable cationic lipid or permanent cationic lipid comprises a head group with a specific structure. In some embodiments, the additional lipid (e.g., an additional lipid (e.g., SORT lipid)) comprises a head group having the following structural formula: Where L is the connector; Z + The part is positively charged, and X -To counteract ions. In some embodiments, the adapter is a biodegradable adapter. The biodegradable adapter can be degraded under physiological pH and temperature conditions. The biodegradable adapter can be degraded by proteins or enzymes from the subject. In some embodiments, the positively charged portion is a quaternary ammonium ion or a quaternary ammonium.

[0795] In some embodiments of the lipid composition, the SORT (another ionizable cationic lipid or permanent cationic) lipid has the following structural formula: , where R 1 and R 2 Each is an independent alternative to C6-C. 24 Alkyl or optionally substituted C6-C 24 Alkenyl group.

[0796] In some embodiments of the lipid composition, the additional lipid (e.g., another lipid (e.g., SORT lipid)) has the following structural formula: .

[0797] In some embodiments of the lipid composition, the additional lipid (e.g., another lipid (e.g., SORT lipid)) comprises a linker (L). In some embodiments, L is... ,in:

[0798] p and q are each independently 1, 2, or 3; and

[0799] R 4 It can be a C1-C6 alkyl group with optional substitution.

[0800] In some embodiments of the lipid composition, the additional lipid (e.g., another lipid (e.g., SORT lipid)) has the following structural formula: (IA),

[0801] in:

[0802] R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 Alkenyl or any other group substitution form;

[0803] R3, R3' and R3'' are each independently a C1-C6 alkyl or a substituted C1-C6 alkyl;

[0804] R4 is a C1-C6 alkyl or a substituted C1-C6 alkyl; and

[0805] X − It is a monovalent anion.

[0806] In some embodiments of the lipid composition, the additional lipid (e.g., another lipid (e.g., SORT lipid)) is phosphatidylcholine (e.g., 14:0 EPC). In some embodiments, the phosphatidylcholine compound is further defined as: (IA),

[0807] in:

[0808] R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 Alkenyl or any other group substitution form;

[0809] R3, R3', and R3'' are each independently a C1-C6 alkyl or a substituted C1-C6 alkyl; and

[0810] X − It is a monovalent anion.

[0811] In some embodiments of the lipid composition, the additional lipid (e.g., an additional lipid (e.g., SORT lipid)) is a phosphorocholine lipid. In some embodiments, the additional lipid (e.g., an additional lipid (e.g., SORT lipid)) is ethylphosphocholine. Ethylphosphocholine can be, for example but not limited to, 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:1 EPC), 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (18:1 EPC), 1,2-distearate-sn-glycerol-3-ethylphosphocholine (18:0 EPC), 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (16:0 EPC), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:0 EPC), 1,2-dilauroyl-sn-glycerol-3-ethylphosphocholine (12:0 EPC), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-ethylphosphocholine (16:0-18:0 EPC).

[0812] In some embodiments of the lipid composition, the lipid has the following structural formula: (S-I'), where:

[0813] R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 Alkenyl or any other group substitution form;

[0814] R3, R3' and R3'' are each independently a C1-C6 alkyl or a substituted C1-C6 alkyl;

[0815] X − It is a monovalent anion.

[0816] For example, but not limited to, another lipid in the previous structure (e.g., another lipid (e.g., SORT lipid)) is 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., chloride salt).

[0817] In some embodiments of the lipid composition, the additional lipid (e.g., another lipid (e.g., SORT lipid)) has the following structural formula: (S-II'), where:

[0818] R4 and R4' are each independently an alkyl group. (C6-C24) alkenyl (C6-C24) Or a substituted form of any group;

[0819] R4'' is an alkyl group. (C≤24) alkenyl (C≤24) Or a substituted form of any group;

[0820] R4''' is an alkyl group (C1-C8) Alkenyl (C2-C8) Or any substituted form of a group; and

[0821] X2 is a monovalent anion.

[0822] For example, but not limited to, another lipid in the previous structural formula (e.g., another lipid (e.g., SORT lipid)) is dimethyl dioctadecylammonium (DDAB).

[0823] In some embodiments of the lipid composition, the additional lipid (e.g., another lipid (e.g., SORT lipid)) is:

[0824] 1,2-Dioleoyl-sn-glycerol-3-phosphate (18:1 PA).

[0825] In some embodiments of the lipid composition, the additional lipids are selected from the lipids shown in Tables 16A and 16B.

[0826] Table 16A. Exemplary other lipids (e.g., SORT lipids)

[0827]

[0828]

[0829] Table 16B. Exemplary other lipids (e.g., SORT lipids)

[0830]

[0831] In some embodiments, the permanent cationic lipid is selected from 1,2-dilauroyl-sn-glycerol-3-ethylphosphocholine (12:0 EPC), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (16:0 EPC), 1,2-distearatel-sn-glycerol-3-ethylphosphocholine (18:0 EPC), 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (18:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-ethylphosphocholine (16:0-18:0 EPC), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:1 EPC), and dimethyl dioctadecylammonium (18:0 EPC). DDAB), 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), 1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP), 1,2-dioleoyl-3-trimethylammonium-propane (18:1 TAP, DOTAP), or 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA). In some embodiments, ethylphosphocholine is 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (16:0 EPC) or 2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:0 EPC).

[0832] In some embodiments, the SORT lipids are 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EPC), 1,2-distearatel-sn-glycero-3-ethylphosphocholine (18:0 EPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:0 EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:1 EPC), and dimethyl dioctadecylammonium (18:0 EPC). DDAB), 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), 1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP), 1,2-dioleoyl-3-trimethylammonium-propane (18:1 TAP, DOTAP) or 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-sn-glycerol-3-phosphate (18:1 PA), 1,2-distearate-sn-glycerol-3-phosphate (18:0 PA), 1,2-dipalmitoyl-sn-glycerol-3-phosphate (16:0 PA), 1,2-dimyristoyl-sn-glycerol-3-phosphate (14:0 PA) or 1,2-dilauroyl-sn-glycerol-3-phosphate (12:0 PA).

[0833] In some embodiments, the SORT lipids are selected from the groups shown in Tables 16A and 16B. In some embodiments, the SORT lipids are present in the composition at a molar percentage of about 5% to about 65%. In some embodiments, the SORT lipids are present in the composition at a molar percentage of about 5% to about 35%.

[0834] In some embodiments of the lipid composition, SORT lipids are present in the composition at a molar percentage of about 5% to about 50%.

[0835] In some embodiments of the lipid composition, SORT lipids are present in the composition at a molar percentage of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%.

[0836] In some embodiments of the lipid composition, SORT lipids are present in the composition in molar percentages of about 5% to about 60%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 60%, about 15% to about 50%, about 15% to about 40%, about 15% to about 30%, about 15% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, or about 20% to about 25%.

[0837] In some embodiments of the lipid composition, the SORT lipids are present in a molar percentage of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 55%. In some embodiments of the lipid composition, the ionizable lipids are present in a molar percentage of at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, or at most about 25%.

[0838] assist lipids

[0839] In some embodiments, the auxiliary lipid is a phospholipid. Phospholipids, as defined herein, are any lipid containing a phosphate group. The lipid component of the lipid nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may be assembled into one or more lipid bilayers. Typically, phospholipids may include a phospholipid moiety and one or more fatty acid moieties. The phospholipid moiety may be selected from the non-limiting group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0840] This also includes non-natural substances, including natural substances with modifications and substitutions (including branching, oxidation, cyclization, and alkynes). For example, phospholipids can be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups where one or more double bonds are replaced by triple bonds). Under suitable reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition upon exposure to azides. Such reactions can be used to functionalize lipid bilayers of nanoparticle compositions to facilitate membrane permeation or cell recognition, or to conjugate nanoparticle compositions to useful components, such as targeting or imaging portions (e.g., dyes).

[0841] Phospholipids that may be used or potentially used in compositions and methods may be selected from: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl- sn-glycerol-3-phosphate choline (DPPC), 1,2-enoacyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 diether PC), 1-oleoyl-2-cholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16) Lyso PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidanoyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenoyl-sn-glycerol-3-phosphate choline, 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0 PE), 1,2-diphydanoyl-sn-glycerol-3-phosphate choline (4ME 16:0 PC), 1,2-diphydanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (sodium salt) (4ME 16:0 PG), 1,2-diphydanoyl-sn-glycerol-3-phosphate-L-serine (sodium salt) (4ME 16:0 PS), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium (DOPG) and sphingomyelin.

[0842] In some implementations, phospholipids may contain one or two long chains (e.g., C6-C). 24 The phospholipid may contain alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate groups, and optionally small organic molecules. Small organic molecules may be amino acids, sugars, or amino-substituted alkoxy groups, such as choline or ethanolamine. In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is distearylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, wherein zwitterionic lipids refer to lipids and lipid-like molecules that carry both positive and negative charges.

[0843] In some embodiments, the cofactor lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE). In some embodiments, the cofactor lipid is 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC). In some embodiments, the cofactor lipid is present in the composition at a molar percentage of about 5% to about 35%.

[0844] In some embodiments of the lipid composition, the auxiliary lipid is present in the composition at a molar percentage of about 7.5% to about 30%.

[0845] In some embodiments of the lipid composition, the auxiliary lipid is present in the composition in a molar percentage of about 5%, about 7.5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%.

[0846] In some embodiments of the lipid composition, the auxiliary lipid is present in the composition in a molar percentage of about 5% to about 50%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 60%, about 15% to about 50%, about 15% to about 40%, about 15% to about 30%, about 15% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, or about 10% to about 25%.

[0847] In some embodiments of the lipid composition, the auxiliary lipid is present at a molar percentage of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30%. In some embodiments of the lipid composition, the ionizable lipid is present at a molar percentage of at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, or at most about 30%.

[0848] Structural lipids

[0849] Lipid nanoparticles may comprise one or more structured lipids. The structured lipids may be steroids or steroid derivatives. In some embodiments of the lipid composition, the lipid composition further comprises a steroid or a steroid derivative. In some embodiments, the steroid or steroid derivative includes any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" is a class of compounds having a tetracyclic 17-carbon ring structure, which may further comprise one or more substituents, including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or double bonds between two or more carbon atoms. In one aspect, the cyclic structure of the steroid comprises three fused cyclohexyl rings and one fused cyclopentyl ring, as shown in the following formula: In some embodiments, the steroid derivative comprises the aforementioned ring structure having one or more non-alkyl substituted components. In some embodiments, the steroid or steroid derivative is a sterol, wherein the formula is further defined as: In some embodiments, the steroid or steroid derivative is a cholesterane or a cholesterane derivat...

Claims

1. A lipid nanoparticle (LNP) composition, said LNP composition comprising: (i) pathogen-associated antigens or polynucleotides encoding pathogen-associated antigens; and (ii) Selective organ-targeted (SORT) lipids.

2. A lipid nanoparticle (LNP) composition, said LNP composition comprising: (i) pathogen-associated antigens or polynucleotides encoding pathogen-associated antigens; and (ii) Permanent cationic lipids.

3. A lipid nanoparticle (LNP) composition, said LNP composition comprising: (i) pathogen-associated antigens or polynucleotides encoding pathogen-associated antigens; and (ii) Ionizable dendritic polymer lipids.

4. The LNP composition according to claim 2 or claim 3, wherein the LNP composition further comprises: (iii) Selective organ-targeted (SORT) lipids; (iv) Auxiliary lipids; Sterols; and / or Polyethylene glycol-conjugated lipids (PEG lipids).

5. A lipid nanoparticle (LNP) composition, said LNP composition comprising: (i) pathogen-associated antigens or polynucleotides encoding pathogen-associated antigens; (ii) Selective organ-targeted (SORT) lipids; (iii) Ionizable cationic lipids; and (iv) Auxiliary lipids; Sterols; and / or Polyethylene glycol-conjugated lipids (PEG lipids).

6. The LNP composition according to claim 3, wherein the ionizable dendritic lipid has the formula D-IV.

7. The LNP composition according to any one of claims 2 to 3, wherein the LNP composition further comprises selective organ-targeting (SORT) lipids.

8. The LNP composition according to any one of claims 1 to 7, wherein the LNP composition comprises a polynucleotide encoding a pathogen-associated antigen.

9. The LNP composition according to claim 8, wherein the polynucleotide is chemically modified.

10. The LNP composition according to any one of claims 1 to 9, wherein the polynucleotide is mRNA.

11. The LNP composition according to any one of claims 1 to 9, wherein the polynucleotide is DNA.

12. The LNP composition according to any one of claims 1 to 11, wherein the polynucleotide encodes a pathogen-associated antigen shown in Table 3, Table 4 or Table 5.

13. The LNP composition according to any one of claims 1 to 11, wherein the polynucleotide encodes a pathogen-associated antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the antigens shown in Tables 3, 4, or 5.

14. The LNP composition according to any one of claims 1 to 13, wherein the polynucleotide encodes an influenza virus antigen, a coronavirus antigen, an RSV antigen, a PIV3 antigen, or an hMPV antigen.

15. The LNP composition according to any one of claims 1 or 4 to 14, wherein the SORT lipid is a permanent cationic lipid.

16. The LNP composition according to claim 15, wherein the SORT lipid is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

17. The LNP composition according to claim 15, wherein the SORT lipid is dimethyl dioctadecylammonium (DDAB).

18. The LNP composition according to claim 15, wherein the SORT lipid is ethyl phosphocholine.

19. The LNP composition according to claim 18, wherein the ethyl phosphocholine is 1,2-dipalmitoyl-sn-glycerol-3-ethyl phosphocholine (16:0 EPC).

20. The LNP composition according to claim 18, wherein the ethyl phosphocholine is 1,2-dimyristoyl-sn-glycerol-3-ethyl phosphocholine (14:0 EPC).

21. The LNP composition according to any one of claims 1 or 4 to 14, wherein the SORT lipid is 1,2-dioleoyl-3-dimethylammonium-propane (DODAP).

22. The LNP composition according to any one of claims 1 or 4 to 14, wherein the SORT lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate (18:1 PA).

23. The LNP composition according to any one of claims 1 to 22, wherein the SORT lipid is present in the composition at a molar percentage of about 5% to about 50%.

24. The LNP composition of claim 16, wherein the DOTAP is present in the composition in a molar percentage of about 30% to about 50%.

25. The LNP composition of claim 18, wherein the ethyl phosphocholine is present in the composition in a molar percentage of about 30% to about 50%.

26. The LNP composition of claim 21, wherein the DODAP is present in the composition in a molar percentage of about 5% to about 30%.

27. The LNP composition of claim 22, wherein the 18:1 PA is present in the composition in a molar percentage of about 5% to about 30%.

28. The LNP composition according to any one of claims 5 to 27, wherein the ionizable cationic lipid is a dendritic polymer lipid.

29. The LNP composition according to any one of claims 5 to 27, wherein the ionizable cationic lipid is a dendritic polymer lipid according to formula (I) or formula (X).

30. The LNP composition according to any one of claims 3 to 29, wherein the dendritic polymer is: (4A3-SC7) 31. The LNP composition according to any one of claims 3 to 29, wherein the dendritic polymer is: (5A2-SC8) 32. The LNP composition according to any one of claims 5 to 31, wherein the ionizable cationic lipid is present in the composition in a molar percentage of about 10% to about 25%.

33. The LNP composition according to any one of claims 4 to 32, wherein the auxiliary lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE).

34. The LNP composition according to any one of claims 4 to 32, wherein the auxiliary lipid is 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC).

35. The LNP composition according to any one of claims 4 to 34, wherein the auxiliary lipid is present in the LNP composition in a molar percentage of about 7.5% to about 30%.

36. The LNP composition according to any one of claims 4 to 35, wherein the sterol is cholesterol.

37. The LNP composition according to any one of claims 4 to 35, wherein the sterol is sitosterol.

38. The LNP composition of claim 36, wherein the cholesterol is present in the LNP composition in a molar percentage of about 20% to about 50%.

39. The LNP composition according to any one of claims 4 to 38, wherein the PEG lipid is present in the LNP composition in a molar percentage of about 0.5% to about 10%.

40. The LNP composition according to any one of claims 1 to 39, wherein the LNP composition comprises mRNA with a lipid:mRNA ratio (weight / weight) between about 5:1 and about 40:

1.

41. The LNP composition according to any one of claims 1 to 40, wherein the lipid:mRNA ratio (weight / weight) is about 40:

1.

42. The LNP composition according to any one of claims 1 to 40, wherein the lipid:mRNA ratio (weight / weight) is about 36:

1.

43. The LNP composition according to any one of claims 1 to 40, wherein the lipid:mRNA ratio (weight / weight) is about 30:

1.

44. The LNP composition according to any one of claims 1 to 43, wherein the LNP composition comprises: The molar percentage of DOPE is approximately 5% to approximately 25%. The molar percentage of cholesterol is approximately 20% to approximately 50%. DMG-PEG with a molar percentage of approximately 0.5% to approximately 10%; Ionizable cationic lipids comprising approximately 10% to approximately 25% in molar percentage; and The molar percentage is approximately 30% to approximately 40% of 16:0 EPC.

45. The LNP composition according to any one of claims 1 to 43, wherein the LNP composition comprises: The molar percentage of DOPE is approximately 5% to approximately 25%. The molar percentage of cholesterol is approximately 20% to approximately 50%. DMG-PEG with a molar percentage of approximately 0.5% to approximately 10%; Ionizable cationic lipids comprising approximately 10% to approximately 25% in molar percentage; and The molar percentage of DODAP is approximately 10% to approximately 30%.

46. ​​The LNP composition according to any one of claims 1 to 43, wherein the LNP composition comprises: The molar percentage of DOPE is approximately 5% to approximately 25%. The molar percentage of cholesterol is approximately 20% to approximately 50%. DMG-PEG with a molar percentage of approximately 0.5% to approximately 10%; Ionizable cationic lipids comprising approximately 10% to approximately 25% in molar percentage; and The molar percentage is approximately 5% to approximately 20% of 18:1 PA.

47. The LNP composition according to any one of claims 1 to 43, wherein the LNP composition comprises: The molar percentage of DOPE is approximately 5% to approximately 25%. The molar percentage of cholesterol is approximately 20% to approximately 50%. DMG-PEG with a molar percentage of approximately 0.5% to approximately 10%; Ionizable cationic lipids comprising approximately 10% to approximately 25% in molar percentage; and The molar percentage of DOTAP is approximately 30% to approximately 50%.

48. The LNP composition according to any one of claims 1 to 43, wherein the LNP composition comprises: DOPE with a molar percentage of approximately 15%; The molar percentage of cholesterol is approximately 30%. The molar percentage of DMG-PEG is approximately 3%; The molar percentage of ionizable cationic lipids is approximately 15%; and The molar percentage is approximately 30% of 16:0 EPC.

49. The LNP composition according to any one of claims 1 to 43, wherein the LNP composition comprises: DOPE with a molar percentage of approximately 20%; The molar percentage of cholesterol is approximately 40%. The molar percentage of DMG-PEG is approximately 3%; The molar percentage of ionizable cationic lipids is approximately 15%; and The molar percentage of DODAP is approximately 15%.

50. The LNP composition according to any one of claims 1 to 43, wherein the LNP composition comprises: DOPE with a molar percentage of approximately 20%; The molar percentage of cholesterol is approximately 40%. The molar percentage of DMG-PEG is approximately 5%; The molar percentage of ionizable cationic lipids is approximately 20%; and The molar percentage is approximately 5% of 18:1 PA.

51. The LNP composition according to any one of claims 1 to 43, wherein the LNP composition comprises: The molar percentage of DOPE is approximately 10%. The molar percentage of cholesterol is approximately 20%. The molar percentage of DMG-PEG is approximately 3%; The molar percentage of ionizable cationic lipids is approximately 10%; and DOTAP has a molar percentage of approximately 50%.

52. The LNP composition according to any one of claims 44 to 51, wherein the ionizable cationic lipid is a dendritic polymer lipid.

53. The LNP composition according to claim 52, wherein the dendritic polymer lipid is 4A3-SC7.

54. The LNP composition according to any one of claims 1 to 53, wherein the LNP composition comprises a Tris buffer, optionally with a pH of about 6 to about 9.

55. The LNP composition according to any one of claims 1 to 54, wherein the LNP composition comprises sucrose, optionally at a concentration (by weight) or molar percentage of about 5% to about 15%.

56. The LNP composition according to any one of claims 1 to 55, wherein the LNP composition comprises a citrate buffer, optionally with a pH of about 4 to about 6.

57. The LNP composition according to any one of claims 1 to 56, wherein the LNP composition comprises: (a) 15 mM Tris buffer, optionally with a pH of about 6 to about 9; and / or (b) Sucrose with a concentration (by weight) or molar percentage of about 5% to about 15%.

58. The LNP composition according to any one of claims 1 to 56, wherein the LNP composition comprises 10 mM citrate buffer, optionally with a pH of about 4 to about 6.

59. A pharmaceutical composition comprising a therapeutically effective amount of the LNP composition according to any one of claims 1 to 58.

60. A pharmaceutical composition comprising the LNP composition according to any one of claims 1 to 58 and comprising a pharmaceutically acceptable excipient.

61. A vaccine comprising the LNP composition according to any one of claims 1 to 58 or the pharmaceutical composition according to claim 59 or claim 60.

62. The vaccine of claim 61, wherein the vaccine comprises an adjuvant.

63. The vaccine of claim 62, wherein the adjuvant comprises squalene.

64. A method of delivering an LNP composition to a subject in need, the method comprising administering to the subject in need an LNP composition according to any one of claims 1 to 58, a pharmaceutical composition according to claim 59 or 60, and / or a vaccine according to any one of claims 61 to 63.

65. A method for preventing an infectious disease caused by a pathogen in a subject of need, the method comprising administering to the subject of need an LNP composition according to any one of claims 1 to 58, a pharmaceutical composition according to claim 59 or 60, and / or a vaccine according to any one of claims 61 to 63.

66. A method for immunizing a subject in need against pathogen infection and / or disease or condition caused by a pathogen, the method comprising administering to the subject a vaccine according to any one of claims 61 to 63.

67. The method of claim 65 or claim 66, wherein the pathogen is a virus, optionally including influenza virus, coronavirus, RSV, PIV3, hMPV or MeV.

68. The method of claim 65 or claim 66, wherein the pathogen is bacteria.

69. The method according to any one of claims 64 to 68, wherein administration to the subject in need comprises intravenous administration.

70. The method according to any one of claims 64 to 68, wherein the administration to the subject comprises intrathecal administration.

71. The method according to any one of claims 64 to 68, wherein the administration to the subject comprises intramuscular administration.

72. The method according to any one of claims 64 to 68, wherein the administration to the subject comprises intradermal or subcutaneous administration.

73. The method according to any one of claims 64 to 68, wherein the administration to the subject comprises intranasal administration.

74. The method according to any one of claims 64 to 73, wherein the method induces an immune response in the subject in need, optionally wherein the immune response is greater than the immune response following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG).

75. The method according to any one of claims 64 to 74, wherein the method induces an antibody response in the desired subject, optionally wherein the antibody response is greater than the antibody response following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG).

76. The method according to any one of claims 64 to 75, wherein the method causes T cells to proliferate in the subject of need, optionally wherein the T cell proliferation is greater than that following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG).

77. The method according to any one of claims 64 to 76, wherein the method activates T cells in the subject in need, optionally wherein the T cell activation is greater than that following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG).

78. The method of claim 76 or claim 77, wherein the T cell is a T helper cell or a cytotoxic T cell.

79. The method according to any one of claims 64 to 78, wherein the method induces a cytokine response in the subject in need.

80. Use of the LNP composition according to any one of claims 1 to 58, the pharmaceutical composition according to claim 59 or 60, or the vaccine according to any one of claims 61 to 63 for the manufacture of a medicament for the vaccination of subjects in need against pathogen infection or for the manufacture of a medicament for the treatment of a disease or condition caused by a pathogen.

81. The LNP composition according to any one of claims 1 to 58, the pharmaceutical composition according to claim 59 or 60, or the vaccine according to any one of claims 61 to 63, for use in manufacturing a medicament for vaccinating subjects in need against pathogen infection, or for use in manufacturing a medicament for treating a disease or condition caused by a pathogen.

82. The LNP composition according to any one of claims 1 to 58, the pharmaceutical composition according to claim 59 or 60, or the vaccine according to any one of claims 61 to 63, for use in vaccinating subjects in need against pathogen infection, or for treating diseases or conditions caused by pathogens.

83. A kit comprising an LNP composition according to any one of claims 1 to 58, a pharmaceutical composition according to claim 59 or 60, or a vaccine according to any one of claims 61 to 63, and instructions for use.

84. A lipid nanoparticle (LNP) composition, said LNP composition comprising: (iii) cancer antigens or polynucleotides encoding cancer antigens; and (iv) Selective organ-targeted (SORT) lipids.

85. A lipid nanoparticle (LNP) composition, said LNP composition comprising: (iii) cancer antigens or polynucleotides encoding cancer antigens; and (iv) Permanent cationic lipids.

86. A lipid nanoparticle (LNP) composition, said LNP composition comprising: (v) cancer antigens or polynucleotides encoding cancer antigens; and (vi) Ionizable dendritic polymer lipids.

87. The LNP composition according to claim 2 or claim 3, wherein the LNP composition further comprises (vii) Selective organ-targeted (SORT) lipids; (viii) Helper lipids; Sterols; and / or Polyethylene glycol-conjugated lipids (PEG lipids).

88. A lipid nanoparticle (LNP) composition, said LNP composition comprising: (v) Cancer antigens or polynucleotides encoding cancer antigens; (vi) Selective organ-targeted (SORT) lipids; (vii) Ionizable cationic lipids; and (viii) Helper lipids; Sterols; and / or Polyethylene glycol-conjugated lipids (PEG lipids).

89. The LNP composition according to claim 3, wherein the ionizable dendritic lipid has the formula D-IV.

90. The LNP composition according to any one of claims 2 to 3, wherein the LNP composition further comprises selective organ-targeting (SORT) lipids.

91. The LNP composition according to any one of claims 1 to 7, wherein the LNP composition comprises a polynucleotide encoding a cancer antigen.

92. The LNP composition according to claim 8, wherein the polynucleotide is chemically modified.

93. The LNP composition according to any one of claims 1 to 9, wherein the polynucleotide is mRNA.

94. The LNP composition according to any one of claims 1 to 9, wherein the polynucleotide is DNA.

95. The LNP composition according to any one of claims 1 to 11, wherein the polynucleotide encodes an antigen shown in Table 6, Table 7 or Table 8.

96. The LNP composition according to any one of claims 1 to 11, wherein the polynucleotide encodes an antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the antigens shown in Tables 6, 7, or 8.

97. The LNP composition according to any one of claims 1 to 96, wherein the polynucleotide encodes a lung cancer antigen, a prostate cancer antigen, or a neoantigen.

98. The LNP composition according to any one of claims 84 or 88 to 14, wherein the SORT lipid is a permanent cationic lipid.

99. The LNP composition according to claim 15, wherein the SORT lipid is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

100. The LNP composition according to claim 15, wherein the SORT lipid is dimethyl dioctadecylammonium (DDAB).

101. The LNP composition according to claim 15, wherein the SORT lipid is ethyl phosphocholine.

102. The LNP composition according to claim 18, wherein the ethyl phosphocholine is 1,2-dipalmitoyl-sn-glycerol-3-ethyl phosphocholine (16:0 EPC).

103. The LNP composition according to claim 18, wherein the ethyl phosphocholine is 1,2-dimyristoyl-sn-glycerol-3-ethyl phosphocholine (14:0 EPC).

104. The LNP composition according to any one of claims 84 or 88 to 14, wherein the SORT lipid is 1,2-dioleoyl-3-dimethylammonium-propane (DODAP).

105. The LNP composition according to any one of claims 84 or 88 to 14, wherein the SORT lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate (18:1 PA).

106. The LNP composition according to any one of claims 84 or 88 to 22, wherein the SORT lipid is present in the composition in a molar percentage of about 5% to about 50%.

107. The LNP composition of claim 16, wherein the DOTAP is present in the composition in a molar percentage of about 30% to about 50%.

108. The LNP composition of claim 18, wherein the ethyl phosphocholine is present in the composition in a molar percentage of about 30% to about 50%.

109. The LNP composition of claim 21, wherein the DODAP is present in the composition in a molar percentage of about 5% to about 30%.

110. The LNP composition of claim 22, wherein the 18:1 PA is present in the composition in a molar percentage of about 5% to about 30%.

111. The LNP composition according to any one of claims 2 or 5 to 27, wherein the ionizable cationic lipid is a dendritic polymer lipid.

112. The LNP composition according to any one of claims 2 or 5 to 27, wherein the ionizable cationic lipid is a dendritic polymer lipid according to formula (I) or formula (X).

113. The LNP composition according to any one of claims 3 to 29, wherein the dendritic polymer is (4A3-SC7) 114. The LNP composition according to any one of claims 3 to 29, wherein the dendritic polymer is (5A2-SC8) 115. The LNP composition according to any one of claims 5 to 31, wherein the ionizable cationic lipid is present in the composition in a molar percentage of about 10% to about 25%.

116. The LNP composition according to any one of claims 4 to 32, wherein the auxiliary lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE).

117. The LNP composition according to any one of claims 4 to 32, wherein the auxiliary lipid is 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC).

118. The LNP composition according to any one of claims 4 to 34, wherein the auxiliary lipid is present in the LNP composition in a molar percentage of about 7.5% to about 30%.

119. The LNP composition according to any one of claims 4 to 118, wherein the sterol is cholesterol.

120. The LNP composition according to any one of claims 4 to 118, wherein the sterol is sitosterol.

121. The LNP composition of claim 36, wherein the cholesterol is present in the LNP composition in a molar percentage of about 20% to about 50%.

122. The LNP composition according to any one of claims 4 to 38, wherein the PEG lipid is present in the LNP composition in a molar percentage of about 0.5% to about 10%.

123. The LNP composition according to any one of claims 1 to 39, wherein the LNP composition comprises mRNA with a lipid:mRNA ratio (weight / weight) between about 5:1 and about 40:

1.

124. The LNP composition according to any one of claims 1 to 40, wherein the lipid:mRNA ratio (weight / weight) is about 40:

1.

125. The LNP composition according to any one of claims 1 to 40, wherein the lipid:mRNA ratio (weight / weight) is about 36:

1.

126. The LNP composition according to any one of claims 1 to 40, wherein the lipid:mRNA ratio (weight / weight) is about 30:

1.

127. The LNP composition according to any one of claims 1 to 126, wherein the LNP composition comprises: The molar percentage of DOPE is approximately 5% to approximately 25%. The molar percentage of cholesterol is approximately 20% to approximately 50%. DMG-PEG with a molar percentage of approximately 0.5% to approximately 10%; Ionizable cationic lipids comprising approximately 10% to approximately 25% in molar percentage; and The molar percentage is approximately 30% to approximately 40% of 16:0 EPC.

128. The LNP composition according to any one of claims 1 to 126, wherein the LNP composition comprises: The molar percentage of DOPE is approximately 5% to approximately 25%. The molar percentage of cholesterol is approximately 20% to approximately 50%. DMG-PEG with a molar percentage of approximately 0.5% to approximately 10%; Ionizable cationic lipids comprising approximately 10% to approximately 25% in molar percentage; and The molar percentage of DODAP is approximately 10% to approximately 30%.

129. The LNP composition according to any one of claims 84 to 126, wherein the LNP composition comprises: The molar percentage of DOPE is approximately 5% to approximately 25%. The molar percentage of cholesterol is approximately 20% to approximately 50%. DMG-PEG with a molar percentage of approximately 0.5% to approximately 10%; Ionizable cationic lipids comprising approximately 10% to approximately 25% in molar percentage; and The molar percentage is approximately 5% to approximately 20% of 18:1 PA.

130. The LNP composition according to any one of claims 1 to 126, wherein the LNP composition comprises: The molar percentage of DOPE is approximately 5% to approximately 25%. The molar percentage of cholesterol is approximately 20% to approximately 50%. DMG-PEG with a molar percentage of approximately 0.5% to approximately 10%; Ionizable cationic lipids comprising approximately 10% to approximately 25% in molar percentage; and The molar percentage of DOTAP is approximately 30% to approximately 50%.

131. The LNP composition according to any one of claims 1 to 126, wherein the LNP composition comprises: DOPE with a molar percentage of approximately 15%; The molar percentage of cholesterol is approximately 30%. The molar percentage of DMG-PEG is approximately 3%; The molar percentage of ionizable cationic lipids is approximately 15%; and The molar percentage is approximately 30% of 16:0 EPC.

132. The LNP composition according to any one of claims 1 to 126, wherein the LNP composition comprises: DOPE with a molar percentage of approximately 20%; The molar percentage of cholesterol is approximately 40%. The molar percentage of DMG-PEG is approximately 3%; The molar percentage of ionizable cationic lipids is approximately 15%; and The molar percentage of DODAP is approximately 15%.

133. The LNP composition according to any one of claims 1 to 126, wherein the LNP composition comprises: DOPE with a molar percentage of approximately 20%; The molar percentage of cholesterol is approximately 40%. The molar percentage of DMG-PEG is approximately 5%; The molar percentage of ionizable cationic lipids is approximately 20%; and The molar percentage is approximately 5% of 18:1 PA.

134. The LNP composition according to any one of claims 1 to 126, wherein the LNP composition comprises: The molar percentage of DOPE is approximately 10%. The molar percentage of cholesterol is approximately 20%. The molar percentage of DMG-PEG is approximately 3%; The molar percentage of ionizable cationic lipids is approximately 10%; and DOTAP has a molar percentage of approximately 50%.

135. The LNP composition according to any one of claims 127 to 134, wherein the ionizable cationic lipid is a dendritic polymer lipid.

136. The LNP composition according to claim 135, wherein the dendritic polymer lipid is 4A3-SC7.

137. The LNP composition according to any one of claims 1 to 136, wherein the LNP composition comprises a Tris buffer, optionally with a pH of about 6 to about 9.

138. The LNP composition according to any one of claims 1 to 54, wherein the LNP composition comprises sucrose, optionally at a concentration (by weight) or molar percentage of about 5% to about 15%.

139. The LNP composition according to any one of claims 1 to 138, wherein the LNP composition comprises a citrate buffer, optionally with a pH of about 4 to about 6.

140. The LNP composition according to any one of claims 1 to 139, wherein the LNP composition comprises: (a) 15 mM Tris buffer, optionally with a pH of about 6 to about 9; and / or (b) Sucrose with a concentration (by weight) or molar percentage of about 5% to about 15%.

141. The LNP composition according to any one of claims 1 to 139, wherein the LNP composition comprises 10 mM citrate buffer, optionally with a pH of about 4 to about 6.

142. A pharmaceutical composition comprising a therapeutically effective amount of the LNP composition according to any one of claims 1 to 141.

143. A pharmaceutical composition comprising the LNP composition according to any one of claims 1 to 58 and comprising a pharmaceutically acceptable excipient.

144. A vaccine comprising an LNP composition according to any one of claims 1 to 141 or a pharmaceutical composition according to claim 142 or claim 143.

145. The vaccine of claim 144, wherein the vaccine comprises an adjuvant.

146. The vaccine of claim 145, wherein the adjuvant comprises squalene.

147. A method of delivering an LNP composition to a subject in need, the method comprising administering to the subject in need an LNP composition according to any one of claims 1 to 141, a pharmaceutical composition according to claim 142 or 143, and / or a vaccine according to any one of claims 144 to 146.

148. A method for preventing cancer in a subject of need, the method comprising administering to the subject of need an LNP composition according to any one of claims 1 to 141, a pharmaceutical composition according to claim 142 or 143, and / or a vaccine according to any one of claims 144 to 146.

149. A method of treating cancer or reducing the severity of cancer in a subject in need, the method comprising administering to the subject in need an LNP composition according to any one of claims 1 to 141, a pharmaceutical composition according to claim 142 or 143, and / or a vaccine according to any one of claims 144 to 146.

150. A method for inhibiting the growth, proliferation, progression, and / or metastasis of cancer cells in a subject in need, the method comprising administering to the subject in need an LNP composition according to any one of claims 1 to 141, a pharmaceutical composition according to claim 142 or 143, and / or a vaccine according to any one of claims 144 to 146.

151. A method for immunizing a subject in need against cancer and / or cancerous diseases or conditions, the method comprising administering to the subject a vaccine according to any one of claims 144 to 146.

152. The method according to any one of claims 147 to 151, wherein administration to the subject in need comprises intravenous administration.

153. The method according to any one of claims 147 to 151, wherein the administration to the subject comprises intrathecal administration.

154. The method according to any one of claims 147 to 151, wherein the administration to the subject comprises intramuscular administration.

155. The method according to any one of claims 147 to 151, wherein the administration to the subject comprises intradermal or subcutaneous administration.

156. The method according to any one of claims 147 to 151, wherein the administration to the subject comprises intranasal administration.

157. The method according to any one of claims 147 to 156, wherein the method induces an immune response in the subject in need, optionally wherein the immune response is greater than the immune response following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG).

158. The method according to any one of claims 147 to 157, wherein the method induces an antibody response in the desired subject, optionally wherein the antibody response is greater than the antibody response following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG).

159. The method according to any one of claims 147 to 158, wherein the method causes T cells to proliferate in the subject of need, optionally wherein the T cell proliferation is greater than that following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG).

160. The method according to any one of claims 147 to 159, wherein the method activates T cells in the subject in need, optionally wherein the T cell activation is greater than that following administration of composition 2H (50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG).

161. The method of claim 159 or claim 160, wherein the T cell is a T helper cell or a cytotoxic T cell.

162. The method according to any one of claims 147 to 161, wherein the method induces a cytokine response in the subject in need.

163. The method according to any one of claims 147 to 161, wherein the cancer is one or more of the following: acute myeloid leukemia (LAML), adrenocortical carcinoma (ACC), bladder cancer, bladder urothelial carcinoma (BLCA), brain cancer, breast cancer, invasive breast cancer (BRCA), Burkitt lymphoma, cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), chronic lymphocytic leukemia (CLL), colonic adenocarcinoma (COAD), colon cancer, colorectal cancer (CRC), diffuse large B-cell lymphoma (DLBCL), Ewing's sarcoma, glioblastoma multiforme (GBM), glioma, head and neck cancer, head and neck squamous cell carcinoma (HNSC), hepatocellular carcinoma, human cancer (including colorectal cancer, gastric cancer, kidney cancer and ovarian cancer), chromophobe renal cell carcinoma (KICH), clear cell renal cell carcinoma (KIRC). The following are considered cancer types: renal papillary cell carcinoma (KIRP), leukemia, hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung cancer, lung squamous cell carcinoma (LUSC), melanoma, multiple myeloma (MM), non-small cell lung cancer, ovarian adenocarcinoma, ovarian tumor, ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), primary adenocarcinoma, prostate adenocarcinoma (PRAD), prostate cancer (including high-grade prostatic intraepithelial neoplasia (PIN), androgen-dependent and androgen-independent prostate tumors), rectal adenocarcinoma (READ), renal cell carcinoma, salivary gland carcinoma, sarcoma, seminoma, cutaneous melanoma (SKCM), small bowel cancer, gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thyroid adenocarcinoma (THCA), thyroid cancer, uterine carcinosarcoma (UCS), and uterine endometrioid carcinoma (UCEC).

164. The use of the LNP composition according to any one of claims 1 to 141, the pharmaceutical composition according to claim 142 or claim 60, or the vaccine according to any one of claims 61 to 63, for the following purposes: for the manufacture of a medicament for the vaccination of subjects in need against cancer and / or cancerous diseases or conditions, or for the manufacture of a medicament for the prevention of cancer; for the treatment of cancer or the reduction of the severity of cancer; and / or for the inhibition of the growth, proliferation, progression, and / or metastasis of cancer cells.

165. The LNP composition according to any one of claims 1 to 141, the pharmaceutical composition according to claim 59 or 60, or the vaccine according to any one of claims 144 to 146, for use in the manufacture of a medicament for the vaccination of subjects in need against cancer and / or cancerous diseases or conditions, or for the manufacture of a medicament for the prevention of cancer; for the treatment of cancer or the reduction of the severity of cancer; and / or for the inhibition of the growth, proliferation, progression, and / or metastasis of cancer cells.

166. The LNP composition according to any one of claims 1 to 141, the pharmaceutical composition according to claim 59 or 60, or the vaccine according to any one of claims 144 to 146 for use in vaccinating subjects in need against cancer and / or cancerous diseases or conditions, or for the prevention of cancer; for the treatment of cancer or for reducing the severity of cancer; and / or for inhibiting the growth, proliferation, progression, and / or metastasis of cancer cells.

167. A kit comprising an LNP composition according to any one of claims 1 to 141, a pharmaceutical composition according to claim 59 or 60, or a vaccine according to any one of claims 144 to 146, and instructions for use.

168. A lipid nanoparticle (LNP) composition, said LNP composition comprising: (i) Payload; (ii) Ionizable cationic lipids; (iii) Selective organ-targeted (SORT) lipids; and / or (iv) Auxiliary lipids; Sterols; and / or A polyethylene glycol-conjugated lipid (PEG lipid), wherein the composition delivers the payload to cells in the central nervous system (CNS) of a subject.

169. The composition of claim 84, wherein the payload comprises a polynucleotide, optionally mRNA, shRNA or microRNA.

170. The composition of claim 169, wherein the mRNA encodes a polypeptide or protein selected from the groups shown in Tables 9 and 10 and / or SEQ ID NO: 201-207.

171. The composition of claim 170, wherein the mRNA-encoding gene editing system or a component thereof is described.

172. The composition according to any one of claims 84 to 171, wherein the payload comprises a polypeptide or a protein.

173. The composition according to any one of claims 84 to 172, wherein the ionizable cationic lipid is a dendritic polymer lipid.

174. The composition according to any one of claims 84 to 173, wherein the ionizable cationic lipid is a dendritic polymer lipid according to formula (I) or formula (X).

175. The composition according to any one of claims 84 to 174, wherein the ionizable cationic lipid is (4A3-SC7) 176. The composition according to any one of claims 84 to 174, wherein the ionizable cationic lipid is (5A2-SC8).

177. The composition according to any one of claims 84 to 176, wherein the ionizable cationic lipid is present in the composition in a molar percentage of about 10% to about 35%.

178. The composition according to any one of claims 84 to 177, wherein the SORT lipid is selected from the group shown in Tables 16A and 16B.

179. The composition according to any one of claims 84 to 178, wherein the SORT lipid is present in the composition in a molar percentage of about 5% to about 65%.

180. The composition according to any one of claims 84 to 179, wherein the SORT lipid is present in the composition in a molar percentage of about 5% to about 35%.

181. The composition according to any one of claims 84 to 180, wherein the auxiliary lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE).

182. The composition according to any one of claims 84 to 180, wherein the auxiliary lipid is 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC).

183. The composition according to any one of claims 84 to 182, wherein the auxiliary lipid is present in the composition in a molar percentage of about 5% to about 35%.

184. The composition according to any one of claims 84 to 183, wherein the sterol is cholesterol.

185. The composition according to any one of claims 84 to 183, wherein the sterol is sitosterol.

186. The composition according to any one of claims 84 to 185, wherein the cholesterol is present in the composition in a molar percentage of about 20% to about 50%.

187. The composition according to any one of claims 84 to 186, wherein the PEG lipid is present in the composition in a molar percentage of about 0.5% to about 10%.

188. The composition according to any one of claims 84 to 187, wherein the composition comprises mRNA with a lipid:mRNA ratio (weight / weight) between 5:1 and 40:

1.

189. The composition according to any one of claims 84 to 188, wherein the composition comprises: 4A3-SC7 with a molar percentage of approximately 5% to approximately 30%; SORT lipids, with a molar percentage of approximately 5% to approximately 30%; The molar percentage of DOPE is approximately 8% to approximately 23%. Cholesterol with a molar percentage of approximately 15% to approximately 46%; and / or The molar percentage of DMG-PEG is from about 0.5% to about 10%.

190. The composition according to any one of claims 84 to 189, wherein the composition comprises: 4A3-SC7 with a molar percentage of approximately 13% to approximately 20%; SORT with a molar percentage of approximately 10% to approximately 30%; The molar percentage of DOPE is approximately 13% to approximately 20%. Cholesterol with a molar percentage of approximately 30% to approximately 40%; and / or The molar percentage of DMG-PEG is from about 3% to about 5%, wherein the SORT lipid is ethyl phosphocholine.

191. The composition according to any one of claims 84 to 189, wherein the composition comprises: 4A3-SC7 with a molar percentage of approximately 13% to approximately 20%; SORT with a molar percentage of approximately 5% to approximately 30%; The molar percentage of DOPE is approximately 13% to approximately 20%. Cholesterol with a molar percentage of approximately 30% to approximately 40%; and / or The molar percentage of DMG-PEG is from about 3% to about 5%, wherein the SORT lipid is an anionic lipid.

192. The composition according to any one of claims 84 to 191, wherein the composition comprises: 4A3-SC7 contains approximately 19% molar percentage; The molar percentage of 14:0 EPC is approximately 20%; DOPE with a molar percentage of approximately 19%; The molar percentage of cholesterol is approximately 38%; and / or The molar percentage of DMG-PEG is approximately 4%.

193. The composition according to any one of claims 84 to 191, wherein the composition comprises: 4A3-SC7 contains approximately 17% molar percentage; The molar percentage of 16:0 EPC is approximately 30%; DOPE with a molar percentage of approximately 17%; The molar percentage of cholesterol is approximately 33%; and / or The molar percentage of DMG-PEG is approximately 3%.

194. The composition according to any one of claims 84 to 191, wherein the composition comprises: 4A3-SC7 contains approximately 17% molar percentage; The molar percentage is approximately 30% of 14:0 EPC; DOPE with a molar percentage of approximately 17%; The molar percentage of cholesterol is approximately 33%; and / or The molar percentage of DMG-PEG is approximately 3%.

195. The composition according to any one of claims 84 to 191, wherein the composition comprises: 4A3-SC7 has a molar percentage of approximately 23%; The molar percentage of 18:1 PA is approximately 5%; DOPE with a molar percentage of approximately 23%; The molar percentage of cholesterol is approximately 45%; and / or The molar percentage of DMG-PEG is approximately 5%.

196. The composition according to any one of claims 84 to 195, wherein the composition is capable of delivering the mRNA to cells in the central nervous system (CNS) of a subject in an amount that effectively increases the expression and / or function of a gene encoded by the mRNA.

197. The composition according to any one of claims 84 to 196, wherein the cells are endothelial cells.

198. The composition according to any one of claims 84 to 197, wherein the cells are pericytes.

199. The composition according to any one of claims 84 to 198, wherein the cells are smooth muscle cells (SMA).

200. A method for treating and / or preventing CNS disease in a subject of need, wherein the method comprises administering the composition according to any one of claims 84 to 199 to the subject by intrathecal injection.

201. The method of claim 200, wherein the composition comprises a payload, wherein the payload is messenger RNA (mRNA), and wherein the method causes the payload to be delivered to the CNS in an amount that effectively increases the expression and / or function of a gene encoded by the mRNA.

202. The method of claim 200 or 201, wherein the method causes the polypeptide to be expressed in the CNS of the subject.

203. The method according to any one of claims 200 to 202, wherein the method causes the polypeptide to be expressed in the CNS of the subject from 1 hour to 72 hours after administration of the composition to the subject.

204. The method according to any one of claims 200 to 203, wherein the CNS disease is an acid lipase disorder, acid maltase deficiency, acid storage disorder, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Addie pupils, Addie syndrome, adrenoleukodystrophy, agnosia, Eckhardt syndrome, Eckhardt-Gutier syndrome, Alexander disease, Alpert disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, hemangioma, hypoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, or aromatic L-amino acid decarboxylase deficiency. (AADC deficiency), aspartate glucosamineuria, Asperger's syndrome, ataxia, ataxia-telangiectasia (Lewis-Barr syndrome), ataxia and cerebellar or spinocerebellar degeneration, attention deficit hyperactivity disorder, autism, autonomic dysfunction, Bass syndrome, Barten disease, Becker's myotonia, Behçet's disease, Bell's palsy, Bernhardt-Rose syndrome, Binswanger disease, Bloch-Sulzberger syndrome, Bradbury-Eggleston syndrome, Brown-Seckal syndrome, medullary spinal muscular atrophy, CADASIL, Canavan disease, burning neuralgia, cavernous malformation, cavernous hemangioma, central cervical spinal cord syndrome, central spinal cord syndrome, central pontine myelinolysis, ceramide deficiency Deficiency syndrome, cerebellar degeneration, cerebellar hypoplasia, cerebral beriberi, cerebral gigantism, cerebral palsy, brain-ocular-facial-bone syndrome (COFS), cholesterol ester storage disease, chorea, chorea-acanthosis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, cavitary brain, congenital myasthenia gravis, corticobasal degeneration, cranial arteritis, Klebsiella encephalitis, Creutzfeldt-Jakob disease, Cushing's syndrome, cystinemia, giant cell inclusion body disease, oculoclonus-myoclonus syndrome, Dandy-Walker syndrome, Danon disease, Dawson's disease, Demosier syndrome, Degerina-Krempk palsy, dementia, dentate nucleus-cerebellar ataxia, dentate nucleus red nucleus Nuclear atrophy, dermatomyositis, developmental motor disorders, Dweck syndrome, diffuse sclerosis, autonomic dysfunction, writing disorders, reading disorders, dysphagia, motor disorders, myoclonic cerebellar dyssynergia, progressive cerebellar dyssynergia, epilepsy (e.g., Amish infantile epilepsy syndrome [AIES], benign familial infantile epilepsy [BFIS], benign familial neonatal epilepsy [BFNS], childhood absence epilepsy [CAE], childhood-onset epileptic encephalopathy [COEE], Dravet syndrome [DS], early infantile epileptic encephalopathy [EIEE], familial adult myoclonic epilepsy [FAME], familial febrile seizures [FFS], familial focal epilepsy with variable lesions [FFEVF]).Familial infantile myoclonic epilepsy [FIME], familial temporal lobe epilepsy [FTLE], focal epilepsy and speech disorders with or without intellectual disability [FESD], generalized epilepsy with paroxysmal motor disorders [GEPD], generalized epilepsy with febrile seizures plus [GEFS+], idiopathic generalized epilepsy [IGE], juvenile absence epilepsy [JAE], juvenile myoclonic epilepsy [JME], myoclonic-atonic epilepsy [MAE], nocturnal frontal lobe epilepsy [NFLE], progressive myoclonic epilepsy [PME], pyridoxine 5'-phosphate oxidase deficiency [PNPOD], pyridoxine-dependent epilepsy [EPD], and severe myoclonic epilepsy in infancy [SMEI], Fabry disease, and Fahl syndrome. Familial autonomic dysfunction, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Fabry disease, fibromuscular dysplasia, Fisher syndrome, infantile relaxation syndrome, Friedreich ataxia, frontotemporal dementia, fucoside storage disease, galactosylsialic acid storage disease, Gaucher disease, systemic ganglioside storage disease, Gerstmann syndrome, GSS disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion body disease, globular cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, GM1 ganglioside storage disease, GM2 ganglioside storage disease (Tai-Sachs disease), Guillain-Barré syndrome, Hallewarden-Scholes disease, persistent unilateral headache, alternating hemiplegia. Hereditary spastic paraplegia, hereditary ataxic polyneuritis, Holmes-Addie syndrome, holoprosencephaly, Hughes syndrome, Huntington's disease, hydrocephalus, hydrocephalus, Cushing's syndrome, immune-mediated encephalomyelitis, inclusion body myositis, incontinence pigmentosa, infantile hypotonia, infantile axonal dystrophy, occipital cleft with exposed head, Isaac syndrome, Jubert syndrome, Karns-Sell syndrome, Kennedy disease, Kingsburn syndrome, Klein-Levin syndrome, Krippel-Fair syndrome, Krippel-Trenone syndrome (KTS), Klüfer-Busy syndrome, Korsakov amnesia syndrome, Crabbe disease, Kugelberg-Weyland disease, Lambert-Eton myasthenia gravis syndrome, etc. Dow-Cleffner syndrome, lateral femoral cutaneous nerve entrapment syndrome, lateral medullary syndrome, Lewy's disease, Lennox-Gasstor syndrome, Lesch-Niehan syndrome, Levin-Critchley syndrome, Lewy body dementia, lipoprotein storage disease, gyriosis, locked-in syndrome, Lughlick disease, lupus, Lyme disease, Machado-Joseph disease, megalencephaly, alpha-mannoside storage disease, beta-mannoside storage disease, Méridov-Rough syndrome, Menkes disease, lateral femoral cutaneous neuralgia, metachromatic leukodystrophy, microcephaly, Miller-Fischer syndrome, Möbius syndrome, mucopolysaccharidosis type IH (Heller syndrome), mucopolysaccharidosis type IH / S (Heller-Shee syndrome), mucopolysaccharidosis type IS (Shee syndrome)Mucopolysaccharidosis type II (Hunter's syndrome), Mucopolysaccharidosis type III-A (Sornifero syndrome type A), Mucopolysaccharidosis type III-B (Sornifero syndrome type B), Mucopolysaccharidosis type III-C (Sornifero syndrome type C), Mucopolysaccharidosis type III-D (Sornifero syndrome type D), Mucopolysaccharidosis type IV-B (Mochio syndrome type B), Mucopolysaccharidosis type VI (Maroto-Lamy syndrome), Mucopolysaccharidosis type VII (Slay syndrome), Mucopolysaccharidosis type IX (Natovich syndrome), Multiple sclerosis, Muscular dystrophy, Myasthenia gravis, Myelinolytic diffuse sclerosis, Narcolepsy. Neuroacanthocytosis, neurofibromatosis, neuroleptic malignant syndrome, neurosarcoidosis, Niemann-Pick disease, Ōtahara syndrome, olivopontocerebellar atrophy, oculoclonus myoclonus, Osullivan-McLeod syndrome, pantothenic kinase-associated neurodegenerative disease, paraneoplastic syndrome, paresthesia, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal unilateral headache, Paroxysmal-Ross syndrome, Peyre's disease, Pena-Shokel syndrome type II, periventricular leukomalacia, phytanoic acid storage disease, Pick's disease, piriformis syndrome, polymyositis, Pompe disease, post-poliomyelitis syndrome, posterior cortical atrophy, primary dentate nucleus atrophy, primary lateral sclerosis, primary... Progressive aphasia, prion disease, progressive hemifacial atrophy, progressive motor ataxia, progressive multifocal leukoencephalopathy, progressive sclerotic gray matter dystrophy, progressive supranuclear palsy, prosopagnosia, Ramsey Hunt syndrome type I, Ramsey Hunt syndrome type II, Rasmussen encephalitis, Rifsum disease, Rett syndrome, Reye's syndrome, Riley-Day syndrome, Sandhof disease, Hilde disease, Setterberg disease, Shay-Dreg syndrome, Sjögren's syndrome, spasticity, spina bifida, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olshefsky syndrome, striatal nigra degeneration Sexual dysfunction, Sturge-Weber syndrome, tardive dyskinesia, Tau proteinosis, Ty-Sachs disease, thoracic outlet syndrome, thyrotoxic myopathy, painful tic, Todd's paralysis, trigeminal neuralgia, tropical spastic paraplegia, Troy syndrome, vascular dementia, von Eknomer disease, von Hippel-Lindau disease (VHL), von Recklinghausen disease, Wallenberg syndrome, Wernig-Hoffmann disease, Wernicke-Korsakov syndrome, West syndrome, Whipple's disease, Williams syndrome, Wilson's disease, Wollman's disease, X-linked spinal-bulbar muscular atrophy, Zelweg syndrome, multiple sclerosis with cerebral atrophy, Lewy body dementia (LBD), or Anglemann syndrome.

205. A method of delivering a payload to cells in the CNS of a subject, wherein the method comprises administering the composition according to any one of claims 84 to 199 to the subject by intrathecal injection.

206. A kit comprising the composition according to any one of claims 84 to 199.

207. A pharmaceutical composition comprising the composition according to any one of claims 168 to 199 and comprising a pharmaceutically acceptable excipient and / or diluent.

208. Use of the composition according to any one of claims 84 to 199 for the treatment of CNS diseases.

209. Use of the composition according to any one of claims 84 to 199 for the treatment of CNS diseases by intrathecal administration.

210. The composition according to any one of claims 84 to 199 or the pharmaceutical composition according to claim 33 for treating CNS disease in a subject in need.

211. The use according to any one of claims 208 to 210 or the composition for said use, wherein said CNS disease is acid lipase disorder, acid maltase deficiency, acid storage disease, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Addie pupils, Addie syndrome, adrenoleukodystrophy, agnosia, Eckhardt syndrome, Eckhardt-Gutier syndrome, Alexander disease, Alpert disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, hemangioma, hypoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, arachidonic L-amino acids. Amino acid decarboxylase deficiency (AADC deficiency), aspartate glucosamineuria, Asperger's syndrome, ataxia, ataxia-telangiectasia (Lewis-Barr syndrome), ataxia and cerebellar or spinocerebellar degeneration, attention deficit hyperactivity disorder, autism, autonomic dysfunction, Bass syndrome, Barten disease, Becker's myotonia, Behçet's disease, Bell's palsy, Bernhardt-Rose syndrome, Binswanger disease, Bloch-Sulzberger syndrome, Bradbury-Eggleston syndrome, Brown-Seckal syndrome, medullary spinal muscular atrophy, CADASIL, Canavan disease, burning neuralgia, cavernous malformation, cavernous hemangioma, central cervical spinal cord syndrome, central spinal cord syndrome, central pontine myelinolysis, and so on. Transaminas deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral beriberi, cerebral gigantism, cerebral palsy, brain-ocular-facial-skeletal syndrome (COFS), cholesterol ester storage disease, chorea, chorea-acanthosis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, cavitary brain, congenital myasthenia gravis, corticobasal degeneration, cranial arteritis, Klebsiella encephalitis, Creutzfeldt-Jakob disease, Cushing's syndrome, cystinemia, giant cell inclusion body disease, oculoclonus-myoclonus syndrome, Dandy-Walker syndrome, Danon disease, Dawson's disease, Demosier syndrome, Degerina-Klempke palsy, dementia, dentate nucleus-cerebellar ataxia, dentate nucleus Nucleotid atrophy, dermatomyositis, developmental motor disorders, Dweck syndrome, diffuse sclerosis, autonomic dysfunction, writing disorders, reading disorders, dysphagia, motor disorders, myoclonic cerebellar dyssynergia, progressive cerebellar dyssynergia, epilepsy (e.g., Amish infantile epilepsy syndrome [AIES], benign familial infantile epilepsy [BFIS], benign familial neonatal epilepsy [BFNS], childhood absence epilepsy [CAE], childhood-onset epileptic encephalopathy [COEE], Dravet syndrome [DS], early infantile epileptic encephalopathy [EIEE], familial adult myoclonic epilepsy [FAME], familial febrile seizures [FFS], familial focal epilepsy with variable lesions [FFEVF]).Familial infantile myoclonic epilepsy [FIME], familial temporal lobe epilepsy [FTLE], focal epilepsy and speech disorders with or without intellectual disability [FESD], generalized epilepsy with paroxysmal motor disorders [GEPD], generalized epilepsy with febrile seizures plus [GEFS+], idiopathic generalized epilepsy [IGE], juvenile absence epilepsy [JAE], juvenile myoclonic epilepsy [JME], myoclonic-atonic epilepsy [MAE], nocturnal frontal lobe epilepsy [NFLE], progressive myoclonic epilepsy [PME], pyridoxine 5'-phosphate oxidase deficiency [PNPOD], pyridoxine-dependent epilepsy [EPD], and severe myoclonic epilepsy in infancy [SMEI], Fabry disease, and Fahl syndrome. Familial autonomic dysfunction, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Fabry disease, fibromuscular dysplasia, Fisher syndrome, infantile relaxation syndrome, Friedreich ataxia, frontotemporal dementia, fucoside storage disease, galactosylsialic acid storage disease, Gaucher disease, systemic ganglioside storage disease, Gerstmann syndrome, GSS disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion body disease, globular cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, GM1 ganglioside storage disease, GM2 ganglioside storage disease (Tai-Sachs disease), Guillain-Barré syndrome, Hallewarden-Scholes disease, persistent unilateral headache, alternating hemiplegia. Hereditary spastic paraplegia, hereditary ataxic polyneuritis, Holmes-Addie syndrome, holoprosencephaly, Hughes syndrome, Huntington's disease, hydrocephalus, hydrocephalus, Cushing's syndrome, immune-mediated encephalomyelitis, inclusion body myositis, incontinence pigmentosa, infantile hypotonia, infantile axonal dystrophy, occipital cleft with exposed head, Isaac syndrome, Jubert syndrome, Karns-Sell syndrome, Kennedy disease, Kingsburn syndrome, Klein-Levin syndrome, Krippel-Fair syndrome, Krippel-Trenone syndrome (KTS), Klüfer-Busy syndrome, Korsakov amnesia syndrome, Crabbe disease, Kugelberg-Weyland disease, Lambert-Eton myasthenia gravis syndrome, etc. Dow-Cleffner syndrome, lateral femoral cutaneous nerve entrapment syndrome, lateral medullary syndrome, Lewy's disease, Lennox-Gasstor syndrome, Lesch-Niehan syndrome, Levin-Critchley syndrome, Lewy body dementia, lipoprotein storage disease, gyriosis, locked-in syndrome, Lughlick disease, lupus, Lyme disease, Machado-Joseph disease, megalencephaly, alpha-mannoside storage disease, beta-mannoside storage disease, Méridov-Rough syndrome, Menkes disease, lateral femoral cutaneous neuralgia, metachromatic leukodystrophy, microcephaly, Miller-Fischer syndrome, Möbius syndrome, mucopolysaccharidosis type IH (Heller syndrome), mucopolysaccharidosis type IH / S (Heller-Shee syndrome), mucopolysaccharidosis type IS (Shee syndrome)Mucopolysaccharidosis type II (Hunter's syndrome), Mucopolysaccharidosis type III-A (Sornifero syndrome type A), Mucopolysaccharidosis type III-B (Sornifero syndrome type B), Mucopolysaccharidosis type III-C (Sornifero syndrome type C), Mucopolysaccharidosis type III-D (Sornifero syndrome type D), Mucopolysaccharidosis type IV-B (Mochio syndrome type B), Mucopolysaccharidosis type VI (Maroto-Lamy syndrome), Mucopolysaccharidosis type VII (Slay syndrome), Mucopolysaccharidosis type IX (Natovich syndrome), Multiple sclerosis, Muscular dystrophy, Myasthenia gravis, Myelinolytic diffuse sclerosis, Narcolepsy. Neuroacanthocytosis, neurofibromatosis, neuroleptic malignant syndrome, neurosarcoidosis, Niemann-Pick disease, Ōtahara syndrome, olivopontocerebellar atrophy, oculoclonus myoclonus, Osullivan-McLeod syndrome, pantothenic kinase-associated neurodegenerative disease, paraneoplastic syndrome, paresthesia, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal unilateral headache, Paroxysmal-Ross syndrome, Peyre's disease, Pena-Shokel syndrome type II, periventricular leukomalacia, phytanoic acid storage disease, Pick's disease, piriformis syndrome, polymyositis, Pompe disease, post-poliomyelitis syndrome, posterior cortical atrophy, primary dentate nucleus atrophy, primary lateral sclerosis, primary... Progressive aphasia, prion disease, progressive hemifacial atrophy, progressive motor ataxia, progressive multifocal leukoencephalopathy, progressive sclerotic gray matter dystrophy, progressive supranuclear palsy, prosopagnosia, Ramsey Hunt syndrome type I, Ramsey Hunt syndrome type II, Rasmussen encephalitis, Rifsum disease, Rett syndrome, Reye's syndrome, Riley-Day syndrome, Sandhof disease, Hilde disease, Setterberg disease, Shay-Dreg syndrome, Sjögren's syndrome, spasticity, spina bifida, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olshefsky syndrome, striatal nigra degeneration Sexual dysfunction, Sturge-Weber syndrome, tardive dyskinesia, Tau proteinosis, Ty-Sachs disease, thoracic outlet syndrome, thyrotoxic myopathy, painful tic, Todd's paralysis, trigeminal neuralgia, tropical spastic paraplegia, Troy syndrome, vascular dementia, von Eknomer disease, von Hippel-Lindau disease (VHL), von Recklinghausen disease, Wallenberg syndrome, Wernig-Hoffmann disease, Wernicke-Korsakov syndrome, West syndrome, Whipple's disease, Williams syndrome, Wilson's disease, Wollman's disease, X-linked spinal-bulbar muscular atrophy, Zelweg syndrome, multiple sclerosis with cerebral atrophy, Lewy body dementia (LBD), or Anglemann syndrome.

212. A composition or method as described herein.

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