Composition for mRNA therapy

Abstract

To provide an enhanced RNA delivery system for more effective, easily scalable, and stable delivery of RNA therapeutics (such as cancer vaccines). [0010] Disclosed herein is an mRNA therapeutic composition comprising one or more polynucleotides encoding one or more tumor antigenic, immunogenic, or signaling polypeptides formulated in a lipid-reconstituted plant messenger pack (LPMP) comprising natural lipids and ionizable lipids. The disclosure also includes a method of making the mRNA therapeutic composition comprising reconstituting a membrane comprising purified PMP lipids in the presence of ionizable lipids to produce LPMPs comprising ionizable lipids, and loading the LPMPs with one or more polynucleotides encoding one or more tumor antigenic or immunogenic polypeptides.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 291,686, filed December 20, 2021, U.S. Provisional Patent Application No. 63 / 320,664, filed March 16, 2022, and U.S. Provisional Patent Application No. 63 / 401,214, filed August 26, 2022, all of which are incorporated by reference in their entireties herein. [Background technology]

[0002] Vaccines on the market or in development (e.g., cancer vaccines) are typically based on whole microorganisms, protein antigens, peptides, polysaccharides or deoxyribonucleic acid (DNA) vaccines and combinations thereof. The use of RNA polynucleotides as therapeutic agents is a new and emerging field.

[0003] Therefore, there is a need to develop enhanced RNA delivery systems for more effective, easily scalable, and stable delivery of RNA therapeutics, such as cancer vaccines. Summary of the Invention

[0004] In one aspect, provided herein is an mRNA therapeutic composition comprising one or more polynucleotides encoding one or more antigenic (e.g., tumor antigenic) or signaling polypeptides. The one or more polynucleotides are formulated in a lipid reconstituted plant messenger pack (LPMP) comprising natural lipids and ionizable lipids. The ionizable lipids have the following characteristics listed below: (i) at least two ionizable amines; (ii) at least three lipid tails, each of said lipid tails being at least 6 carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) ionizable amine and heteroorganic groups separated by a chain of at least two atoms; and (v) an N:P ratio of at least 10.

[0005] In another aspect, a method for making an mRNA therapeutic composition is provided herein. The method includes reconstituting a membrane containing purified PMP lipids in the presence of ionizable lipids to produce a lipid-reconstituted plant messenger pack (LPMP) containing ionizable lipids. The ionizable lipids have the following characteristics: (i) at least two ionizable amines; (ii) at least three lipid tails, each of said lipid tails being at least 6 carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) ionizable amine and heteroorganic groups separated by a chain of at least two atoms; and (v) an N:P ratio of at least 10.

[0006] The method further includes loading the LPMP with one or more polynucleotides encoding one or more antigenic (eg, tumor antigenic) or signaling polypeptides.

[0007] In some embodiments, the polynucleotide is a polynucleotide construct that encodes one or more wild-type or engineered antigens (or antibodies to antigens). The antigens can be derived from a tumor, e.g., tumor-specific antigens, tumor-associated antigens, tumor neoantigens, or a combination thereof.

[0008] In some embodiments, the polypeptide encoded by the polynucleotide is selected from the group consisting of p53, ART-4, BAGE, ss-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, CLAUDIN-12, C-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, (HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A2. E-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Plac-1, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or S ART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, WTWT-1, or combinations thereof.

[0009] In some embodiments, the polypeptide encoded by the polynucleotide is CD2, CD3, CD4, CD8, CD11b, CD14, CD16, CD19, CD20, CD22, CD25, CD27, CD33, CD37, CD38, CD40, CD44, CD45, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-0, 62, BTLA, CAIX, carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbBl, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, HLA, HLA-DR, ICOS, IGF1R, integrin αν, integrin ανβ, LAG-3, Lewis Antigenic (e.g., tumor antigenic) or signaling polypeptides, including Y, mesothelin, c-MET, MN carbonic anhydrase IX, MUC1, MUC16, Nectin-4, KGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, syndecan-1, TACI, TAG-72, tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and variants thereof.

[0010] You can also find a new source of information on your website. Introduction to IL-1α、IL-1 β、IL-1ra、IL-2、FILL-3、FILL-4、FILL-5、FILL-6、FILL-7、FILL-8、FILL-9、FILL-10、FILL-11、FILL-12、FILL-13、FILL-14、FILL IL-16、IL-17A、IL-17B、IL-17C、IL-17D、IL-17E、IL-17F、IL-18、IL-19、IL-20、IL-21、IL-22、IL-23. L-24、IL-25、IL-26、IL-27、IL-28A / B、IL-29、IL-30、IL-31、IL-32、IL-33、IL-35、TGF-β、GM-CSF、M-CS F、G-CSF、TNF-α、TNF-β、LAF、TCGF、BCGF、TRF、BAF、BDG、MP、LIF、OSM、TMF、PDGF、IFN-α、IFN-β、、IFN- Foxp3, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8,1CXCCL11 2、CXCL13、CXCL14、CXCL15、CXCL16、CCL1、CCL2、CCL3、CCL4、CCL5、CCL6、CCL7、CCL8、CCL9 / 10、CCL11 CL12、CCL13、CCL14、CCL15、CCL16、CCL17、CCL18、CCL19、CCL20、CCL21、CCL22、CCCL23 L26、CCL27、CCL28、XCL1、XCL2、CX3CL1 If you want to change your mind, These types of information should also be used to describe the data.

[0011] In some embodiments, the polypeptide encoded by the polynucleotide is an IL-2 peptide, IL-2-Ra, tdTomato, Cre recombinase, GFP, eGFP, anti-CD19, CD20, CAR-T, anti-HER2, etanercept (Enbrel), Humira, erythropoietin, epogen, filgrastim, Keytruda, rituximab, romiplostim, sargramostim, or a fragment or subunit thereof. In one embodiment, the polypeptide is an IL-2 peptide, or a fragment or subunit thereof. In one embodiment, the polypeptide is an erythropoietin or epogen, or a fragment or subunit thereof.

[0012] In some embodiments, the polypeptide encoded by the polynucleotide is an IL-15 peptide, IL-15-Ra, or a fragment or subunit thereof. In one embodiment, the polypeptide is an IL-15 peptide, or a fragment or subunit thereof.

[0013] In some embodiments, the tumor antigenic polypeptide comprises a tumor antigen selected from the group consisting of carcinoma, sarcoma, melanoma, lymphoma, leukemia, and combinations thereof. In one embodiment, the tumor antigenic polypeptide comprises a lung cancer antigen.

[0014] In some embodiments, the polynucleotide may be an mRNA, an siRNA or siRNA precursor, a microRNA (miRNA) or miRNA precursor, a plasmid, a dicer substrate small interfering RNA (dsiRNA), a small hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), a peptide nucleic acid (PNA), a morpholino, a locked nucleic acid (LNA), a piwi-interacting RNA (piRNA), a ribozyme, a deoxyribozyme (DNAzyme), an aptamer, a circular RNA (circRNA), a guide RNA (gRNA), or a DNA molecule encoding any of these RNAs. In one embodiment, the polynucleotide is an mRNA.

[0015] In one embodiment, the polynucleotide is an mRNA encoding an IL-2 molecule comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of an IL-2 molecule provided in any one of Tables I-III.

[0016] In one embodiment, the polynucleotide is an mRNA encoding an IL-15 or IL-15RA molecule comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of an IL-15 molecule provided in Table IV. In one embodiment, the polynucleotide is an mRNA encoding an IL-15 molecule comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of an IL-15 or IL-15RA molecule provided in Table IV.

[0017] In some embodiments, the mRNA is derived from (a) a DNA molecule, or (b) an RNA molecule. In the mRNA, T is optionally replaced with U.

[0018] In some embodiments, the mRNA is derived from a DNA molecule. The DNA molecule may further comprise a promoter. In some embodiments, the promoter is a T7 promoter, a T3 promoter, or a SP6 promoter. In some embodiments, the promoter is located in the 5'UTR.

[0019] In some embodiments, the mRNA is derived from an RNA molecule. The RNA molecule may be a self-replicating RNA molecule.

[0020] In some embodiments, the mRNA is an RNA molecule. The RNA molecule may further comprise a 5' cap. The 5' cap may have a cap1 structure, a cap1(m6A) structure, a cap2 structure, a cap3 structure, a cap0 structure, or any combination thereof.

[0021] In some embodiments, the polynucleotide is an mRNA encoding an IL-2 molecule. In one embodiment, the IL-2 molecule comprises a naturally occurring IL-2 molecule, a fragment of a naturally occurring IL-2 molecule, or a variant thereof. In one embodiment, the IL-2 molecule comprises a variant of a naturally occurring IL-2 molecule (e.g., an IL-2 variant described herein), or a fragment thereof.

[0022] In some embodiments, the polynucleotide is an mRNA encoding an IL-15 molecule. In one embodiment, the IL-15 molecule comprises a naturally occurring IL-15 molecule, a fragment of a naturally occurring IL-15 molecule, or a variant thereof. In one embodiment, the IL-15 molecule comprises a variant of a naturally occurring IL-15 molecule (e.g., an IL-15 variant described herein), or a fragment thereof. In one embodiment, the polynucleotide is an mRNA encoding an IL-15 superagonist, an IL-15 molecule, an IL-15RA molecule, or a combination thereof.

[0023] In some embodiments, the mRNA comprises a 5' untranslated region (UTR) and / or a 3' UTR.

[0024] In some embodiments, the mRNA comprises a 5'UTR. The 5'UTR may comprise a Kozak sequence.

[0025] In some embodiments, the mRNA comprises a 3'UTR. In some embodiments, the 3'UTR comprises one or more sequences derived from a split amino-terminal enhancer (AES). In some embodiments, the 3'UTR comprises a sequence derived from mitochondrially encoded 12S rRNA (mtRNRl).

[0026] In some embodiments, the mRNA comprises a poly(A) sequence. In one embodiment, the poly(A) sequence is a 110 nucleotide sequence consisting of a sequence of 30 adenosine residues, a linker sequence of 10 nucleotides, and a sequence of 70 adenosine residues.

[0027] In some embodiments, the polynucleotide is encapsulated by lipid reconstituted plant messenger packs (LPMPs). In some embodiments, the polynucleotide is embedded on the surface of the LPMP. In some embodiments, the polynucleotide is conjugated to the surface of the LPMP.

[0028] In some embodiments, the LPMPs are produced by a method comprising lipid extrusion. In some embodiments, the LPMPs are produced by a method comprising processing a solution comprising a lipid extract of the PMP in a microfluidic device comprising an aqueous phase, thereby producing the LPMP. In some embodiments, the aqueous phase comprises a polynucleotide.

[0029] In some embodiments, the natural lipids of the LPMPs are extracted from lemons or algae.

[0030] In some embodiments, the ionizable lipid of the LPMP is selected from the group consisting of 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LPO1, 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315.

[0031] In one embodiment, the ionizable lipid is C12-200.

[0032] In some embodiments, the ionizable lipid is [ka] (I), where R is a C8-C14 alkyl group.

[0033] In some embodiments, the reconstitution is performed in the presence of a sterol, thereby producing LPMPs that include a natural lipid, an ionizable lipid, and a sterol. In some embodiments, the sterol is cholesterol or sitosterol.

[0034] In some embodiments, the reconstitution is carried out in the presence of a PEGylated lipid (or a PEG-lipid conjugate), thereby producing an LPMP that includes a natural lipid, an ionizable lipid, and a PEG-lipid conjugate. In some embodiments, the PEG-lipid conjugate is C14-PEG2k, C18-PEG2k, or DMPE-PEG2k. In some embodiments, the PEG-lipid conjugate is PEG-DMG or PEG-PE. In some embodiments, the PEG-DMG is PEG2000-DMG or PEG2000-PE.

[0035] In some embodiments, the LPMP further comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate.

[0036] In some embodiments, the LPMP is about 20 mol % to about 50 mol % of an ionizable lipid; about 20 mol % to about 60 mol % of natural lipids; about 7 mol % to about 20 mol % of a sterol, and The mRNA therapeutic composition of any one of the preceding embodiments, comprising about 0.5 mol % to about 3 mol % of a polyethylene glycol (PEG)-lipid conjugate.

[0037] In some embodiments, the LPMP is about 35 mole % ionizable lipids; Approximately 50 mol % natural lipids, about 12.5 mole % of said sterol, and The mRNA therapeutic composition of embodiment 39, comprising about 2.5 mol % of a polyethylene glycol (PEG)-lipid conjugate.

[0038] In one embodiment, the LPMP comprises a molar ratio of ionizable lipids:natural lipids:sterols:PEG lipids of about 35:50:12.5:2.5 In one embodiment, the LPMP comprises a molar ratio of ionizable lipids:natural lipids:sterols:PEG lipids of about 35:20:42.5:2.5.

[0039] In some embodiments, the LPMP is Natural lipids extracted from lemons or algae, C12-200, Cholesterol, and Contains DMPE-PEG2k.

[0040] In one embodiment, the LPMP is Natural lipids extracted from lemons. C12-200, Cholesterol, and The LPMP may comprise C12-200: lemon lipid: cholesterol: DMPE-PEG2k in a molar ratio of about 35:50:12.5:2.5.

[0041] In one embodiment, the LPMP is Natural lipids extracted from algae, C12-200, Cholesterol, and The LPMP may comprise C12-200:algal lipid:cholesterol:DMPE-PEG2k in a molar ratio of about 35:20:42.5:2.5.

[0042] In some embodiments, the LPMP is a lipophilic moiety selected from the group consisting of lipoplexes, liposomes, lipid nanoparticles, polymeric carriers, exosomes, lamellar bodies, micelles, and emulsions. In one embodiment, the LPMP is a liposome selected from the group consisting of cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes. In one embodiment, the LPMP is a lipid nanoparticle.

[0043] In some embodiments, the LPMPs have a size of less than about 200 nm. In one embodiment, the LPMPs have a size of less than about 150 nm. In one embodiment, the LPMPs have a size of less than about 100 nm. In one embodiment, the LPMPs have a size of about 55 nm to about 80 nm.

[0044] In some embodiments, an mRNA therapeutic composition has a total lipid:polynucleotide weight ratio of about 50:1 to about 10:1. In one embodiment, an mRNA therapeutic composition has a total lipid:polynucleotide weight ratio of about 44:1 to about 24:1. In one embodiment, an mRNA therapeutic composition has a total lipid:polynucleotide weight ratio of about 40:1 to about 28:1. In one embodiment, an mRNA therapeutic composition has a total lipid:polynucleotide weight ratio of about 38:1 to about 30:1. In one embodiment, an mRNA therapeutic composition has a total lipid:polynucleotide weight ratio of about 37:1 to about 33:1.

[0045] In some embodiments, the mRNA therapeutic composition, e.g., the aqueous phase, further comprises a HEPES or TRIS buffer. The HEPES or TRIS buffer may have a pH of about 7.0 to about 8.5. The HEPES or TRIS buffer may have a concentration of about 7 mg / mL to about 15 mg / mL. The aqueous phase may further comprise about 2.0 mg / mL to about 4.0 mg / mL NaCl.

[0046] In some embodiments, the mRNA therapeutic composition, e.g., the aqueous phase, comprises water, PBS, or a citrate buffer. In one embodiment, the aqueous phase comprises a citrate buffer having a pH of about 3.2.

[0047] In some embodiments, the aqueous phase and lipid solution are mixed in a volume ratio of 3:1.

[0048] In some embodiments, the mRNA therapeutic composition further comprises one or more cryoprotectants. The one or more cryoprotectants may be sucrose, glycerol, or a combination thereof. In one embodiment, the mRNA therapeutic composition comprises a combination of sucrose at a concentration of about 70 mg / mL to about 110 mg / mL and glycerol at a concentration of about 50 mg / mL to about 70 mg / mL.

[0049] In some embodiments, the mRNA therapeutic composition is a lyophilized composition. The lyophilized mRNA therapeutic composition may include one or more lyoprotectants. The lyophilized mRNA therapeutic composition may include poloxamer, potassium sorbate, sucrose, or any combination thereof. In one embodiment, the lyophilized mRNA therapeutic composition includes poloxamer, for example, poloxamer 188.

[0050] In some embodiments, the mRNA therapeutic composition is a lyophilized composition. In one embodiment, the lyophilized mRNA therapeutic composition comprises about 0.01 to about 1.0% w / w polynucleotide. In one embodiment, the lyophilized mRNA therapeutic composition comprises about 1.0 to about 5.0% w / w lipid. In one embodiment, the lyophilized mRNA therapeutic composition comprises about 0.5 to about 2.5% w / w TRIS buffer. In one embodiment, the lyophilized mRNA therapeutic composition comprises about 0.75 to about 2.75% w / w NaCl. In one embodiment, the lyophilized mRNA therapeutic composition comprises about 85 to about 95% w / w sugar, e.g., sucrose. In one embodiment, the lyophilized mRNA therapeutic composition comprises about 0.01 to about 1.0% w / w poloxamer, e.g., poloxamer 188. In one embodiment, the lyophilized mRNA therapeutic composition comprises about 1.0 to about 5.0% w / w potassium sorbate.

[0051] In another aspect, provided herein is a method of delivering an mRNA therapeutic in a subject, comprising administering to the subject an mRNA therapeutic composition as discussed in the above aspect of the invention.

[0052] In another aspect, provided herein is a method of inducing an immune response in a subject, comprising administering to the subject an mRNA therapeutic composition as discussed in the above aspect of the invention.

[0053] In another aspect, provided herein is a method of treating or preventing cancer in a subject, comprising administering to the subject an mRNA therapeutic composition as discussed in the above aspect of the invention.

[0054] In these aspects of the present invention relating to the method of delivering mRNA therapeutics, the method of inducing immune responses, and the method of treating or preventing cancer, the mRNA therapeutic composition can be administered orally, intravenously, intradermally, intramuscularly, intranasally, intraocularly, or rectally, and / or subcutaneously. In certain embodiments, the mRNA therapeutic composition is administered orally, intravenously, intramuscularly, and / or subcutaneously.

[0055] In some embodiments, an mRNA therapeutic composition is administered at a dosage level sufficient to deliver from about 0.001 mg / kg to about 0.5 mg / kg (e.g., from about 0.005 mg / kg to about 0.5 mg, from about 0.006 mg / kg to about 0.5 mg / kg, or from 0.01 mg / kg to about 0.4 mg / kg) of polynucleotide (e.g., mRNA) to a subject. In some embodiments, the mRNA therapeutic composition is administered at a dosage level sufficient to deliver about 0.006 mg / kg, about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, or about 0.4 mg / kg of the polynucleotide (e.g., mRNA) to the subject. In some embodiments, the mRNA therapeutic composition is administered at a dosage level sufficient to deliver about 0.0001 mg / kg to about 0.0005 mg / kg (e.g., about 0.0003 mg / kg to about 0.002 mg / kg) of the polynucleotide (e.g., mRNA) to the subject.

[0056] In some embodiments, the mRNA therapeutic composition is administered to the subject one, two, three, four or more times. In some embodiments, the mRNA therapeutic composition is administered to the subject one or two times. In some embodiments, the mRNA therapeutic composition is administered to the subject four times.

[0057] In some embodiments, the methods further comprise administering to the subject an additional therapeutic agent.

[0058] In some embodiments, the additional therapeutic agent is an anti-cancer therapeutic agent.

[0059] In some embodiments, the additional therapeutic agent is an immunogenic therapeutic agent.

[0060] In some embodiments, the additional therapeutic agent is a signal transduction therapeutic agent.

[0061] In some embodiments, the additional therapeutic agent is a therapeutic agent for treating and / or preventing chronic pain. In one embodiment, the additional therapeutic agent is an opioid analgesic such as buprenorphine, a nonsteroidal anti-inflammatory drug (NSAID) such as meloxicam SR, or a combination thereof.

[0062] In some embodiments, the additional therapeutic agent is administered prior to, concurrently with, or following administration of the mRNA therapeutic composition.

[0063] definition As used herein, the terms "effective amount," "effective concentration," or "effective concentration" refer to an amount of an LPMP or nucleic acid composition sufficient to produce a recited result or to reach a target level (e.g., a predetermined level or threshold level) in or on a target organism.

[0064] As used herein, the term "therapeutic agent" refers to an agent that can act on an animal, e.g., a mammal (e.g., a human), an animal pathogen, or a pathogen vector, such as an antifungal, antibacterial, virucidal, antiviral, insecticidal, nematicidal, antiparasitic, or insect repellent.

[0065] As defined herein, the terms "nucleic acid" and "polynucleotide" are interchangeable and refer to RNA or DNA that is linear or branched, single-stranded or double-stranded, or a hybrid thereof, regardless of length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 150, 200, 250, 500, 1000, or more nucleic acids). The term also encompasses RNA / DNA hybrids. Nucleotides are typically linked in nucleic acids by phosphodiester bonds, but the term "nucleic acid" also encompasses nucleic acid analogs with other types of bonds or backbones (e.g., phosphoramide, phosphorothioate, phosphorodithioate, O-methyl phosphoramidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threos nucleic acid (TNA), and peptide nucleic acid (PNA) bonds or backbones, among others). Nucleic acids may be single-stranded, double-stranded, or contain portions of both single-stranded and double-stranded sequences. Nucleic acids may contain any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including, for example, adenine, thymine, cytosine, guanine, uracil, and modified or non-standard bases, including, for example, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5 hydroxymethylcytosine.

[0066] As used herein, the terms "peptide," "protein," or "polypeptide" encompass any chain of naturally or non-naturally occurring amino acids (either D- or L-amino acids), regardless of length (e.g., at least 2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100, or more amino acids), the presence or absence of post-translational modifications (e.g., glycosylation or phosphorylation), or the presence of one or more non-amino acyl groups (e.g., sugars, lipids, etc.) covalently attached to the peptide, including, for example, naturally occurring proteins, synthetic, or recombinant polypeptides and peptides, hybrid molecules, peptoids, or peptidomimetics.

[0067] As used herein, the "percent identity" between two sequences is determined by the BLAST 2.0 algorithm, as described in Altschul et al., (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0068] As used herein, the term "plant" refers to whole plants, plant organs, plant tissues, seeds, plant cells, seeds, and their progeny. Plant cells include, but are not limited to, cells derived from seeds, suspension cultures, embryos, ciliary regions, callus tissues, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant parts include, but are not limited to, the following: roots, stems, shoots, leaves, pollen, seeds, fruits, harvested products, tumor tissues, sap (e.g., xylem sap and phloem sap), and differentiated and undifferentiated tissues, including various forms of cells and cultures (e.g., single cells, protists, embryos, and callus tissues). Plant tissues may be within a plant or within a plant organ, tissue, or cell culture.

[0069] As used herein, the term "modified PMP" or "modified LPMP" refers to a composition comprising a plurality of PMPs or LPMPs comprising one or more heterologous agents (e.g., PMPs or LPMPs comprising one or more exogenous lipids, e.g., ionizable lipids, e.g., ionizable lipids and sterols and / or PEGylated lipids) that can increase cellular uptake (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake) of the PMP or LPMP, or a portion or component thereof, can enable or increase delivery of a heterologous functional agent (e.g., agrochemical or therapeutic agent) to a cell by the PMP or LPMP, and / or can enable or increase loading (e.g., loading efficiency or loading capacity) of a heterologous functional agent (e.g., agrochemical or therapeutic agent) compared to an unmodified PMP or LPMP. The PMP or LPMP may be modified in vitro or in vivo.

[0070] As used herein, the term "unmodified PMP" or "unmodified LPMP" refers to a composition comprising a plurality of PMPs or LPMPs that lack a heterologous cell uptake agent that can increase cellular uptake of the PMPs (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake).

[0071] As used herein, the term "cellular uptake" refers to the uptake of a PMP or LPMP or a portion or component thereof (e.g., a polynucleotide carried by a PMP or LPMP) by a cell, such as an animal cell, a plant cell, a bacterial cell, or a fungal cell. For example, uptake can include the transfer of a portion of a PMP (e.g., an LPMP) or a component thereof from the extracellular environment to or across a cell membrane, cell wall, extracellular matrix, or into the intracellular environment of a cell. Cellular uptake of a PMP (e.g., an LPMP) can occur through active or passive cellular mechanisms. Cellular uptake includes aspects in which the entire PMP (e.g., an LPMP) is taken up by the cell, for example, by endocytosis. In some embodiments, one or more polynucleotides are exposed to the cytoplasm of the target cell after endocytosis and endosomal escape. In some embodiments, modified LPMPs (e.g., LPMPs comprising ionizable lipids, e.g., LPMPs comprising ionizable lipids and sterols and / or PEGylated lipids) have an increased rate of endosomal escape compared to unmodified LPMPs. Cellular uptake also includes aspects in which a PMP (e.g., an LPMP) fuses with a membrane of a target cell. In some embodiments, one or more polynucleotides are exposed to the cytoplasm of a target cell after membrane fusion. In some embodiments, an LPMP has an increased rate of fusion with a membrane of a target cell (e.g., is more fusogenic) compared to unmodified LPMPs.

[0072] As used herein, the term "cell permeabilizing agent" refers to an agent that alters the properties (e.g., permeability) of the cell wall, extracellular matrix, or cell membrane of a cell (e.g., an animal cell, a plant cell, a bacterial cell, or a fungal cell) in a manner that promotes increased cellular uptake compared to a cell not contacted with the agent.

[0073] As used herein, the term "plant extracellular vesicle", "plant EV" or "EV" refers to an enclosed lipid bilayer structure that naturally occurs in plants. Optionally, the plant EV includes one or more plant EV markers. As used herein, the term "plant EV marker" refers to a component that is naturally associated with a plant, such as a plant protein, a plant nucleic acid, a plant small molecule, a plant lipid, or a combination thereof, including but not limited to any of the plant EV markers listed in the appendix. In some cases, the plant EV marker is an identification marker of a plant EV but is not an insecticide. In some cases, the plant EV marker is both an identification marker of a plant EV and an insecticide (e.g., either associated with or encapsulated by multiple PMPs or LPMPs, or not directly associated with or encapsulated by multiple PMPs or LPMPs).

[0074] As used herein, the term "plant messenger pack" or "PMP" refers to lipid structures (e.g., lipid bilayer, monolayer, multilayer structures, e.g., vesicular lipid structures) that are about 5-2000 nm in diameter (e.g., at least 5-1000 nm, at least 5-500 nm, at least 400-500 nm, at least 25-250 nm, at least 50-150 nm, or at least 70-120 nm) that are derived from a plant source or segment, part, or extract thereof (e.g., enriched, isolated, or purified) and that contain lipid or non-lipid components (e.g., peptides, nucleic acids, or small molecules) associated with the plant source or segment, part, or extract thereof, and that have been enriched, isolated, or purified from a plant, plant part, or plant cell, concentrate or isolate that has been stripped of one or more contaminants or undesirable components from the source plant. PMPs may be highly purified preparations of naturally occurring EVs. Preferably, at least 1% of contaminants or undesirable components are removed from the source plant (e.g., at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100% of one or more contaminants or undesirable components from the source plant, e.g., plant cell wall components, pectin, plant organelles (e.g., mitochondria, chloroplasts such as plastids, leucoplasts or amyloplasts, and nuclei), plant chromatin (e.g., plant chromosomes), or plant molecular aggregates (e.g., protein aggregates, protein-nucleic acid aggregates, lipoprotein aggregates, or lipid-protein structures). Preferably, the PMP is at least 30% pure (e.g., at least 40% pure, at least 50% pure, at least 60% pure, at least 70% pure, at least 80% pure, at least 90% pure, at least 99% pure, or 100% pure) compared to one or more contaminants or undesirable components from the source plant as measured by weight (w / w), spectroscopic imaging (% transmittance), or conductivity (S / m).

[0075] Lipid reconstituted PMP (LPMP) is used herein. The terms "lipid reconstituted PMP" and "LPMP" refer to PMPs derived from lipid structures (e.g., lipid bilayers, monolayers, multilayers, e.g., vesicular lipid structures) derived from plant sources (e.g., enriched, isolated or purified), and the lipid structures are disrupted (e.g., disrupted by lipid extraction) as described herein, and reconstituted or reconstituted in liquid phase (e.g., liquid phase containing cargo) using standard methods, e.g., reconstituted by methods including lipid film hydration and / or solvent injection to produce LPMPs. The methods may further include, if desired, sonication, freeze / thaw processing, and / or lipid extrusion, e.g., to reduce the size of the reconstituted LPMPs. Alternatively, LPMPs may be produced using microfluidic devices (such as NanoAssemblr® IGNITE™ microfluidic devices (Precision NanoSystems)).

[0076] As used herein, the term "cationic lipid" refers to a positively charged amphipathic molecule (e.g., a lipid or lipidoid) that contains a cationic group (e.g., a cationic head group).

[0077] As used herein, the term "ionizable lipid" refers to an amphipathic molecule (e.g., a lipid or lipidoid, e.g., a synthetic lipid or lipidoid) that contains a group (e.g., a head group) that can be ionized under a given condition (e.g., pH), e.g., dissociated to generate a species having one or more charges. By way of example, an ionizable lipid can carry a net positive charge at a selected pH, such as physiological pH (e.g., a pH of about 7.0). In this scenario, a molecule that contains a charged group (e.g., a positively charged lipid, i.e., a cationic lipid) can be considered an ionizable lipid.

[0078] Surprisingly, it has been found that ionizable lipids that contain alkyl chains with multiple unsaturation sites, for example, at least two or three unsaturation sites, are particularly useful for forming lipid particles with increased membrane fluidity.A number of ionizable lipids and related analogs suitable for use herein are described in U.S. Patent Publication Nos. 20060083780 and 20060240554, U.S. Patent Nos. 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613 and 5,785,992, and PCT Publication No. 96 / 10390, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0079] In some embodiments, ionizable lipid is ionizable so that it can dissociate depending on pH and exist in positively charged form.Ionization of ionizable lipid affects the surface charge of lipid nanoparticles that contain ionizable lipid under different pH conditions.The surface charge of lipid nanoparticles can in turn affect its plasma protein absorption, blood clearance and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)) and its ability to form endosomolytic non-bilayer structure (Hafez, IM, et al., Gene Ther 8: 1188-1196 (2001)), which can affect intracellular delivery of nucleic acid.

[0080] In some embodiments, the ionizable lipid is, for example, a lipid that is generally neutral at physiological pH (e.g., pH about 7) but can carry a net charge at acidic or basic pH. In one embodiment, the ionizable lipid is a lipid that is generally neutral at pH about 7 but can carry a net charge at acidic pH. In one embodiment, the ionizable lipid is a lipid that is generally neutral at pH about 7 but can carry a net charge at basic pH.

[0081] In some embodiments, ionizable lipids do not include cationic lipids or anionic lipids, which generally carry a net charge at physiological pH (eg, a pH of about 7).

[0082] As used herein, the term "lipidoid" refers to a molecule that has one or more characteristics of a lipid.

[0083] As used herein, the term "stable LPMP formulation" refers to a formulation that is stable over a period of time (e.g., at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days, or at least 90 days), and optionally, over a defined temperature range (e.g., at least 24°C (e.g., at a temperature of at least 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C), at least 20°C (e.g., at least 20°C, 21°C, 22°C, or 23 ... at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 300%, 350%, 40%, 45%, 50 ... %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or optionally, at a defined temperature range (e.g., at least 24° C. (e.g., at least 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., or 30° C.), at least 20° C. (e.g., at least 20° C., 21° C., 22° C., or 23° C.), at least 4° C. (e.g., at least 5° C., 10° C., or 15° C.), at least −20° C. (e.g., at least −20° C., −15° C., −10° C., −5° C., or retains at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of its activity (e.g., cell wall permeation activity and / or activity of the mRNA formulated within the LPMP) compared to the initial activity of the LPMP (e.g., upon production or formulation) at a temperature of at least 0° C. or 0° C., or at least 0° C. (e.g., at least −80° C., −70° C., −60° C., −50° C., −40° C., or −30° C.);Refers to the LPMP composition. [Brief description of the drawings]

[0084] [Figure 1] Figure 1 is a photograph and bar graph showing erythropoietin (EPO) levels in pg / mL in serum of Ai9 (Tomato Red loxP) mice orally treated with reconstituted LPMP (recPMP) derived from lemon and containing Cre recombinase mRNA. The photograph shows the fluorescence of the marker in the mice. [Diagram 2] FIG. 2 is a bar graph showing the percentage of in vivo immune cells (CD-4 cells, CD-8 cells, or B cells) transfected from the spleens of Ai9 (Tomato Red loxP) mice treated with recLPMPs derived from lemon and containing CRE recombinase mRNA, lipid nanoparticle (LNP) control, or phosphate buffered saline (PBS) control. [Figure 3A] FIG. 3A is a bar graph showing the size (nm) and polydispersity index (PDI) of lipid nanoparticles (LNPs) and reconstituted LPMPs (recPMPs) of Example 2. [Figure 3B] FIG. 3B is a bar graph showing the percent encapsulation efficiency of RNA cargo in the LNPs and recPMPs of Example 2. [Figure 4] 4 is a plot showing serum erythropoietin (EPO) expression following repeated dosing of reconstituted Lemon LPMP containing EPO mRNA in cynomolgus monkeys. Reconstituted Lemon LPMP was administered intramuscularly (IM) at a dose of 0.1 mg / kg on day 1 and 0.05 mg / kg on day 8. Concentrations of human erythropoietin (hEPO) are shown prior to dosing and 6, 12, and 24 hours after dosing. [Diagram 5]5 is a plot showing EPO expression in serum after repeated dosing of reconstituted Lemon LPMP containing EPO mRNA in cynomolgus monkeys. Reconstituted Lemon LPMP was administered IM at doses of 0.1 mg / kg on day 1, 0.05 mg / kg on day 8, and 0.05 mg / kg on day 15. Concentrations of hEPO are shown prior to dosing and 6, 12, and 24 hours after dosing. 50% of the animals were premedicated with 0.2 mg / kg (IV injection) of buprenorphine and meloxicam SR prior to doses 2 and 3. [Figure 6] 6 is a plot showing EPO expression in serum after repeated administration of reconstituted Lemon LPMP containing EPO mRNA in cynomolgus monkeys. Reconstituted Lemon LPMP was administered IM at doses of 0.1 mg / kg on day 1, 0.05 mg / kg on day 8, and 0.01 mg / kg on day 15. Concentrations of hEPO are shown prior to administration and 6, 12, and 24 hours after administration. 50% of the animals were premedicated with 0.2 mg / kg (IV injection) of buprenorphine and meloxicam SR prior to dose 2, and 90% of the animals were premedicated prior to dose 3. [Figure 7] 7 is a plot showing EPO expression in serum after repeated dosing of reconstituted Lemon LPMP containing EPO mRNA in cynomolgus monkeys. Reconstituted Lemon LPMP was administered subcutaneously (SubQ) at doses of 0.1 mg / kg on day 1, 0.05 mg / kg on day 8, and 0.01 mg / kg on day 15. Concentrations of hEPO are shown prior to dosing and 6, 12, and 24 hours after dosing. 50% of the animals were pre-medicated with 0.2 mg / kg (IV injection) of buprenorphine and meloxicam SR prior to dose 2, and 90% of the animals were pre-medicated prior to dose 3. [Figure 8]8 is a plot showing EPO expression in serum after repeated dosing of reconstituted Lemon LPMP containing EPO mRNA in cynomolgus monkeys. Reconstituted Lemon LPMP was administered SubQ at doses of 0.1 mg / kg on day 1, 0.05 mg / kg on day 8, and 0.05 mg / kg on day 15. Concentrations of hEPO are shown prior to dosing and at 6, 12, and 24 hours after dosing. 50% of the animals were premedicated with 0.2 mg / kg (IV injection) of buprenorphine and meloxicam SR prior to doses 2 and 3. [Figure 9] FIG. 9 is a bar graph showing the expression of EPO in serum following oral delivery to mice of reconstituted lemon LPMPs containing EPO mRNA. [Figure 10] FIG. 10 is a set of bar graphs showing the frequency of Tomato Red positive cells (in vivo transfected immune cells) in the parent populations of splenocytes, lung cells, and bone marrow cells (circulating immune cells and progenitor cells) in Tomato Red loxP mouse cells treated intravenously (IV) with reconstituted Lemon LPMP containing Cre recombinase. [Figure 11] FIG. 11 is a pair of bar graphs showing the percentage of GFP-positive immune cells (CD4 T cells, CD8 T cells, and B cells) in cynomolgus monkeys treated intramuscularly (IM; top panel) or subcutaneously (SubQ; bottom panel) with reconstituted Lemon LPMPs containing GFP mRNA. [Figure 12A] FIG. 12A shows tumor volumes measured on days following implantation of MC38 tumor cells in mice following administration of 5 μg of recLemon LPMP / IL-2 mRNA formulations around the first and second tumors. [Figure 12B] FIG. 12B shows tumor volumes measured on days following implantation of MC38 tumor cells in mice following all four peritumoral administrations of the recLemon LPMP / IL-2 mRNA formulation. [Figure 12C]FIG. 12C shows mouse survival by days after implantation of MC38 tumor cells following all four peritumoral administrations of the recLemon LPMP / IL-2 mRNA formulation, with reduced survival in mice receiving buffer. [Figure 13A] FIG. 13A shows IL-2 levels in serum of mice 4 hours after the fourth peritumoral administration of a 5 μg dose of the recLemon LPMP / IL-2 mRNA formulation. [Figure 13B] FIG. 13B shows the levels of IL4 in the serum of mice 4 hours after the fourth peritumoral administration of a 5 μg dose of the recLemon LPMP / IL-2 mRNA formulation. [Figure 13C] FIG. 13C shows IL5 levels in serum of mice 4 hours after the fourth peritumoral administration of a 5 μg dose of the recLemon LPMP / IL-2 mRNA formulation. [Figure 13D] FIG. 13D shows the levels of IFNγ in the serum of mice 4 hours after the fourth peritumoral administration of a 5 μg dose of the recLemon LPMP / IL-2 mRNA formulation. [Figure 13E] FIG. 13E shows the levels of TNFα in the serum of mice 4 hours after the fourth peritumoral administration of a 5 μg dose of the recLemon LPMP / IL-2 mRNA formulation. [Figure 13F] FIG. 13F shows the levels of IL6 in the serum of mice 4 hours after the fourth peritumoral administration of a 5 μg dose of the recLemon LPMP / IL-2 mRNA formulation. [Figure 13G] FIG. 13G shows the levels of CXCL1(KC) in serum of mice 4 hours after the fourth peritumoral administration of a 5 μg dose of the recLemon LPMP / IL-2 mRNA formulation. [Figure 14A] FIG. 14A shows the levels of IL6 in the serum of mice 48 hours after the second peritumoral administration of a 5 μg dose of recLemon LPMP / IL-2 mRNA formulation. [Figure 14B]FIG. 14B shows the levels of IFNγ in the serum of mice 48 hours after the second peritumoral administration of a 5 μg dose of recLemon LPMP / IL-2 mRNA formulation. [Figure 14C] FIG. 14C shows the levels of TNFα in the serum of mice 48 hours after the second peritumoral administration of a 5 μg dose of recLemon LPMP / IL-2 mRNA formulation. [Figure 15] Figure 15 shows circulating human IL-2 levels 6 hours after dosing. Intramuscular injection; recLemon LPMP / mRNA = 0.4 mg / kg (equivalent to 10 μg of IL-2); N = 9 mice per group 6 hours after dosing and N = 6 mice per group 2 days after dosing. [Figure 16A] Figure 16A shows the levels of circulating anti-tumor cytokines 6 hours after dosing. Intramuscular injection; recLemon LPMP / mRNA = 0.4 mg / kg (equivalent to 10 μg of IL-2); N = 9 mice per group 6 hours after dosing and N = 6 mice per group 2 days after dosing. [Figure 16B] Figure 16B shows the levels of circulating anti-tumor cytokines two days after dosing (Figure 16B). Intramuscular injection; recLemon LPMP / mRNA = 0.4 mg / kg (equivalent to 10 μg of IL-2); N = 9 mice per group 6 hours after dosing and N = 6 mice per group 2 days after dosing. [Figure 17] Figure 17 shows the T cell profile 6 days after dosing. The graph shows that a single dose of recLemon LPMP / IL-2 mRNA formulation was potent and induced sustained T cell effects over 6 days after dosing. Intramuscular injection; 0.4 mg / kg recLemon LPMP / mRNA (equivalent to 10 μg IL-2); N=3 mice per group. [Figure 18]Figure 18 shows IL-15 serum concentrations after dosing. The graph shows the systemic effect: a single intramuscular dose of recLemon LPMP / IL-15 mRNA formulation increased systemic protein levels up to 72 hours after dosing. Intramuscular injection; recLemon LPMP / mRNA = 0.4 mg / kg (equivalent to 10 μg of IL-15); N = 3 mice per group. [Figure 19] Figure 19 shows cytokine and T cell profiles in blood 6 days after administration. The graph shows the cytokine effect: a single intramuscular administration of recLemon LPMP / IL-15 mRNA formulation increased the frequency of IFNγ producing T cells in the blood 6 days after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.4 mg / kg (equivalent to 10 μg of IL-15); N = 3 mice per group. [Figure 20] Figure 20 shows the T cell profile in the spleen 6 days after administration. The graph shows the cellular effect: a single intramuscular administration of recLemon LPMP / IL-15 mRNA formulation resulted in an increase in the number of T cells and NK cells in the spleen 6 days after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.4 mg / kg (equivalent to 10 μg of IL-15); N = 3 mice per group. [Figure 21] Figures 21A-21B show cytokines / chemokines in blood 4, 6, 24, 48, and 72 hours after administration. Graphs show cytokine effects: A single intramuscular administration of recLemon LPMP / IL-15 mRNA formulation increased the frequency of inflammatory cytokines and chemokines (IL-6 and IP-10) in the blood of mice within 24 hours after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.4 mg / kg (equivalent to 10 μg of IL-15); N = 3 mice per group. [Figure 22A]Figure 22A shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15) in mice was able to increase IL-2Ra expression on NK cells in the spleen of mice 10 days after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.4 mg / kg (equivalent to 10 μg of IL-15); N = 3 mice per group. [Figure 22B] Figure 22B shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15) in mice was able to increase IL-2Ra expression on IFNγ+ NK cells in the spleen of mice 10 days after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.4 mg / kg (equivalent to 10 μg of IL-15); N = 3 mice per group. [Figure 23] Figure 23 shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15) in NHPs was able to increase IL-2Ra (CD25) expression on NK cells and T cells in the blood of NHPs 24 hours after administration (day 1). Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 24A] Figure 24 shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15) was able to increase granzyme B / perforin and IFNγ expression in T cells (Figure 24A) and NK cells (Figure 24B) in the blood of NHPs 24 hours after administration (day 1). Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 24B]Figure 24 shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15) was able to increase granzyme B / perforin and IFNγ expression in T cells (Figure 24A) and NK cells (Figure 24B) in the blood of NHPs 24 hours after administration (day 1). Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Diagram 25] Figure 25 shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15 and IL-2) resulted in proliferation of T and NK cells in the blood of NHPs 4 days after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 26A] Figure 26 shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15 and IL-2) resulted in increased levels of granzyme B / perforin and IFNγ expression in T cells (Figure 26A) and NK cells (Figure 26B) in the blood of NHPs 4 days after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 26B] Figure 26 shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15 and IL-2) resulted in increased levels of granzyme B / perforin and IFNγ expression in T cells (Figure 26A) and NK cells (Figure 26B) in the blood of NHPs 4 days after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 27]Figure 27 shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15) resulted in increased IL-2Ra (CD25) expression on NK cells and T cells in the blood of NHPs 4 days after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 28A] Figure 28A shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15) resulted in increased levels of granzyme B / perforin and IFNγ expression in T cells in the blood of NHPs 8 days after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 28B] Figures 28A-B show that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15) resulted in increased levels of granzyme B / perforin and IFNγ expression in NK cells in the blood of NHPs at day 8 post-administration. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 29] Figure 29 shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15 and IL-2) resulted in increased levels of IL-2Ra (CD25) on NK cells in the blood of NHPs 8 days after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Diagram 30]Figure 30 shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15) resulted in proliferation of T and NK cells in the blood of NHPs 8 days after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Diagram 31] Figure 31 shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15 and IL-2) resulted in proliferation of T and NK cells in the blood of NHPs 13 days after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Diagram 32] Figure 32 shows that a single intramuscular administration of recLemon LPMP / mRNA formulation (IL-15) resulted in increased levels of IL-2Ra (CD25) on T cells and NK cells in the blood of NHPs 13 days after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 33A] 33A-B are line graphs showing blood cytokine levels of IL-15 measured 4 hours, 6 hours, 24 hours, 4 days (96 hours), 8 days (192 hours), and 13 days (312 hours) after a single intramuscular administration of recLemon LPMP / mRNA formulation. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 33B]Figure 33B is a line graph showing blood cytokine levels of IL-2 measured 4 hours, 6 hours, 24 hours, 4 days (96 hours), 8 days (192 hours), and 13 days (312 hours) after a single intramuscular administration of recLemon LPMP / mRNA formulation. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Diagram 34] Figure 34 is a line graph showing the proliferation of CD56+ NK cells in the blood of NHPs over 13 days post-treatment following a single intramuscular dose of recLemon LPMP / mRNA formulation (IL-15 and IL-2) compared to naive controls. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 35A] Figure 35A is a line graph showing blood cytokine levels of inflammatory cytokines, including IP-10, measured 4 hours, 6 hours, 24 hours, 4 days (96 hours), 8 days (192 hours), and 13 days (312 hours) after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 35B] Figure 35B is a line graph showing blood cytokine levels of inflammatory cytokines, including IFNγ, measured 4 hours, 6 hours, 24 hours, 4 days (96 hours), 8 days (192 hours), and 13 days (312 hours) after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 35C]Figure 35C is a line graph showing blood cytokine levels of inflammatory cytokines, including IL-6, measured 4 hours, 6 hours, 24 hours, 4 days (96 hours), 8 days (192 hours), and 13 days (312 hours) after administration. Intramuscular injection; recLemon LPMP / mRNA = 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; 0.006 mg / kg IL-2; N = 3 NHPs per group except for control (N = 1). [Figure 36A] Figure 36A is a comparison of doses and animal models between mice as measured by systemic IL-15 production 6 hours after administration. Both bar graphs show comparable IL-15 production between various animal models based on the dose given. Intramuscular injection; recLemon LPMP / mRNA = 0.5 mg / kg IL-15; 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; N = 3 NHPs or mice per group. [Figure 36B] Figure 36B is a comparison of doses and animal models between non-human primates through a measure of systemic IL-15 production 6 hours after administration. Both bar graphs show comparable IL-15 production between various animal models based on the dose given. Intramuscular injection; recLemon LPMP / mRNA = 0.5 mg / kg IL-15; 0.02 mg / kg IL-15; 0.006 mg / kg IL-15; N = 3 NHPs or mice per group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] Featured herein are mRNA therapeutic compositions (e.g., RNA, e.g., mRNA nucleic acid cancer vaccines) that can safely instruct the body's cellular machinery to produce almost any cancer protein of interest or fragments thereof. The mRNA therapeutic compositions can be used, for example, to induce a balanced immune response against cancer, including both cellular and humoral immunity, without risking the possibility of insertional mutagenesis. These mRNA therapeutic compositions include one or more polynucleotides (e.g., RNA, such as messenger RNA (mRNA)) that encode one or more antigenic (e.g., tumor antigenic) polypeptides or signaling polypeptides, formulated in lipid-reconstituted plant messenger packs (LPMPs) that include natural lipids and ionizable lipids. PMPs are lipid assemblies that are generated in whole or in part from plant extracellular vesicles (EVs), or segments, parts, or extracts thereof. LPMPs are PMPs that are derived from lipid structures, which are disrupted and reconstituted or reconstituted in a liquid phase.

[0086] The present disclosure also includes a method of making an mRNA therapeutic composition comprising reconstituting a membrane comprising purified PMP lipids in the presence of an ionizable lipid to produce an LPMP comprising an ionizable lipid, and loading the LPMP with one or more polynucleotides encoding one or more antigenic (e.g., tumor antigenic) or signaling polypeptides.

[0087] Lipid Reconstituted Plant Messenger Pack (LPMP) Plant Messenger Pack (PMP) PMPs are lipid (e.g., lipid bilayer, monolayer, or multilayer) structures that contain plant EVs, or segments, parts, or extracts thereof (e.g., lipid extracts). Plant EVs refer to enclosed lipid bilayer structures that occur naturally in plants and are approximately 5-2000 nm in diameter. Plant EVs can be derived from various plant biosynthetic pathways. In nature, plant EVs can be found in intracellular and extracellular compartments of plants, such as the plant apoplast, a compartment located outside the cell membrane and formed by the continuous cell wall and extracellular space. Alternatively, PMPs can be concentrated plant EVs found in cell culture media upon secretion from plant cells. Plant EVs can be isolated from plants, thereby resulting in PMPs by various methods further described herein. Additionally, PMPs can optionally include therapeutic agents that can be introduced in vivo or in vitro.

[0088] PMPs may include plant EVs, or segments, parts, or extracts thereof. Optionally, PMPs may also include exogenous lipids (e.g., sterols (e.g., cholesterol or sitosterol), ionizable lipids, and / or PEGylated lipids) in addition to lipids derived from plant EVs. In some embodiments, plant EVs are about 5-1000 nm in diameter. For example, PMPs may be about 5-50 nm, about 50-100 nm, about 100-150 nm, about 150-200 nm, about 200-250 nm, about 250-300 nm, about 300-350 nm, about 350-400 nm, about 400-450 nm, about 450-500 nm, about 500-550 nm, about 550-600 nm, about 600-650 nm, about 650-700 nm, about The plant EVs may have an average diameter of 700-750 nm, about 750-800 nm, about 800-850 nm, about 850-900 nm, about 900-950 nm, about 950-1000 nm, about 1000-1250 nm, about 1250-1500 nm, about 1500-1750 nm, or about 1750-2000 nm, or a segment, part, or extract thereof. In some cases, the PMP comprises a plant EV, or a segment, part, or extract thereof, having an average diameter of about 5-950 nm, about 5-900 nm, about 5-850 nm, about 5-800 nm, about 5-750 nm, about 5-700 nm, about 5-650 nm, about 5-600 nm, about 5-550 nm, about 5-500 nm, about 5-450 nm, about 5-400 nm, about 5-350 nm, about 5-300 nm, about 5-250 nm, about 5-200 nm, about 5-150 nm, about 5-100 nm, about 5-50 nm, or about 5-25 nm. In certain cases, the plant EV, or a segment, part, or extract thereof, has an average diameter of about 50-200 nm. In certain cases, the plant EV, or a segment, part, or extract thereof, has an average diameter of about 50-300 nm. In certain cases, the plant EVs, or segments, parts, or extracts thereof, have an average diameter of about 200-500 nm, In certain cases, the plant EVs, or segments, parts, or extracts thereof, have an average diameter of about 30-150 nm.

[0089] In some cases, the PMP may comprise a plant EV, or a segment, portion, or extract thereof, having an average diameter of at least 5 nm, at least 50 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 350 nm, at least 400 nm, at least 450 nm, at least 500 nm, at least 550 nm, at least 600 nm, at least 650 nm, at least 700 nm, at least 750 nm, at least 800 nm, at least 850 nm, at least 900 nm, at least 950 nm, or at least 1000 nm. In some cases, PMPs include plant EVs, or segments, parts, or extracts thereof, having an average diameter of less than 1000 nm, less than 950 nm, less than 900 nm, less than 850 nm, less than 800 nm, less than 750 nm, less than 700 nm, less than 650 nm, less than 600 nm, less than 550 nm, less than 500 nm, less than 450 nm, less than 400 nm, less than 350 nm, less than 300 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, or less than 50 nm. Various methods standard in the art (e.g., dynamic light scattering) can be used to measure the particle diameter of plant EVs, or segments, parts, or extracts thereof.

[0090] In one case, the PMP is 77 nm 2 ~3.2×10 6 nm 2 (For example, 77 to 100 nm 2 , 100~1000nm 2 , 1000~1×10 4 nm 2 , 1×10 4 ~1×10 5 nm 2 , 1×10 5 ~1×10 6 nm 2 , or 1 × 10 6 ~3.2×10 6 nm 2 In some cases, the PMP may comprise a plant EV, or a segment, part, or extract thereof, having an average surface area of ​​65 nm 3~5.3×10 8 nm 3 (For example, 65 to 100 nm 3 , 100~1000nm 3 , 1000~1×10 4 nm 3 , 1×10 4 ~1×10 5 nm 3 , 1×10 5 ~1×10 6 nm 3 , 1×10 6 ~1×10 7 nm 3 , 1×10 7 ~1×10 8 nm 3 , 1×10 8 ~5.3×10 8 nm 3 In some cases, the PMP may comprise a plant EV, or a segment, part, or extract thereof, having an average volume of at least 77 nm 2 (e.g., at least 77 nm 2 , at least 100 nm 2 , at least 1000 nm 2 , at least 1 × 10 4 nm 2 , at least 1 × 10 5 nm 2 , at least 1 × 10 6 nm 2 , or at least 2 × 10 6 nm 2 In some cases, the PMP may comprise a plant EV, or a segment, part, or extract thereof, having an average surface area of ​​at least 65 nm 3 (e.g., at least 65 nm 3 , at least 100 nm 3 , at least 1000 nm 3 , at least 1 × 10 4 nm 3 , at least 1 × 10 5 nm 3 , at least 1 × 10 6 nm 3 , at least 1 × 10 7 nm 3, at least 1 × 10 8 nm 3 , at least 2 × 10 8 nm 3 , at least 3 × 10 8 nm 3 , at least 4 × 10 8 nm 3 , or at least 5 × 10 8 nm 3 The plant EVs, or segments, parts, or extracts thereof, may comprise a plant EV having an average volume of about 1000 mg / kg or more.

[0091] In some cases, PMPs may have the same size as plant EVs or segments, extracts, or portions thereof. Alternatively, PMPs may have a different size than the initial plant EVs from which they are generated. For example, PMPs may have a diameter of about 5-2000 nm in diameter. For example, PMPs may have a diameter of about 5-50 nm, about 50-100 nm, about 100-150 nm, about 150-200 nm, about 200-250 nm, about 250-300 nm, about 300-350 nm, about 350-400 nm, about 400-450 nm, about 450-500 nm, about 500-550 nm, about 550-600 nm, about 600-650 nm, about 650 The nanoparticles may have an average diameter of about 700 nm, about 700 to 750 nm, about 750 to 800 nm, about 800 to 850 nm, about 850 to 900 nm, about 900 to 950 nm, about 950 to 1000 nm, about 1000 to 1200 nm, about 1200 to 1400 nm, about 1400 to 1600 nm, about 1600 to 1800 nm, or about 1800 to 2000 nm. In some cases, the PMP may have an average diameter of at least 5 nm, at least 50 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 350 nm, at least 400 nm, at least 450 nm, at least 500 nm, at least 550 nm, at least 600 nm, at least 650 nm, at least 700 nm, at least 750 nm, at least 800 nm, at least 850 nm, at least 900 nm, at least 950 nm, at least 1000 nm, at least 1200 nm, at least 1400 nm, at least 1600 nm, at least 1800 nm, or about 2000 nm. The particle diameter of the PMP can be measured using various methods (e.g., dynamic light scattering) that are standard in the art. In some cases, the size of the PMP is determined after loading with a therapeutic agent or other modifications to the PMP.

[0092] In one case, the PMP is 77 nm 2 ~1.3×10 7 nm 2 (For example, 77 to 100 nm 2 , 100~1000nm 2 , 1000~1×104 nm 2 , 1×10 4 ~1×10 5 nm 2 , 1×10 5 ~1×10 6 nm 2 , or 1 × 10 6 ~1.3×10 7 nm 2 In some cases, the PMP may have an average surface area of ​​65 nm 3 ~4.2×10 9 nm 3 (For example, 65 to 100 nm 3 , 100~1000nm 3 , 1000~1×10 4 nm 3 , 1×10 4 ~1×10 5 nm 3 , 1×10 5 ~1×10 6 nm 3 , 1×10 6 ~1×10 7 nm 3 , 1×10 7 ~1×10 8 nm 3 , 1×10 8 ~1×10 9 nm 3 , or 1 × 10 9 ~4.2×10 9 nm 3 In some cases, the PMP may have an average volume of at least 77 nm 2 (e.g., at least 77 nm 2 , at least 100 nm 2 , at least 1000 nm 2 , at least 1 × 10 4 nm 2 , at least 1 × 10 5 nm 2 , at least 1 × 10 6 nm 2 , or at least 1 × 10 7 nm 2 In some cases, the PMP has an average surface area of ​​at least 65 nm 3 (e.g., at least 65 nm3 , at least 100 nm 3 , at least 1000 nm 3 , at least 1 × 10 4 nm 3 , at least 1 × 10 5 nm 3 , at least 1 × 10 6 nm 3 , at least 1 × 10 7 nm 3 , at least 1 × 10 8 nm 3 , at least 1 × 10 9 nm 3 , at least 2 × 10 9 nm 3 , at least 3 × 10 9 nm 3 , or at least 4 × 10 9 nm 3 ) average volume.

[0093] In some cases, the PMP may include an intact plant EV. Alternatively, the PMP may include a segment, portion, or extract of the total surface area of ​​a vesicle of a plant EV (e.g., a segment, portion, or extract that includes less than 100% (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 10%, 5%, or 1%) of the total surface area of ​​the vesicle). The segment, portion, or extract may be of any shape, such as a circumferential segment, a spherical segment (e.g., a hemisphere), a curved segment, a straight segment, or a flat segment. When the segment is a spherical segment of a vesicle, the spherical segment may represent one resulting from the division of a spherical vesicle along a pair of parallel lines or one resulting from the division of a spherical vesicle along a pair of non-parallel lines. Thus, the PMPs may contain intact plant EVs, plant EV segments, parts, or extracts, or a mixture of intact plant EVs and plant EV segments. Those skilled in the art will understand that the ratio of intact plant EVs to segmented plant EVs will depend on the particular isolation method used. For example, grinding or blending a plant, or parts thereof, may produce a PMP that contains a higher percentage of plant EV segments, parts, or extracts than non-destructive extraction methods such as vacuum infiltration.

[0094] When the PMPs comprise segments, parts, or extracts of plant EVs, the EV segments, parts, or extracts have an average surface area that is smaller than the average surface area of ​​intact vesicles (e.g., 77 nm 2 , 100 nm 2 , 1000nm 2 , 1×10 4 nm 2 , 1×10 5 nm 2 , 1×10 6 nm 2 , or 3.2 × 10 6 nm 2 In some cases, the EV segments, portions, or extracts may have an average surface area of ​​less than 70 nm. 2 , 60nm 2 , 50 nm2 , 40nm 2 , 30 nm 2 , 20nm 2 , or 10 nm 2 In some cases, the PMPs have an average volume that is smaller than the average volume of an intact vesicle (e.g., 65 nm 3 , 100 nm 3 , 1000nm 3 , 1×10 4 nm 3 , 1×10 5 nm 3 , 1×10 6 nm 3 , 1×10 7 nm 3 , 1×10 8 nm 3 , or 5.3 × 10 8 nm 3 The plant EVs, or segments, parts or extracts thereof, may comprise plant EVs having an average volume of less than 100 μg / ml.

[0095] Where a PMP comprises an extract of a plant EV, for example where a PMP comprises lipids extracted from a plant EV (e.g., with chloroform), the PMP may comprise at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% of lipids extracted from the plant EV (e.g., with chloroform). A plurality of PMPs may comprise plant EV segments and / or plant EV extracted lipids or mixtures thereof.

[0096] Generating PMP PMPs may be produced from plant EVs, or segments, parts or extracts thereof (e.g., lipid extracts) that occur naturally in a plant, or part thereof, including plant tissues or plant cells. An exemplary method for producing PMPs includes (a) providing an initial sample from a plant or part thereof, the plant or part thereof including EVs, and (b) isolating a crude PMP fraction from the initial sample, the crude PMP fraction having a reduced level of at least one contaminant or undesirable component from the plant or part thereof compared to the level in the initial sample. The method may further include an additional step (c) including purifying the crude PMP fraction, thereby producing a plurality of pure PMPs, the plurality of pure PMPs having a reduced level of at least one contaminant or undesirable component from the plant or part thereof compared to the level in the crude EV fraction. Each production step is discussed in further detail below. Exemplary methods for isolating and purifying PMPs can be found, for example, in Rutter and Innes, Plant Physiol. 173(1):728-741, 2017; Rutter et al, Bio. Protoc. 7(17): e2533, 2017; Regente et al, J of Exp. Biol. 68(20):5485-5496, 2017; Mu et al, Mol. Nutr. Food Res., 58, 1561-1573, 2014, and Regente et al, FEBS Letters . 583: 3363-3366, 2009, each of which is incorporated herein by reference.

[0097] In some cases, the plurality of PMPs can be prepared by (a) providing an initial sample from a plant or part thereof, the plant or part thereof including EVs; (b) isolating a crude PMP fraction from the initial sample, the crude PMP fraction comprising a reduced level (e.g., a level that is at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100% reduced) of at least one contaminant or undesirable component from the plant or part thereof compared to the level in the initial sample. and (c) purifying the crude PMP fraction, thereby producing a plurality of pure PMPs, the plurality of pure PMPs having a reduced level (e.g., at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100% reduced) of at least one contaminant or undesirable component from the plant or part thereof as compared to the level in the crude EV fraction.

[0098] PMP can comprise plant EVs, or segments, parts, or extracts thereof, that are produced from various plants.PMP can be produced from any genus of plant (vascular or non-vascular), including but not limited to angiosperms (monocotyledonous and dicotyledonous), gymnosperms, ferns, Selaginellaceae, horsetail, archaic scoparium, lycopodidae, algae (e.g., unicellular or multicellular, e.g., archaic plastids), or mosses.In certain cases, PMP can be produced using vascular plants, e.g., monocotyledonous or dicotyledonous plants or gymnosperms. For example, PMPs may be detected in alfalfa, apple, Arabidopsis, banana, barley, canola, castor seed, chicory, chrysanthemum, clover, cocoa, coffee, cotton, cottonseed, corn, crambe, cranberry, cucumber, dendrobium, yam, eucalyptus, fescue, flax, gladiolus, lilac, linseed, millet, muskmelon, mustard, oats, oil palm, rapeseed, papaya, peanuts, pineapple, ornamental plants, Phaseolus, potato, rapeseed, rice, rye, ryegrass, safflower, sesame, sorghum, soybean, sugar beet, sugarcane, sunflower, strawberry, tobacco, tomato, turfgrass, wheat Or plant crops such as lettuce, celery, broccoli, cauliflower, cucurbits, fruit and nut trees such as apple, pear, peach, orange, grapefruit, lemon, lime, almond, pecan, walnut, hazel, vines such as grapes, kiwi, hops, shrub fruit trees such as raspberry, blackberry, gooseberry, and forest trees such as bramble, ash, pine, fir, maple, oak, chestnut, poplar, along with alfalfa, canola, castor seed, corn, cotton, crambe, flax, linseed, mustard, oil palm, rapeseed, peanut, potato, rice, safflower, sesame, soybean, sugar beet, sunflower, tobacco, tomato, or wheat.

[0099] PMPs may be produced using whole plants (e.g., whole rosettes or whole seedlings) or, alternatively, from one or more plant parts (e.g., leaves, seeds, roots, fruits, vegetables, pollen, phloem sap, or xylem sap). For example, PMPs may be produced using shoot vegetative organs / structures (e.g., leaves, stems, or tubers), roots, flowers and floral organs / structures (e.g., pollen, bracts, bracts, sepals, petals, stamens, carpels, anthers, or ovules), seeds (including embryos, endosperm, or seed coats), fruits (mature ovaries), sap (e.g., phloem or xylem sap), plant tissues (e.g., vascular tissue, crushed tissue, tumor tissue, etc.), and cells (e.g., single cells, protists, embryos, callus tissue, guard cells, egg cells, etc.), or progeny thereof. For example, the isolating step may include (a) providing a plant, or a part thereof. In some examples, the plant part is an Arabidopsis leaf. The plant may be at any stage of development. For example, PMPs can be produced using seedlings, such as 1-week-old, 2-week-old, 3-week-old, 4-week-old, 5-week-old, 6-week-old, 7-week-old, or 8-week-old seedlings (e.g., Arabidopsis seedlings). Other exemplary PMPs can include PMPs produced using roots (e.g., ginger root), fruit juice (e.g., grapefruit juice), vegetables (e.g., broccoli), pollen (e.g., olive pollen), phloem sap (e.g., Arabidopsis phloem sap), or xylem sap (e.g., tomato plant xylem sap).

[0100] In some embodiments, the PMPs are produced from algae or lemons.

[0101] PMPs can be produced using plants, or parts thereof, by a variety of methods. Any method that allows the release of the EV-containing apoplastic fraction of the plant, or the extracellular fraction (e.g., cell culture medium) that contains PMPs, including otherwise secreted EVs, is suitable for the present method. EVs can be isolated from the plant or parts of the plant by either disruptive (e.g., grinding or mixing the plant, or any part of the plant) or non-disruptive (washing or vacuum infiltration of the plant or any part of the plant) methods. For example, the plant, or parts thereof, can be vacuum infiltrated, ground, mixed, or a combination thereof, to isolate EVs from the plant or part of the plant, thereby producing PMPs. For example, the isolation step can include vacuum infiltrating the plant (e.g., vesicle isolation buffer) to release and collect the apoplastic fraction. Alternatively, the isolation step can include grinding or mixing the plant to release EVs, thereby producing PMPs.

[0102] When isolating plant EVs and thereby generating PMPs, the PMPs can be separated or collected into a crude PMP fraction (e.g., an apoplastic fraction). For example, the separation step can include separating a plurality of PMPs into a crude PMP fraction using centrifugation (e.g., differential centrifugation or ultracentrifugation) and / or filtration to separate the plant PMP-containing fraction from larger contaminants, including plant tissue debris or plant cells. Thus, the crude PMP fraction will have a reduced number of large contaminants, including plant tissue debris or plant cells, compared to the initial sample from the plant or plant part. Depending on the method used, the crude PMP fraction may further include a reduced level of plant cell organelles (e.g., nuclei, mitochondria, or chloroplasts) compared to the initial sample from the plant or plant part.

[0103] In some cases, the isolation step may include separating the PMPs into a crude PMP fraction using centrifugation (e.g., differential centrifugation or ultracentrifugation) and / or filtration to separate the PMP-containing fraction from the plant cells or cell debris. In such cases, the crude PMP fraction will have a reduced number of plant cells or cell debris compared to the initial sample from the source plant or plant part.

[0104] The crude PMP fraction can be further purified by additional purification methods to produce a plurality of pure PMPs. For example, the crude PMP fraction can be separated from other plant components by ultracentrifugation, for example, using a density gradient (iodixanol or sucrose), and / or using other approaches to remove aggregated components (e.g., precipitation or size exclusion chromatography). The resulting pure PMP may have a reduced level of contaminants or other undesirable components from the source plant (e.g., one or more non-PMP components such as protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures, nuclei, cell wall components, organelles, or combinations thereof) compared to one or more fractions produced during a previous separation step, or compared to a pre-established threshold level, for example, a commercially available release specification. For example, a pure PMP may have a reduced level (e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%, or about 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold) of a plant organelle or cell wall component as compared to the level in the initial sample. In some cases, a pure PMP is substantially free (e.g., has undetectable levels) of one or more non-PMP components, such as protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures), nuclei, cell wall components, organelles, or combinations thereof. A PMP may be, for example, 1×10 9 , 5x10 9 , 1x10 10 , 5x1010 , 5x10 10 , 1x10 11 , 2x10 11 , 3x10 11 , 4x10 11 , 5x10 11 , 6x10 11 , 7x10 11 , 8x10 11 , 9x10 11 , 1x10 12 , 2x10 12 , 3x10 12 , 4x10 12 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 , 9x10 12 , 1×10 13 , or 1 × 10 13 Concentrations of more than PMP / mL may be used.

[0105] For example, protein aggregates may be removed from the PMPs. For example, the PMPs may be taken through a range of pH (e.g., measured using a pH probe) to precipitate protein aggregates in the solution. The pH may be adjusted, for example, to pH 3, pH 5, pH 7, pH 9, or pH 11, for example, by the addition of sodium hydroxide or hydrochloric acid. Once the solution is at the specified pH, it may be filtered to remove particles. Alternatively, the PMPs may be aggregated using the addition of a charged polymer, such as Polymin-P or Plastol 2640. Briefly, Polymin-P or Plastol 2640 is added to the solution and mixed with an impeller. The solution may then be filtered to remove particles. Alternatively, the aggregates may be solubilized by increasing the salt concentration. For example, NaCl may be added to the PMPs, for example, until 1 mol / L. The solution may then be filtered to isolate the PMPs. Alternatively, the aggregates may be solubilized by increasing the temperature. For example, PMPs can be heated under mixing for 5 minutes until the solution reaches a uniform temperature, for example, 50°C. The PMP mixture can then be filtered to isolate PMPs. Alternatively, soluble contaminants from the PMP solution can be separated by a size-exclusion chromatography column according to standard procedures, with PMPs eluting in the first fraction, while proteins and ribonucleoproteins, as well as some lipoproteins, are eluted later. The efficiency of protein aggregate removal can be determined by measuring and comparing protein concentrations before and after removal of protein aggregates via BCA / Bradford protein quantification.

[0106] Any of the production methods described herein can be supplemented with any quantitative or qualitative methods known in the art to characterize or identify PMPs at any step of the production process. PMPs can be characterized by various analytical methods to estimate PMP yield, PMP concentration, PMP purity, PMP composition, or PMP size. PMPs can be evaluated by a number of methods known in the art that allow PMPs to be visualized, quantified, or qualitatively characterized (e.g., composition identification), such as microscopy (e.g., transmission electron microscopy), dynamic light scattering, nanoparticle tracking, spectroscopy (e.g., Fourier transform infrared analysis), or mass spectrometry (protein and lipid analysis). In certain cases, methods (e.g., mass spectrometry) can be used to identify plant EV markers present on PMPs, such as the markers disclosed in the appendix. PMPs can be further labeled or stained to aid in the analysis and characterization of PMP fractions. For example, PMPs can be stained using 3,3'-dihexyloxacarbocyanine iodide (DIOC6), a fluorescent lipophilic dye, PKH67 (Sigma Aldrich), Alexa Fluor® 488 (Thermo Fisher Scientific), or DyLight™ 800 (Thermo Fisher). In the absence of sophisticated forms of nanoparticle tracking, this relatively simple approach can be used to quantify total membrane content and indirectly measure the concentration of PMPs (Rutter and Innes, Plant Physiol. 173(1): 728-741, 2017; Rutter et al, Bio. Protoc. 7(17): e2533, 2017). For more precise measurements and to assess the size distribution of PMPs, nanoparticle tracking can be used.

[0107] During the production process, the PMPs can be optionally prepared such that the PMPs are at an increased concentration (e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%, or about 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or greater than 100-fold) compared to the EV levels in the control or initial sample. The PMPs can comprise from about 0.1% to about 100%, such as any one of about 0.01% to about 100%, about 1% to about 99.9%, about 0.1% to about 10%, about 1% to about 25%, about 10% to about 50%, about 50% to about 99%, or about 75% to about 100% of the PMP composition. In some cases, the composition comprises at least 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more PMP, for example, as measured by weight / volume, percentage of PMP protein composition, and / or percentage of lipid composition (e.g., by measuring fluorescently labeled lipid). In some cases, the concentrated agent is used as a commercial product, for example, the end user may use a diluted agent having a substantially lower concentration of active ingredient. In some embodiments, the composition is formulated as an agricultural concentrate, for example, an ultra-low volume concentrate.

[0108] Lipid Reconstituted Plant Messenger Pack (LPMP) Lipid reconstituted PMP (LPMP) is used herein. LPMP refers to PMP derived from lipid structures (e.g., lipid bilayer, monolayer, multilayer structures, e.g., vesicular lipid structures) derived from plant sources (e.g., enriched, isolated or purified), and the lipid structures are disrupted (e.g., disrupted by lipid extraction) as described herein, and reconstituted or reconstituted in liquid phase (e.g., liquid phase containing cargo) using standard methods, e.g., reconstituted by methods including lipid film hydration and / or solvent injection to generate LPMP. The method may further include, if desired, ultrasonication, freeze / thaw processing, and / or lipid extrusion, e.g., to reduce the size of the reconstituted LPMP. Alternatively, LPMP may be generated using a microfluidic device (such as NanoAssemblr® IGNITE™ microfluidic device (Precision NanoSystems)).

[0109] In some embodiments, LPMPs are produced by a process comprising: (a) providing a plurality of purified PMPs (e.g., PMPs purified as described in Section IA herein); (b) treating the plurality of PMPs to produce a lipid membrane; (c) reconstituting the lipid membrane with an organic solvent or combination of solvents, thereby producing a lipid solution; and (d) treating the lipid solution of step (c) in a microfluidic device comprising an aqueous phase, thereby producing LPMPs.

[0110] In some cases, processing the plurality of PMPs to produce a lipid membrane includes extracting lipids from the plurality of PMPs, for example, extracting lipids using the Bligh-Dyer method (Bligh and Dyer, J Biolchem ​​Physiol, 37: 911-917, 1959). The extracted lipids may be provided as a stock solution, for example, a solution in chloroform:methanol. Producing a lipid membrane may include, for example, evaporation of the solvent by a stream of inert gas (e.g., nitrogen).

[0111] Natural lipids LPMPs may contain 10%-100% lipids derived from lipid structures derived from plant sources (e.g., lemon or algae), for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% lipids derived from lipid structures derived from plant sources. LPMPs may contain all or a fraction of the lipid species present in lipid structures derived from plant sources (e.g., lemon or algae), for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% lipid species present in lipid structures derived from plant sources. LPMPs may contain none, a fraction, or all of the protein species present in lipid structures derived from plant sources (e.g., lemons or algae), for example, 0%, less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, less than 100%, or 100% of the protein species present in lipid structures derived from plant sources (e.g., lemons or algae). In some cases, the lipid bilayer of an LPMP does not contain protein. In some cases, the lipid structures of an LPMP contain reduced amounts of protein compared to lipid structures derived from plant sources.

[0112] In some embodiments, the natural lipids of the LPMPs are extracted from lemons or algae.

[0113] exogenous lipids LPMPs may be modified to contain a heterologous agent (e.g., a cell penetrating agent) that can increase cellular uptake (e.g., animal cell uptake (e.g., mammalian cell uptake, e.g., human cell uptake), plant cell uptake, bacterial cell uptake, or fungal cell uptake) compared to unmodified LPMPs. For example, the modified LPMPs may include a plant cell penetrating agent, such as an ionizable lipid (e.g., may be loaded, e.g., encapsulated or conjugated, with a plant cell penetrating agent) or may be formulated with a plant cell penetrating agent (e.g., may be suspended or resuspended in a solution containing a plant cell penetrating agent). Each of the modified LPMPs may include at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionizable lipids.

[0114] LPMPs may include one or more exogenous lipids, e.g., lipids that are exogenous to the plant (e.g., derived from a source that is not the plant or part of the plant from which the LPMP is produced). The lipid composition of LPMPs may include 0%, less than 1%, or at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% exogenous lipids. In some examples, exogenous lipids (e.g., ionizable lipids) are added in an amount of 25% or 40% (w / w) of the total lipids in the preparation. In some examples, exogenous lipids are added to the preparation before step (b), e.g., mixed with the PMP lipids extracted before step (b).

[0115] Exemplary exogenous lipids include ionizable lipids.

[0116] Exogenous lipids may also include cationic lipids.

[0117] In some cases, the exogenous lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), DLin-MC3-DMA (MC3), dioleoyl-3-trimethylammonium propane (DODAP), DC-cholesterol, DOTAP, ethyl PC, GL67, DLin-KC2-DMA (KC2), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, lipid 5 (Moderna), cationic sulfonamide amino lipids, amphipathic zwitterionic amino lipids, DODAC, DOBAQ, YSK05, DOBAT, The ionizable or cationic lipid may be selected from DOBAQ, DOPAT, DOMAPQ, DOAAQ, DMAP-BLP, DLinDMA, DODMA, DOTMA, DSDMA, DOSPA, DODAC, DOBAQ, DMRIE, DOTAP-cholesterol, GL67A, and 98N12-5, or combinations thereof.

[0118] In some embodiments, the exogenous lipid may be an ionizable lipid or a cationic lipid selected from C12-200, MC3, DODAP, DC-cholesterol, DOTAP, ethyl PC, GL67, KC2, MD1, OF2, EPC, ZA3-Ep10, TT3, LPO1, 5A2-SC8, lipid 5 (Moderna), cationic sulfonamide amino lipid, and amphipathic zwitterionic amino lipid, or a combination thereof. In some embodiments, the ionizable lipid is selected from C12-200, MC3, DODAP, and DC-cholesterol, or a combination thereof. In some cases, the ionizable lipid is an ionizable lipid. In some embodiments, the ionizable lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200) or (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA (MC3). In some cases, the exogenous lipid is a cationic lipid. In some embodiments, the cationic lipid is DC-cholesterol or dioleoyl-3-trimethylammonium propane (DOTAP).

[0119] In some cases, the LPMP comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% ionizable lipids.

[0120] In some cases, the LPMP comprises a molar ratio of at least 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, or greater than 90% ionizable lipid, e.g., 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% ionizable lipid, e.g., about 30%-75% ionizable lipid (e.g., about 30%-75% ionizable lipid). In some embodiments, the LPMP comprises 25% C12-200. In some embodiments, the LPMP comprises a molar ratio of 35% C12-200. In some embodiments, the LPMP comprises a molar ratio of 50% C12-200. In some embodiments, the LPMP comprises 40% MC3. In some embodiments, the LPMP comprises a molar ratio of 50% C12-200. In some embodiments, the LPMP comprises 20% or 40% DC-cholesterol. In some embodiments, the LPMP comprises 25% or 40% DOTAP.

[0121] The agent can increase the uptake of the LPMP as a whole, or can increase the uptake of a portion or component of the LPMP carried by the LPMP (e.g., an mRNA therapeutic agent). The degree to which cellular uptake is increased can vary depending on the plant or part of the plant to which the composition is delivered, the LPMP formulation, and other modifications made to the LPMP, for example, a modified LPMP can have at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increased cellular uptake (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake) compared to an unmodified LPMP. In some cases, the increased cellular uptake is at least 2-fold, 4-fold, 5-fold, 10-fold, 100-fold, or 1000-fold increased cellular uptake compared to an unmodified LPMP.

[0122] In some embodiments, LPMPs modified with ionizable lipids encapsulate negatively charged polynucleotides more efficiently than LPMPs that are not modified with ionizable lipids. In some aspects, LPMPs modified with ionizable lipids have altered biodistribution compared to LPMPs that are not modified with ionizable lipids. In some aspects, LPMPs modified with ionizable lipids have altered (e.g., increased) fusion with the endosomal membrane of target cells compared to LPMPs that are not modified with ionizable lipids.

[0123] Ionizable lipids In some embodiments, the ionizable lipid has the following characteristics: (i) at least two ionizable amines (e.g., at least two, at least three, at least four, at least five, at least six, or more than six ionizable amines, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 ionizable amines); (ii) at least three lipid tails (e.g., at least three, at least four, at least five, at least six, or more than six lipid tails, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 lipid tails) (each of the lipid tails is independently at least 6 carbon atoms in length (e.g., at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or more than 18 carbon atoms in length, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 carbon atoms in length); (iii) an acid dissociation constant (pKa) of about 4.5 to about 7.5 (e.g., a pKa of about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 (e.g., a pKa of about 6.5 to about 7.5 (e.g., a pKa of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5)); (iv) ionizable amine and heteroorganic groups, and (v) having at least one (e.g., one, two, three, four, or all five) of the following N:P (ionizable lipid amines:mRNA phosphate) ratios of at least 10:

[0124] In some embodiments, the ionizable lipid is not selected from 1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, Lipid 5 (Moderna), and 98N12-5.

[0125] In some embodiments, the ionizable lipid is selected from the group consisting of 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315.

[0126] In some embodiments, the ionizable lipid is an ionizable amine and a heteroorganic group. In some embodiments, the heteroorganic group is hydroxyl. In some embodiments, the heteroorganic group comprises a hydrogen bond donor. In some embodiments, the heteroorganic group comprises a hydrogen bond acceptor. In some embodiments, the heteroorganic group is -OH, -SH, -(CO)H, -CO2H, -NH2, -CONH2, optionally substituted C1-C6 alkoxy, or fluorine.

[0127] In some embodiments, the ionizable lipid is an ionizable amine and a heteroorganic group separated by a chain of at least two atoms.

[0128] In some embodiments, the ionizable lipid has the following formula I: [ka] (I) (wherein R is C8-C 14 is an alkyl group).

[0129] In some embodiments, the lipid membrane of the LPMP comprises at least 35% lipids of formula I, e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, or more than 90% lipids of formula I, e.g., 35%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% lipids of formula I.

[0130] In some cases, the LPMP comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% ionizable lipids.

[0131] In some cases, the LPMP comprises a molar ratio of at least 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, or greater than 90% ionizable lipid, e.g., 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% ionizable lipid, e.g., about 25%-75% ionizable lipid (e.g., about 25%-75% ionizable lipid).

[0132] The ionizable lipids described herein may comprise an amine core as described herein substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) lipid tails. In some embodiments, the ionizable lipids described herein comprise at least three lipid tails. The lipid tails are selected from the group consisting of C8-C 18 Hydrocarbons (e.g., C6-C 18 -Alkyl or C6-C 18 -alkanoyl). The amine core may be substituted at the nitrogen atom with one or more lipid tails (e.g., one hydrogen atom attached to the nitrogen atom may be replaced with a lipid tail).

[0133] In some embodiments, the amine core is [ka] It has the structure:

[0134] In some embodiments, the amine core is [ka] It has the structure:

[0135] In some embodiments, the amine core is [ka] It has the structure:

[0136] In some embodiments, the amine core is [ka] It has the structure:

[0137] In some embodiments, the amine core is [ka] It has the structure:

[0138] In some embodiments, the amine core is [ka] It has the structure:

[0139] In some embodiments, the amine core is [ka] It has the structure:

[0140] In some embodiments, the amine core is [ka] It has the structure:

[0141] Other lipids suitable for use in the mRNA therapeutic compositions and methods of making and using same include those described in International Patent Publication No. WO 2016 / 118725, which is incorporated by reference in its entirety.

[0142] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0143] Other lipids suitable for use in the mRNA therapeutic compositions and methods of making and using same include those described in International Patent Publication No. WO 2016 / 118724, which is incorporated by reference in its entirety.

[0144] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0145] Other lipids suitable for use in the mRNA therapeutic compositions and methods of making and using them include lipids having the formula 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharma- ceutically acceptable salts thereof.

[0146] Other lipids suitable for use in the mRNA therapeutic compositions and methods of making and using them include those described in International Patent Publications 2013 / 063468 and 2016 / 205691, each of which is incorporated by reference in its entirety.

[0147] In some embodiments, the mRNA therapeutic compositions and methods of making and using same comprise a lipid of the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein R L Each instance of is independently an optionally substituted C6-C40 alkenyl.

[0148] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0149] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0150] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0151] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0152] Other lipids suitable for use in the mRNA therapeutic compositions and methods of making and using the same include those described in International Patent Publication No. 2015 / 184256, which is incorporated herein by reference in its entirety. In some embodiments, the mRNA therapeutic compositions and methods of making and using the same comprise a lipid of the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein each X is independently O or S, each Y is independently O or S, each m is independently 0 to 20, each n is independently 1 to 6, and each R Aare independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen, and each R is independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen.

[0153] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] (Target 23), and pharma- ceutically acceptable salts thereof.

[0154] Other lipids suitable for use in the mRNA therapeutic compositions and methods of making and using same include those described in International Patent Publication No. WO 2016 / 004202, which is incorporated by reference in its entirety.

[0155] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0156] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0157] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0158] Other lipids suitable for use in mRNA therapeutic compositions and methods of making and using same include those described in U.S. Provisional Patent Application No. 62 / 758,179, which is incorporated by reference in its entirety.

[0159] In some embodiments, the mRNA therapeutic compositions and methods of making and using same comprise a lipid of the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein each R 1 and R 2 is independently H or a C1-C6 aliphatic group, each m is independently an integer having a value of 1 to 4, each A is independently a covalent bond or an arylene group, and each L 1 is independently an ester, thioester, disulfide, or anhydride group, and each L 2 is independently a C2-C10 aliphatic group, and each X 1 is independently H or OH, and each R 3 are independently C6-C20 aliphatic.

[0160] In some embodiments, the mRNA therapeutic compositions and methods of making and using same comprise a lipid of the following formula: [ka] (Compound 1), or a pharma- ceutically acceptable salt thereof.

[0161] In some embodiments, the mRNA therapeutic compositions and methods of making and using same comprise a lipid of the following formula: [ka] (Compound 2), or a pharma- ceutically acceptable salt thereof.

[0162] In some embodiments, the mRNA therapeutic compositions and methods of making and using same comprise a lipid of the following formula: [ka] (Compound 3), or a pharma- ceutically acceptable salt thereof.

[0163] Other lipids suitable for use in the mRNA therapeutic compositions and methods of making and using same include those described in J. McClellan, MC King, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated by reference herein in their entireties.

[0164] In certain embodiments, the lipids of the mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0165] Other lipids suitable for use in the mRNA therapeutic compositions and methods of making and using same include those described in International Patent Publication No. WO 2015 / 199952, which is incorporated by reference in its entirety.

[0166] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0167] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0168] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0169] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0170] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0171] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0172] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0173] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0174] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0175] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0176] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0177] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0178] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0179] Other lipids suitable for use in the mRNA therapeutic compositions and methods of making and using same include those described in International Patent Publication No. WO 2017 / 004143, which is incorporated by reference in its entirety.

[0180] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0181] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0182] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0183] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0184] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0185] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0186] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0187] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0188] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0189] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0190] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0191] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0192] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0193] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0194] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0195] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0196] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0197] Other lipids suitable for use in the mRNA therapeutic compositions and methods of making and using same include those described in International Patent Publication No. WO 2017 / 075531, which is incorporated by reference in its entirety.

[0198] In some embodiments, the mRNA therapeutic compositions and methods of making and using same comprise a lipid of the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a- , or -NR a C(=O)O-, L 1 or L 2The other of the above is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, ,NR a C(=O)NR a -, -OC(=O)NR a -OR-NR a C(=O)O- or a direct bond, G 1 and G 2 are each independently an unsubstituted C-C 12 Alkylene or C1-C 12 Alkenylene, G 3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1-C 12 is alkyl, R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 alkenyl, R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and R 4 is C1-C 12 is alkyl, R 5 is H or C1-C6 alkyl and x is 0, 1 or 2.

[0199] Other lipids suitable for use in the mRNA therapeutic compositions and methods of making and using same include those described in International Patent Publication No. 2017 / 117528, which is incorporated by reference in its entirety. In some embodiments, the mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0200] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0201] In some embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0202] Other lipids suitable for use in the mRNA therapeutic compositions and methods of making and using same include those described in International Patent Publication No. WO 2017 / 049245, which is incorporated by reference in its entirety.

[0203] In some embodiments, the lipid of the mRNA therapeutic composition and methods of making and using same have the following formula: [ka] [ka] [ka] [ka] In any one of these four formulas, R4 is independently -(CH2) n Q and -(CH2) n CHQR, where Q is -OR, -OH, -O(CH2)n and heterocycle, wherein n is 1, 2, or 3.

[0204] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0205] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0206] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0207] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0208] Other lipids suitable for use in the mRNA therapeutic compositions and methods of making and using same include those described in International Patent Publication Nos. 2017 / 173054 and 2015 / 095340, which are incorporated herein by reference in their entireties. In certain embodiments, the mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0209] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0210] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0211] In certain embodiments, mRNA therapeutic compositions and methods of making and using same include: [ka] and pharma- ceutically acceptable salts thereof.

[0212] In some embodiments, the LPMPs described herein may comprise, be formulated as described, or comprise or consist of a composition described in WO2016118724, WO2016118725, WO2016187531, WO2017176974, WO2018078053, WO2019027999, WO2019036030, WO2019089828, WO2019099501, WO2020072605, WO2020081938, WO2020118041, WO2020146805, or WO2020219876, each of which is incorporated by reference in its entirety.

[0213] Other lipids and other drugs The exogenous lipid may be a cell-penetrating agent, may increase the delivery of the polypeptide to the cell by the LPMP, and / or may increase the loading (e.g., loading efficiency or loading capacity) of the polypeptide. Further exemplary exogenous lipids include sterols and PEGylated lipids.

[0214] LPMPs may be modified with other components (e.g., lipids, e.g., sterols, e.g., cholesterol, or small molecules) to further alter the functional and structural characteristics of the LPMP. For example, LPMPs may be further modified with stabilizing molecules that increase the stability of the LPMP (e.g., stable at room temperature for at least one day and / or stable at 4°C for at least one week).

[0215] In some embodiments, the LPMP is modified with a sterol, such as sitosterol, sitostanol, β-sitosterol, 7α-hydroxycholesterol, pregnenolone, cholesterol (e.g., ovine cholesterol or cholesterol isolated from plants), stigmasterol, campesterol, fucosterol, or any sterol analog (e.g., glycoside, ester, or peptide). In some examples, the exogenous sterol is added to the preparation before step (b), e.g., mixed with the PMP lipid extracted before step (b). The exogenous sterol may be added in an amount of, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% (w / w) of the total lipids and sterols in the preparation.

[0216] In some embodiments, the sterol is cholesterol or sitosterol. In some cases, the LPMP comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60% sterol (e.g., cholesterol or sitosterol), for example, 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or 50%-60% molar ratio of sterol. In some embodiments, the LPMP comprises about 35%-50% sterol (e.g., cholesterol or sitosterol), for example, about 36%, 38.5%, 42.5%, or 46.5% molar ratio of sterol. In some embodiments, the LPMP comprises about 20%-40% molar ratio of sterol.

[0217] In some embodiments, LPMPs that are modified with sterols have altered stability (e.g., increased stability) compared to LPMPs that are not modified with sterols. In some aspects, LPMPs that are modified with sterols have a faster fusion rate with the membrane of a target cell compared to LPMPs that are not modified with sterols.

[0218] In some cases, the LPMP comprises an exogenous lipid and an exogenous sterol.

[0219] In some embodiments, the LPMP is modified with a PEGylated lipid. The length of the polyethylene glycol (PEG) can vary from 1 kDa to 10 kDa, and in some aspects, a PEG with a length of 2 kDa is used. In some embodiments, the PEGylated lipid is C14-PEG2k, C18-PEG2k, or DMPE-PEG2k. In some cases, the LPMP is at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 0%, 30%, 40%, 50%, or more than 50% PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k), e.g., 0.1%-0.5%, 0.5%-1%, 1%-1.5%, 1.5%-2.5%, 2.5%-3.5%, 3.5%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, or 30%-50% molar ratio of PEGylated lipid. In some embodiments, the LPMPs comprise about 0.1%-10% molar ratio of PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k), e.g., about 1%-3% PEGylated lipid, e.g., about 1.5% or about 2.5% PEGylated lipid. In some embodiments, LPMPs modified with PEGylated lipids have altered stability (e.g., increased stability) compared to LPMPs not modified with PEGylated lipids. In some embodiments, LPMPs modified with PEGylated lipids have altered particle size compared to LPMPs not modified with PEGylated lipids. In some embodiments, LPMPs modified with PEGylated lipids are less likely to be phagocytosed compared to LPMPs not modified with PEGylated lipids. The addition of PEGylated lipids may also affect stability in the GI tract and enhance particle transmigration through mucus. PEG may be used as a method to attach targeting moieties.

[0220] In some embodiments, the LPMP is modified with an ionizable lipid (e.g., C12-200 or MC3) and one or both of a sterol (e.g., cholesterol or sitosterol) and a PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k).

[0221] In some embodiments, the modified LPMP comprises a molar ratio of about 5%-50% LPMP lipid (e.g., about 10%-20% LPMP lipid, e.g., about 10%, 12.5%, 16%, or 20% LPMP lipid), about 30%-75% ionizable lipid (e.g., about 35% or about 50% ionizable lipid), about 35%-50% sterol (e.g., about 36%, 38.5%, 42.5%, or 46.5% sterol), and about 0.1%-10% PEGylated lipid (e.g., about 1%-3% PEGylated lipid, e.g., about 1.5% or about 2.5% PEGylated lipid).

[0222] In some embodiments, the modified LPMP comprises a molar ratio of about 5% to 60% LPMP lipid (e.g., about 10% to 20%, 20%-30%, 30%-40%, 40%-50%, or 50% to 60% LPMP lipid, e.g., about 10%, 12.5%, 16%, 20%, 30%, 40%, 50%, or 60% LPMP lipid), about 25% to 75% ionizable lipid (e.g., about 35% or about 50% ionizable lipid), about 10%-50% sterol (e.g., about 10%, 12.5%, 14%, 16%, 18%, 20%, 36%, 38.5%, 42.5%, or 46.5% sterol), and about 0.1%-10% PEGylated lipid (e.g., about 0.5%-5% PEGylated lipid, e.g., about 1%-3% PEGylated lipid, or about 1.5% or about 2.5% PEGylated lipid).

[0223] In some embodiments, the ionizable lipids, LPMP lipids, sterols, and PEGylated lipids comprise about 25%-75%, about 20%-60%, about 10%-45%, and about 0.5%-5%, respectively, of the lipids in the modified PMP.

[0224] In some embodiments, the ionizable lipids, LPMP lipids, sterols, and PEGylated lipids comprise about 30%-75%, about 20%-50%, about 10%-45%, and about 1%-5%, respectively, of the lipids in the modified PMP.

[0225] In some embodiments, the ionizable lipids, LPMP lipids, sterols, and PEGylated lipids comprise about 35%-75%, about 20%-50%, about 10%-45%, and about 1%-5%, respectively, of the lipids in the modified PMP.

[0226] In some embodiments, the ionizable lipid, the LPMP lipid, the sterol, and the PEGylated lipid are formulated in a molar ratio of about 35:50:12.5:2.5.

[0227] In some embodiments, the ionizable lipid, the LPMP lipid, the sterol, and the PEGylated lipid are formulated in a molar ratio of about 35:50:11.5:3.5.

[0228] In some embodiments, the ionizable lipid, the LPMP lipid, the sterol, and the PEGylated lipid are formulated in a molar ratio of about 35:20:42.5:2.5.

[0229] In some embodiments, LPMPs modified with ionizable lipids (and / or cationic lipids) and sterols and / or PEGylated lipids more efficiently encapsulate negatively charged cargo (e.g., nucleic acids) than LPMPs not modified with ionizable lipids (and / or cationic lipids) and sterols and / or PEGylated lipids. The modified LPMPs may have an encapsulation efficiency for the cargo (e.g., nucleic acid, e.g., RNA or DNA) that is at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or greater than 99%, e.g., 5%-30%, 30%-50%, 50%-70%, 70%-80%, 80%-90%, 90%-95%, or 95%-100%.

[0230] Cellular uptake of modified LPMPs can be measured by a variety of methods known in the art. For example, the LPMP, or a component thereof, can be labeled with a marker (e.g., a fluorescent marker) that can be detected in isolated cells to confirm uptake.

[0231] In some embodiments, the LPMP formulations provided herein include two or more different modified LPMPs, e.g., modified LPMPs derived from different unmodified LPMPs (e.g., unmodified LPMPs from two or more different plant sources), and / or modified LPMPs comprising different species and / or different ratios of ionizable lipids, sterols, and / or PEGylated lipids.

[0232] In some cases, the organic solvent in which the lipid film is dissolved is dimethylformamide:methanol (DMF:MeOH). Alternatively, the organic solvent or solvent combination may be, for example, acetonitrile, acetone, ethanol, methanol, dimethylformamide, tetrahydrofuran, 1-butanol, dimethylsulfoxide, acetonitrile:ethanol, acetonitrile:methanol, acetone:methanol, methyl tert-butyl ether:propanol, tetrahydrofuran:methanol, dimethylsulfoxide:methanol, or dimethylformamide:methanol.

[0233] The aqueous phase may be any suitable solution, such as a citrate buffer (e.g., a citrate buffer having a pH of about 3.2), water, or phosphate buffered saline (PBS). The aqueous phase may further comprise a nucleic acid (e.g., an siRNA or siRNA precursor (e.g., dsRNA), an miRNA or miRNA precursor, an mRNA, or a plasmid (pDNA)) or a small molecule.

[0234] The lipid solution and the aqueous phase may be mixed in the microfluidic device in any suitable ratio. In some examples, the aqueous phase and the lipid solution are mixed in a volume ratio of 3:1.

[0235] LPMPs may optionally include additional agents, such as cell-penetrating agents, therapeutic agents, polynucleotides, polypeptides, or small molecules. LPMPs can carry or associate with additional agents in a variety of ways to enable delivery of agents to target plants, for example, by encapsulating the agent, incorporating the agent into the lipid bilayer structure, or associating the agent with the surface of the lipid bilayer structure (e.g., by conjugation). Nucleic acid molecules can be incorporated into LPMPs either in vivo (e.g., in planta) or in vitro (e.g., in tissue culture, cell culture, or synthetically incorporated).

[0236] Zeta potential LPMPs comprising an ionizable lipid (e.g., C12-200 or MC3) and optionally a cationic lipid (e.g., DC-cholesterol or DOTAP) may have a zeta potential of, for example, greater than -30 mV in the absence of cargo, greater than -20 mV, greater than -5 mV, greater than 0 mV, or about 30 mV in the absence of cargo. In some examples, LPMPs have a negative zeta potential, for example, a zeta potential of less than 0 mV, less than -10 mV, less than -20 mV, less than -30 mV, less than -40 mV, or less than -50 mV in the absence of cargo. In some examples, LPMPs have a positive zeta potential, for example, a zeta potential of greater than 0 mV, greater than 10 mV, greater than 20 mV, greater than 30 mV, greater than 40 mV, or greater than 50 mV in the absence of cargo. In some examples, LPMPs have a zeta potential of about 0.

[0237] The zeta potential of LPMPs can be measured using any method known in the art. Zeta potential is generally measured indirectly, for example, calculated using theoretical models from data obtained using methods and techniques known in the art, such as electrophoretic mobility or dynamic electrophoretic mobility. Electrophoretic mobility is typically measured using microelectrophoresis, electrophoretic light scattering, or tunable resistive pulse sensing. Electrophoretic light scattering is based on dynamic light scattering. Typically, zeta potential is accessible from dynamic light scattering (DLS) measurements, also known as photon correlation spectroscopy or quasi-elastic light scattering.

[0238] Plant EV markers The LPMPs in mRNA therapeutic compositions and methods of making and using them may have a wide range of markers that identify the LPMPs produced using plant EVs and / or include segments, portions, or extracts thereof. As used herein, the term "plant EV marker" refers to a component that is naturally associated with a plant and is incorporated into or on a plant EV in a plant body, such as a plant protein, a plant nucleic acid, a plant small molecule, a plant lipid, or a combination thereof. Examples of plant EV markers can be found, for example, in Rutter and Innes, Plant Physiol. 173(1):728-741, 2017; Raimondo et al., Oncotarget. 6(23): 19514, 2015; Ju et al., Mol. Therapy. 21(7):1345-1357, 2013; Wang et al., Molecular Therapy. 22(3): 522-534, 2014; and Regente et al, J of Exp. Biol. 68(20): 5485-5496, 2017, each of which is incorporated herein by reference.

[0239] Further examples of suitable plant EV markers include those described and listed in International Patent Application Publication No. 2021 / 041301, which is incorporated by reference in its entirety into this specification.

[0240] Drug (e.g., nucleic acid) loading LPMPs are modified to include therapeutic agents (e.g., nucleic acid molecules) to form mRNA therapeutic compositions. LPMPs can carry or associate with such agents in a variety of ways to enable delivery of the agent to a target organism (e.g., a target animal), for example, by encapsulating the agent, incorporating a component into the lipid bilayer structure, or associating a component with the surface of the lipid bilayer structure of the LPMP (e.g., by conjugation). In some cases, the agent is included in the LPMP formulation as described herein.

[0241] The agent may be incorporated or loaded into or onto the LPMP by any method known in the art that allows for a direct or indirect association between the LPMP and the agent. The agent may be incorporated into the LPMP by in vivo methods (e.g., through the generation of the LPMP in planta, e.g., from a transgenic plant containing the agent), or in vitro (e.g., in tissue culture or cell culture), or by both in vivo and in vitro methods.

[0242] In some cases, LPMPs are loaded in vitro. Substances may be loaded onto or within LPMPs (e.g., encapsulated by) using, but not limited to, physical, chemical, and / or biological methods (e.g., in tissue culture or cell culture). For example, agents may be introduced into LPMPs by one or more of electroporation, sonication, passive diffusion, agitation, lipid extraction, or extrusion. In some cases, agents are incorporated into LPMPs using a microfluidic device, for example, a method in which LPMP lipids are provided in an organic phase, a heterogeneous functional agent is provided in an aqueous phase, and the organic and aqueous phases are combined in the microfluidic device to generate an LPMP containing a heterogeneous functional agent. Loaded LPMPs can be evaluated to confirm the presence or level of loaded agents using a variety of methods, such as HPLC (e.g., to evaluate small molecules), immunoblotting (e.g., to evaluate proteins), and / or quantitative PCR (e.g., to evaluate nucleotides). However, it should be understood by those skilled in the art that loading of a substance of interest into an LPMP is not limited to the methods described above.

[0243] In some cases, the agent may be conjugated to the LPMP, where the agent is indirectly or directly linked or bound to the LPMP. For example, one or more agents may be chemically linked to the LPMP, such that the one or more agents are directly bound (e.g., by covalent or ionic bonds) to the lipid bilayer of the LPMP. In some cases, conjugation of various agents to the LPMP can be accomplished by first mixing one or more agents with a suitable crosslinker (e.g., N-ethylcarbodiimide ("EDC"), which is generally utilized as a carboxyl activating agent for amide bonds with primary amines, and also reacts with phosphate groups) in a suitable solvent. After a period of incubation sufficient to allow the agent to attach to the crosslinker, the crosslinker / agent mixture can then be combined with the LPMP and, after a further incubation period, subjected to a sucrose gradient (e.g., 8, 30, 45, and 60% sucrose gradient) to separate the free agent and the free LPMP from the agent conjugated to the LPMP. As part of combining the mixture with a sucrose gradient and accompanying centrifugation step, the LPMP conjugated to the drug is then seen as a band in the sucrose gradient, and the conjugated LPMP can then be collected, washed, and dissolved in a suitable solution for use as described herein.

[0244] In some cases, the LPMP is stably associated with the agent, for example, before and after delivery of the LPMP to the plant. Alternatively, the LPMP may be generated using a microfluidic device (such as the NanoAssemblr® IGNITE™ microfluidic device (Precision NanoSystems)).

[0245] LPMPs can be loaded or formulated with various concentrations of agents depending on the particular agent or use. For example, in some cases, LPMPs can be loaded or formulated such that the LPMP formulations disclosed herein contain about 0.001, 0.01, 0.1, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 95 (or any range between about 0.001 and 95) or more weight percent of agent. In some cases, the LPMP is loaded or formulated such that the LPMP formulation contains about 95, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.0, 0.1, 0.01, 0.001 (or any range between about 95 and 0.001) or less by weight of the agent. For example, the LPMP formulation may contain about 0.001 to about 0.01 weight percent, about 0.01 to about 0.1 weight percent, about 0.1 to about 1 weight percent, about 1 to about 5 weight percent, or about 5 to about 10 weight percent, about 10 to about 20 weight percent of the agent. In some cases, LPMPs may be loaded or LPMPs formulated with about 1, 5, 10, 50, 100, 200, or 500, 1,000, 2,000 (or any range between about 1 and 2,000) or more μg / ml of drug. LPMPs of the present invention may be loaded or LPMPs formulated with about 2,000, 1,000, 500, 200, 100, 50, 10, 5, 1 (or any range between about 2,000 and 1) or less μg / ml of drug.

[0246] In some cases, the LPMP is loaded or the LPMP is formulated such that the LPMP formulation disclosed herein comprises at least 0.001%, at least 0.01%, at least 0.1%, at least 1.0%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% by weight of drug. In some cases, the LPMP may be loaded or formulated with at least 1 μg / ml, at least 5 μg / ml, at least 10 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 200 μg / ml, at least 500 μg / ml, at least 1,000 μg / ml, or at least 2,000 μg / ml of drug.

[0247] In some cases, the LPMPs are formulated with an agent by suspending the LPMPs in a solution containing or consisting of the agent, e.g., suspending or resuspending the LPMPs by vigorous mixing. The agent (e.g., a cell permeation agent, e.g., a nucleic acid, an enzyme, a detergent, an ion, a fluorescent, or a zwitterionic liquid, or an ionizable lipid) may comprise, for example, less than 1% or at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the solution.

[0248] Pharmaceutical preparations The modified LPMPs are formulated into pharmaceutical compositions (i.e., mRNA therapeutic compositions), for example, for administration to animals (e.g., humans). The pharmaceutical compositions may be administered to animals (e.g., humans) together with pharma- ceutically acceptable diluents, carriers, and / or excipients. Depending on the mode of administration and the dosage, the pharmaceutical compositions of the methods described herein are formulated into suitable pharmaceutical compositions to allow easy delivery. A single dose may be in unit dosage form, if desired.

[0249] The LPMP / mRNA therapeutic composition may be formulated, for example, for oral, intravenous (e.g., injection or infusion), intramuscular, or subcutaneous administration to an animal. For injectable formulations, a variety of effective pharmaceutical carriers are known in the art (see, for example, Remington: The Science and Practice of Pharmacy, 22 nd ed., (2012) and ASHP Handbook on Injectable Drugs, 18 th ed., (2014).

[0250] Suitable pharma- ceutically acceptable carriers and excipients are non-toxic to recipients at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzylammonium chloride, resorcinol and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, histidine and lysine, and carbohydrates such as glucose, mannose, sucrose and sorbitol. LPMP / mRNA therapeutic compositions may be formulated according to conventional pharmaceutical practice. The concentration of the compound in the formulation will vary depending on many factors, including the dosage of the active agent (e.g., LPMP and nucleic acid) to be administered, and the route of administration.

[0251] For oral administration to animals, the LPMP / mRNA therapeutic composition can be prepared in the form of an oral formulation. Formulations for oral use can include tablets, caplets, capsules, syrups, or oral liquid dosage forms containing the active ingredient in a mixture with non-toxic pharma- ceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate), granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starch including potato starch, croscarmellose sodium, alginates, or alginic acid), binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol), as well as lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oils, or talc). Other pharma- ceutically acceptable excipients may be colorants, flavoring agents, plasticizers, humectants, buffers, etc. Formulations for oral use may also be provided in unit dosage form as chewable tablets, non-chewable tablets, caplets, capsules (e.g., as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules in which the active ingredient is mixed with water or oil medium). The compositions disclosed herein may also further include immediate release, extended release, or sustained release formulations.

[0252] For parenteral administration to animals, the LPMP / mRNA therapeutic composition may be formulated in the form of a liquid solution or suspension and administered by parenteral routes of administration (e.g., subcutaneous, intravenous, or intramuscular). The pharmaceutical composition may be formulated for injection or infusion. Pharmaceutical compositions for parenteral administration may be formulated using a sterile solution or any pharma- ceutical acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, saline, or cell culture media (e.g., Dulbecco's Modified Eagle Medium (DMEM), alpha modified Eagle Medium (alpha-MEM), and F-12 medium). Formulation methods are known in the art, see, for example, Gibson (ed.) Pharmaceutical Preformulation and Formulation (2nd ed.) Taylor & Francis Group, CRC Press (2009).

[0253] Polynucleotides LPMP / mRNA therapeutic compositions include one or more nucleic acid molecules, e.g., polynucleotides, that encode one or more wild-type or engineered proteins, peptides, or polypeptides. Exemplary polynucleotides, e.g., polynucleotide constructs, include RNA polynucleotides, e.g., mRNAs, that encode antigens.

[0254] Examples of polypeptides that may be used herein include enzymes (e.g., metabolic recombinases, helicases, integrases, RNAses, DNAses, or ubiquitinating proteins), pore-forming proteins, signaling ligands, cell-penetrating peptides, transcription factors, receptors, antibodies, nanobodies, gene editing proteins (e.g., CRISPR-Cas systems, TALENs, or zinc fingers), riboproteins, protein aptamers, or chaperones.

[0255] The polypeptides included herein may include naturally occurring polypeptides or recombinantly produced variants. In some cases, the polypeptides may be functional fragments or variants thereof (e.g., enzymatically active fragments or variants thereof). For example, the polypeptides may be functionally active variants of any of the polypeptides described herein that have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequences of the polypeptides described herein or naturally occurring polypeptides, for example, over a specific region or over the entire sequence. In some cases, a polypeptide may have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) identity to a protein of interest.

[0256] An LPMP / mRNA therapeutic composition may include any number or type (e.g., class) of polypeptides, such as at least about one of one polypeptide, two, three, four, five, ten, fifteen, twenty, or more polypeptides. The appropriate concentration of each polypeptide in an LPMP / mRNA therapeutic composition depends on factors such as efficacy, stability of the polypeptide, the number of distinct polypeptides in the formulation, and the method of application of the formulation. In some cases, each polypeptide in a liquid formulation is about 0.1 ng / mL to about 100 mg / mL. In some cases, each polypeptide in a solid formulation is about 0.1 ng / g to about 100 mg / g.

[0257] Nucleic acid encoding a peptide In some cases, the LPMP / mRNA therapeutic composition comprises a heterologous nucleic acid encoding a polypeptide. The nucleic acid encoding the polypeptide may be from about 10 to about 50,000 nucleotides in length (nt), from about 25 to about 100 nt, from about 50 to about 150 nt, from about 100 to about 200 nt, from about 150 to about 250 nt, from about 200 to about 300 nt, from about 250 to about 350 nt, from about 300 to about 500 nt, from about 10 to about 1000 nt, from about 50 to about 1000 nt, from about 1000 to about 2000 nt, from about 2000 to about 3000 nt, from about 3000 to about 4000 nt, from about 4000 to about 5000 nt, from about 50 to about 5000 nt, or from about 50 to about 6000 nt. It may have an amino acid sequence of from about 1,000 to about 6,000 nt, from about 6,000 to about 7,000 nt, from about 7,000 to about 8,000 nt, from about 8,000 to about 9,000 nt, from about 9,000 to about 10,000 nt, from about 10,000 to about 15,000 nt, from about 10,000 to about 20,000 nt, from about 10,000 to about 25,000 nt, from about 10,000 to about 30,000 nt, from about 10,000 to about 40,000 nt, from about 10,000 to about 45,000 nt, from about 10,000 to about 50,000 nt, or any range therebetween.

[0258] LPMP / mRNA therapeutic compositions may also include active variants of the nucleic acid sequence of interest.In some cases, the nucleic acid variant has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleic acid sequence of interest, for example, over a specified region or over the entire sequence.In some cases, the present invention includes active polypeptides encoded by the nucleic acid variants described herein. In some cases, an active polypeptide encoded by a nucleic acid variant has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a subject polypeptide sequence or a naturally-occurring polypeptide sequence, e.g., over a specified region or over the entire amino acid sequence.

[0259] Certain methods for expressing nucleic acids encoding proteins may involve expression in cells, including insects, yeast, plants, bacteria, or other cells, under the control of a suitable promoter. Expression vectors may include non-transcriptional elements, such as origins of replication, suitable promoters and enhancers, and other 5' or 3' adjacent non-transcriptional sequences, as well as 5' or 3' non-translation sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 virus genome, such as SV40 origin, early promoter, enhancer, splice, and polyadenylation sites, may be used to provide other genetic elements required for the expression of heterologous DNA sequences. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012.

[0260] Genetic modification using recombinant methods is generally known in the art.The nucleic acid sequence encoding the desired gene can be obtained using recombinant methods known in the art, such as, for example, screening a library from cells expressing the gene, deriving the gene from a vector known to contain the gene, or directly isolating it from cells and tissues containing it using standard techniques.Alternatively, the gene of interest can be produced synthetically instead of being cloned.

[0261] Expression of natural or synthetic nucleic acids is typically achieved by operably linking the nucleic acid encoding the gene of interest to a promoter and incorporating the construct into an expression vector. The expression vector may be suitable for replication and expression in bacteria. The expression vector may also be suitable for replication and integration in eukaryotes. A typical cloning vector contains transcription and translation terminators, initiation sequences, and promoters useful for expressing the desired nucleic acid sequence.

[0262] Additional promoter elements, e.g. enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 base pairs (bp) upstream of the start site, although a number of promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, such that promoter function is retained when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function cooperatively or independently to activate transcription.

[0263] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can bring about high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the elongation growth factor 1 alpha (EF-1 alpha). However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0264] Alternatively, the promoter may be an inducible promoter. The use of an inducible promoter provides a molecular switch that can turn on the expression of the operably linked polynucleotide sequence when such expression is desired, or can turn off the expression when such expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionine promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0265] The expression vector to be introduced can also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells that are to be transfected or infected through a viral vector. In other embodiments, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0266] Reporter genes can be used to identify potentially transformed cells and evaluate the functionality of control sequences. Generally, reporter genes are genes that are absent from or expressed by the recipient source and encode a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., FEBS Letters 479:79-82, 2000). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. Generally, the construct with the smallest 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to the reporter gene and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0267] In some cases, an organism may be genetically modified to alter the expression of one or more proteins. The expression of one or more proteins may be modified for a particular time, for example, during the development or differentiation state of the organism. In one example, a composition is provided for altering the expression of one or more proteins, for example, a protein that affects activity, structure, or function. The expression of one or more proteins may be restricted to a particular location or may be widespread throughout the organism.

[0268] mRNA LPMP / mRNA therapeutic compositions may include mRNA molecules, for example, mRNA molecules that code for polypeptides. mRNA molecules can be synthesized and modified (e.g., chemically). mRNA molecules can be chemically synthesized or transcribed in vitro. mRNA molecules can be placed on a plasmid, for example, a viral vector, a bacterial vector, or a eukaryotic expression vector. In some examples, mRNA molecules can be delivered to cells by transfection, electroporation, or transduction (e.g., adenoviral or lentiviral transduction).

[0269] In some cases, the modified RNA agent of the subject described herein has modified nucleoside or nucleotide.Such modifications are known and are described, for example, in International Publication No. WO 2012 / 019168.Additional modifications are described, for example, in International Publication No. WO 2015 / 038892; WO 2015 / 038892; WO 2015 / 089511; WO 2015 / 196130; WO 2015 / 196118 and International Publication No. WO 2015 / 196128 A2, which are incorporated herein by reference in their entirety.

[0270] In some cases, the modified RNA encoding the polypeptide of interest has one or more terminal modifications, such as a 5' cap structure and / or a polyA tail (e.g., 100-200 nucleotides in length). The 5' cap structure can be selected from the group consisting of CapO, Capl, ARCA, inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some cases, the modified RNA also contains a 5' UTR that includes at least one Kozak sequence, and a 3' UTR. Such modifications are known and are described, for example, in WO 2012 / 135805 and WO 2013 / 052523, which are incorporated herein by reference in their entireties. Additional termination modifications are described, for example, in WO 2014 / 164253 and WO 2016 / 011306, WO 2012 / 045075, and WO 2014 / 093924, which are incorporated by reference in their entireties. Chimeric enzymes for synthesizing capped RNA molecules (e.g., modified mRNAs) that may contain at least one chemical modification are described in WO 2014 / 028429, which is incorporated by reference in its entirety.

[0271] In some cases, modified mRNA (mmRNA) may be circularized or ligated to generate a translation-competent molecule that supports the interaction between polyA-binding protein and 5'-end binding protein. The mechanism of circularization or ligation may occur via at least three different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalysis. The newly formed 5'- / 3'-bonds may be intramolecular or intermolecular. Such modifications are described, for example, in WO 2013 / 151736.

[0272] The method of making and purifying modified RNA is known and disclosed in the art.For example, modified RNA is made by using only in vitro transcription (IVT) enzymatic synthesis.The method of making IVT polynucleotide is known in the art and described in WO 2013 / 151666, WO 2013 / 151668, WO 2013 / 151663, WO 2013 / 151669, WO 2013 / 151670, WO 2013 / 151664, WO 2013 / 151665, WO 2013 / 151671, WO 2013 / 151672, WO 2013 / 151667 and WO 2013 / 151736, which are incorporated herein by reference in their entirety. Purification methods include purifying RNA transcripts containing a polyA tail by contacting the sample with a surface linked to multiple thymidines or derivatives thereof and / or multiple uracils or derivatives thereof (polyT / U) under conditions such that the RNA transcripts bind to the surface, and eluting the purified RNA transcripts from the surface using ion (e.g., anion) exchange chromatography (WO 2014 / 152031), which allows for the separation of longer RNAs up to 10,000 nucleotides in length via a scalable method (WO 2014 / 144767), and subjecting the modified mRNA sample to DNAse treatment (WO 2014 / 152030).

[0273] Formulations of modified RNA are known and described, for example, in WO 2013 / 090648. For example, the formulation may be, but is not limited to, nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microparticles, lipidoids, lipoplexes, liposomes, polymers, carbohydrates (including simple sugars), cationic lipids, fibrin gels, fibrin hydrogels, fibrin glues, fibrin sealants, fibrinogen, thrombin, rapidly cleared lipid nanoparticles (reLNPs), and combinations thereof.

[0274] Modified RNAs encoding polypeptides in the fields of human disease, antibodies, viruses, and various in vivo settings are known and are disclosed, for example, in Table 6 of WO 2013 / 151666, WO 2013 / 151668, WO 2013 / 151663, WO 2013 / 151669, WO 2013 / 151670, WO 2013 / 151664, WO 2013 / 151665, WO 2013 / 151736; Tables 6 and 7 of WO 2013 / 151672, Tables 6, 178 and 179 of WO 2013 / 151671, and Tables 6, 185 and 186 of WO 2013 / 151667, which are incorporated herein by reference in their entireties. Any of the foregoing may be synthesized as an IVT polynucleotide, a chimeric polynucleotide or a circular polynucleotide, each of which may contain one or more modified nucleotides or terminal modifications.

[0275] Inhibitory RNA In some cases, the LPMP / mRNA therapeutic composition includes an inhibitory RNA molecule, for example, an inhibitory RNA molecule that acts through the RNA interference (RNAi) pathway. In some cases, the inhibitory RNA molecule reduces the level of gene expression in the plant and / or reduces the level of a protein in the plant. In some cases, the inhibitory RNA molecule inhibits the expression of a plant gene. For example, the inhibitory RNA molecule can include short interfering RNA or its precursor, small hairpin RNA, and / or microRNA or its precursor that targets a gene in the plant. Certain RNA molecules can inhibit gene expression through the biological process of RNA interference (RNAi). RNAi molecules typically contain 15-50 base pairs (e.g., about 18-25 base pairs) and include RNA or RNA-like structures with nucleobase sequences identical (or complementary) or nearly identical (or substantially complementary) to the coding sequence of a target gene expressed in a cell. RNAi molecules include, but are not limited to, short interfering RNA (siRNA), double-stranded RNA (dsRNA), small hairpin RNA (shRNA), meroduplex, dicer substrate, and multivalent RNA interference (US Pat. Nos. 8,084,599, 8,349,809, 8,513,207 and 9,200,276, which are incorporated herein by reference in their entirety).Inhibitory RNA molecules can be chemically synthesized or transcribed in vitro.

[0276] Additional examples of inhibitory RNA molecules include those described in detail in International Patent Application Publication No. 2021 / 041301, which is incorporated by reference in its entirety into this specification.

[0277] Gene editing In some cases, the LPMP / mRNA therapeutic composition may include components of a gene editing system. For example, the agent may introduce modifications (e.g., insertions, deletions (e.g., knockouts), translocations, inversions, point mutations, or other mutations) into the plant's genes. Exemplary gene editing systems include zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and clustered regulatory interspaced short palindromic repeats (CRISPR) systems. ZFN, TALEN, and CRISPR-based methods are described, for example, in Gaj et al., Trends Biotechnol 31(7):397-405, 2013.

[0278] Further description of the components and processes of the gene editing system can be found in International Patent Application Publication No. WO 2021 / 041301, which is incorporated by reference in its entirety.

[0279] mRNA therapeutics LPMP / mRNA therapeutic compositions include one or more polynucleotides (e.g., mRNA) that encode one or more antigenic or signaling polypeptides for therapeutic purposes, such as combating cancer. The one or more polynucleotides (e.g., mRNA) encode one or more antigenic (e.g., tumor antigenic) or signaling polypeptides.

[0280] Tumor antigens and signal transduction therapeutic agents Cancer or tumor includes, but is not limited to, neoplasm, malignancy, metastasis, or any disease or disorder characterized by uncontrolled cell proliferation and thus considered cancerous. Cancer can be primary or metastatic cancer. Specific cancers that can be treated according to the present invention include, but are not limited to, those described below (for a review of such disorders, see Fishman et al, 1985, Medicine, 2d Ed., JB Lippincott Co., Philadelphia). Cancers include, but are not limited to, biliary tract cancer, bladder cancer, brain cancer including glioblastoma and medulloblastoma, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, hematological neoplasms including acute lymphocytic leukemia and myeloid leukemia, multiple myeloma, AIDS-related leukemia and adult T-cell leukemia lymphoma, intraepithelial neoplasms including Bowen's disease and Paget's disease, liver cancer, lung cancer, lymphomas including Hodgkin's disease and lymphocytic lymphoma, neuroblastoma, oral cancer including squamous cell carcinoma, These include ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells, pancreatic cancer, prostate cancer, rectal cancer, sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma, skin cancer, including melanoma, Kaposi's sarcoma, basal cell carcinoma and squamous cell carcinoma, testicular cancer, including germinal tumors such as seminoma, non-seminoma, teratoma, choriocarcinoma, stromal tumor and germ cell tumor, thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma, and renal cancer, including adenocarcinoma and Wilms' tumor. Commonly encountered cancers include breast cancer, prostate cancer, lung cancer, ovarian cancer, colorectal cancer and brain cancer.

[0281] In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial carcinoma, HPV-negative head and neck squamous cell carcinoma (HNSCC), and solid malignancies that are microsatellite high (MSI H) / mismatch repair (MMR) deficient. In some embodiments, the NSCLC lacks EGFR-sensitive mutations and / or ALK translocations. In some embodiments, the solid malignancies that are microsatellite high (MSI H) / mismatch repair (MMR) deficient are selected from the group consisting of colorectal cancer, gastric adenocarcinoma, esophageal adenocarcinoma, and endometrial cancer. In some embodiments, the cancer is selected from cancers of the pancreas, peritoneum, large intestine, small intestine, bile duct, lung, endometrium, ovary, reproductive tract, digestive tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic and lymphatic tissues. In some embodiments, the cancer is colorectal cancer.

[0282] In some embodiments, the antigenic (e.g., tumor antigenic) or signaling polypeptide is selected from the group consisting of p53, ART-4, BAGE, ss-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, CLAUDIN-12, C-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A2. E-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Plac-1, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or S ART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survivin, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, WT-1, or combinations thereof.

[0283] In some embodiments, the tumor antigen is one of the following antigens: CD2, CD3, CD4, CD8, CD11b, CD14, CD16, CD19, CD20, CD22, CD25, CD27, CD33, CD37, CD38, CD40, CD44, CD45, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbBl, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, HLA, HLA-DR, ICOS, IGF1R, integrin αν, integrin ανβ, LAG-3, Lewis Y, mesothelin, c-MET, MN carbonic anhydrase IX, MUC1, MUC16, Nectin-4, KGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, syndecan-1, TACI, TAG-72, tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and variants thereof.

[0284] In some embodiments, the antigenic polypeptide, tumor antigenic polypeptide, or signaling polypeptide is an IL-2 peptide, IL-2-Ra, tdTomato, Cre recombinase, GFP, eGFP, anti-CD19, CD20, CAR-T, anti-HER2, etanercept (Enbrel), Humira, erythropoietin, epogen, filgrastim, Keytruda, rituximab, romiplostim, sargramostim, or a fragment or subunit thereof. In one embodiment, the polypeptide is an IL-2 peptide, or a fragment or subunit thereof. In one embodiment, the polypeptide is an erythropoietin or epogen, or a fragment or subunit thereof.

[0285] In some embodiments, the polypeptide encoded by the polynucleotide is IL-1α, IL-1 β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A / B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, TGF-β, GM-CSF, M-CSF, G-CSF, TNF-α, TNF-β, LAF, TCGF, ​​BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β, IFN-γ, uteroglobin, Fox p3, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CCL1, CC and an antigenic (e.g., tumor antigenic) or signaling polypeptide comprising L2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, XCL1, XCL2, CX3CL1, or a fragment, subunit, or variant thereof.

[0286] In some embodiments, the polypeptide encoded by the polynucleotide is an IL-15 peptide, IL-15-Ra, or a fragment or subunit thereof. In one embodiment, the polypeptide is an IL-15 peptide, or a fragment or subunit thereof.

[0287] In some embodiments, the antigenic polypeptide, tumor antigenic polypeptide, or signaling polypeptide comprises a tumor antigen selected from the group consisting of carcinoma, sarcoma, melanoma, lymphoma, leukemia, and combinations thereof.

[0288] In one embodiment, the tumor antigenic polypeptide comprises a melanoma antigen. In one embodiment, the tumor antigenic polypeptide comprises a prostate cancer antigen. In one embodiment, the tumor antigenic polypeptide comprises a HPV16 positive head and neck cancer antigen. In one embodiment, the tumor antigenic polypeptide comprises a breast cancer antigen. In one embodiment, the tumor antigenic polypeptide comprises an ovarian cancer antigen. In one embodiment, the tumor antigenic polypeptide comprises a lung cancer antigen. In one embodiment, the tumor antigenic polypeptide comprises an NSCLC antigen.

[0289] In some embodiments, the antigen is an autoantigenic polypeptide or immunogenic variant, or an immunogenic fragment thereof. In some embodiments, the autoantigenic polypeptide comprises an antigen that is typically expressed on a cell and is recognized by the immune system as an autoantigen. In some embodiments, the autoantigenic polypeptide comprises a multiple sclerosis antigenic polypeptide, a rheumatoid arthritis antigenic polypeptide, a lupus antigenic polypeptide, a celiac disease antigenic polypeptide, a Sjogren's syndrome antigenic polypeptide, or ankylosing spondylitis antigenic polypeptide, or a combination thereof.

[0290] Immune enhancing factor mRNA In some embodiments, the one or more polynucleotides include an mRNA encoding a polypeptide that stimulates or enhances an immune response to one or more cancer antigens of a subject. The mRNA that enhances an immune response to a cancer antigen of a subject is referred to herein as an immune enhancer mRNA construct or immune enhancer mRNA, including chemically modified mRNA (mmRNA). The immune enhancer enhances the immune response to a target antigen in a subject. The enhanced immune response can be a cellular response, a humoral response, or both. As used herein, a "cellular" immune response is intended to encompass an immune response that involves or is mediated by T cells, while a "humoral" immune response is intended to encompass an immune response that involves or is mediated by B cells. An immune enhancer can be, for example, (i) stimulating type I interferon pathway signaling; (ii) stimulating NFkB pathway signaling; (iii) stimulating an inflammatory response; (iv) stimulating cytokine production, or (v) stimulating the development, activity, or recruitment of dendritic cells; and (vi) Any combination of (i) to (vi) can enhance the immune response.

[0291] As used herein, "stimulating type I interferon pathway signaling" is intended to encompass activating one or more components of the type I interferon signaling pathway (e.g., modifying the phosphorylation or dimerization, etc., of such components to activate the pathway), stimulating transcription from an interferon sensitivity response element (ISRE), and / or stimulating the production or secretion of a type I interferon (e.g., IFN-a, IFN-β, IFN-ε, IFN-K, and / or IFN-co). As used herein, "stimulating NFkB pathway signaling" is intended to encompass activating one or more components of the NFkB signaling pathway (e.g., modifying the phosphorylation or dimerization, etc., of such components to activate the pathway), stimulating transcription from an NFkB site, and / or stimulating the production of a gene product whose expression is controlled by NFkB. As used herein, "stimulating an inflammatory response" is intended to encompass stimulating the production of inflammatory cytokines, including, but not limited to, type I interferon, IL-6, and / or TNFα. As used herein, "stimulating the development, activity, or recruitment of dendritic cells" is intended to encompass directly or indirectly stimulating the maturation, proliferation, and / or functional activity of dendritic cells.

[0292] In some embodiments, the mRNA encodes a polypeptide that stimulates or enhances an immune response in a subject in need thereof (e.g., enhances an immune response in a subject), for example, by inducing adaptive immunity (e.g., stimulating type I interferon production), stimulating an inflammatory response, stimulating FkB signaling, and / or stimulating the development, activity, or recruitment of dendritic cells (DCs) in the subject. In some embodiments, administration of the immune enhancer mRNA to a subject in need thereof enhances cellular immunity (e.g., T cell-mediated immunity), humoral immunity (e.g., B cell-mediated immunity), or both cellular and humoral immunity in the subject. In some embodiments, administration of the immune enhancer mRNA stimulates cytokine production (e.g., inflammatory cytokine production) and enhances the expression of cancer antigen-specific CD8 + Stimulates effector cell responses and antigen-specific CD4 + Stimulates helper cell responses and effector memory CD62L 10 In some embodiments, administration of the immune enhancer mRNA stimulates cytokine production (e.g., inflammatory cytokine production) and stimulates antigen-specific CD8 + Stimulates effector cell responses. In some embodiments, administration of immune enhancer mRNA stimulates cytokine production (e.g., inflammatory cytokine production) and stimulates antigen-specific CD4 + Stimulates a helper cell response. In some embodiments, administration of immune enhancer mRNA stimulates cytokine production (e.g., inflammatory cytokine production) and stimulates effector memory CD62L 10 Increases T cell populations. In some embodiments, administration of immune enhancer mRNA stimulates cytokine production (e.g., inflammatory cytokine production), stimulates B cell activity, or stimulates antigen-specific antibody production.

[0293] In one embodiment, the immune enhancing agent is a cancer antigen-specific CD8 + Increase effector cell responses (cell-mediated immunity). For example, immune enhancing factors include, but are not limited to, CD8+ One or more indicators of antigen-specific CD8+ effector cell activity can be increased, including T cell proliferation and CD8+ T cell cytokine production. For example, in one embodiment, the immune enhancing agent increases the production of IFN-γ, TNFa, and / or IL-2 by antigen-specific CD8+ T cells. In various embodiments, the immune enhancing agent can increase CD8+ T cell cytokine production (e.g., IFN-γ, TNFa, and / or IL-2 production) in response to an antigen by at least 5% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or at least 35% or at least 40% or at least 45% or at least 50% (compared to CD8+ T cell cytokine production in the absence of the immune enhancing agent). For example, T cells obtained from a treated subject can be stimulated in vitro with a cancer antigen, and CD8+ T cell cytokine production can be evaluated in vitro. CD8+ T cell cytokine production can be determined by standard methods known in the art, including, but not limited to, measuring the secretion level of cytokine production (e.g., by ELISA or other suitable methods known in the art for determining the amount of cytokine in the supernatant) and / or determining the percentage of CD8+ T cells that are positive for intracellular staining (ICS) of cytokines. For example, intracellular staining (ICS) of CD8+ T cells for expression of IFN-γ, TNFa, and / or JL-2 can be performed by methods known in the art. In one embodiment, the immune enhancing agent increases the percentage of CD8+ T cells that are positive for one or more cytokines (e.g., IFN-γ, TNFa, and / or IL-2) by at least 5% or at least 10% or at least 15%> or at least 20% or at least 25% or at least 30%> or at least 35% or at least 40% or at least 45% or at least 50% in response to an antigen (compared to the percentage of CD8+ T cells that are positive for cytokines by ICS in the absence of the immune enhancing agent).

[0294] In one embodiment, the immune enhancing agent increases the percentage of CD8+ T cells among the total T cell population (e.g., splenic T cells and / or PBMCs) compared to the percentage of CD8+ T cells in the absence of the immune enhancing agent. For example, the immune enhancing agent can increase the percentage of CD8+ T cells among the total T cell population by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, compared to the percentage of CD8+ T cells in the absence of the immune enhancing agent. The total percentage of CD8+ T cells among the total T cell population can be determined by standard methods known in the art, including but not limited to fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS).

[0295] In one embodiment, the immune enhancing agent increases tumor-specific immune cell response as determined by the reduction of tumor volume in vivo in the presence of the immune enhancing agent, compared to the tumor volume in the absence of the immune enhancing agent. For example, the immune enhancing agent can reduce tumor volume by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, compared to the tumor volume in the absence of the immune enhancing agent. Measurement of tumor volume can be determined by methods well established in the art. In another embodiment, the immune enhancing agent increases B cell activity (humoral immune response), for example, by increasing the amount of antigen-specific antibody production, compared to the antigen-specific antibody production in the absence of the immune enhancing agent. For example, the immune enhancing agent can increase antigen-specific antibody production by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, compared to the antigen-specific antibody production in the absence of the immune enhancing agent. In one embodiment, antigen-specific IgG production is evaluated. Antigen-specific antibody production can be evaluated by methods well established in the art, including but not limited to ELISA, RIA, etc., which measure the level of antigen-specific antibody (e.g., IgG) in a sample (e.g., serum sample).

[0296] In one embodiment, the immune enhancing factor is effector memory CD62L 10 Increases T cell population. For example, immune enhancing factors increase the expression of CD62L among CD8+ T cells. 10 It can increase the total percentage of effector memory CD62L T cells, among other functions. 10T cell populations have been shown to have important functions in intralymphoid trafficking (see, e.g., Schenkel, JM and Masopust, D. (2014) Immunity 41:886-897). In various embodiments, the immune enhancer enhances effector memory CD62L among CD8+ T cells in response to antigen. 10 The total percentage of T cells was calculated by dividing the total number of CD62L among CD8+ T cells in the absence of immune enhancing factors. 10 The effector memory CD62L among CD8+ T cells can be increased by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50% (compared to the total percentage of T cells). 10 The total percentage of T cells can be determined by standard methods known in the art, including but not limited to, fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS).

[0297] The ability of immune enhancer mRNA to enhance the immune response to cancer antigens can be evaluated in mouse model systems known in the art. In one embodiment, an immune competent mouse model system is used. In one embodiment, the mouse model system includes C57 / B16 mice. In another embodiment, the mouse model system includes BalbC mice or CD1 mice (e.g., to evaluate B cell responses, such as antigen-specific antibody responses). In one embodiment, the immune enhancer polypeptide functions downstream of at least one Toll-like receptor (TLR), thereby enhancing the immune response. Thus, in one embodiment, the immune enhancer is not a TLR, but a molecule in the TLR signaling pathway downstream of the receptor itself.

[0298] In one embodiment, the mRNA encoding the immune enhancing agent may include one or more modified nucleobases. Suitable modifications are discussed further below.

[0299] In one embodiment, the mRNA encoding the immune enhancing factor is formulated into the LPMP formulation. In one embodiment, the mRNA encodes a cancer antigen. In one embodiment, the LPMP / mRNA formulation is administered to a subject to enhance the immune response to a cancer antigen in the subject.

[0300] Immune enhancing factor mRNA that stimulates type I interferon In some embodiments, the present disclosure provides immune enhancer mRNAs that encode polypeptides that stimulate or enhance an immune response to an antigen of interest by stimulating or enhancing type I interferon pathway signaling, thereby stimulating or enhancing type I interferon (IFN) production.

[0301] Many components involved in type I IFN pathway signaling have been established, including STING, interferon regulatory factors such as IRF1, IRF3, IRF5, IRF7, IRF8, and IRF9, TBK1, IKKi, MyD88, and TRAM. Additional components involved in type I IFN pathway signaling include TRAF3TRAF6, IRAK-1, IRAK-4, TRIF, IPS-1, TLR-3, TLR-4, TLR-7, TLR-8, TLR-9, RIG-1, DAI, and IFI16.

[0302] Thus, in one embodiment, the immune enhancing factor mRNA encodes any of the aforementioned components involved in type I IFN pathway signaling.

[0303] Antigen-presenting cell promoter In some embodiments, the LPMP / mRNA therapeutic composition can be combined with agents to promote the production of antigen-presenting cells (APCs), for example by converting non-APCs into pseudo-APCs. Antigen presentation is a key step in the initiation, amplification, and duration of an immune response. In this process, fragments of antigens are

[0304] Antigens are presented through major histocompatibility complexes (MHC) or human leukocyte antigens (HLA) to T cells that provide antigen-specific immune responses. For immunoprophylaxis and therapy, enhancing this response is important to improve efficacy. LPMP / mRNA therapeutic compositions can be designed or enhanced to provide efficient antigen presentation. One way to enhance APC processing and presentation is to provide better targeting of LPMP / mRNA therapeutic compositions to antigen-presenting cells (APCs). Another approach involves activating APC cells with immune stimulating agents and / or components. Alternatively, methods to reprogram non-APCs to become APCs may be used with LPMP / mRNA therapeutic compositions. Importantly, most cells that take up mRNA agents and are the targets of their therapeutic action are not APCs. Therefore, designing methods to convert these cells into APCs would be beneficial to efficacy. Provided herein are methods and approaches to deliver LPMP / mRNA therapeutic compositions, such as mRNA vaccines, to cells while promoting the shift of non-APCs to APCs. In some embodiments, mRNA encoding an APC reprogramming molecule is included in or co-administered with an LPMP / mRNA therapeutic composition.

[0305] APC reprogramming molecules, as used herein, are molecules that promote the transition to APC-like phenotype in non-APC cells. APC-like phenotype is a property that allows MHC class II processing. Thus, APC cells with APC-like phenotype are cells that have one or more exogenous molecules (APC reprogramming molecules) and have enhanced MHC class II processing ability compared to the same cells that do not have one or more exogenous molecules. In some embodiments, APC reprogramming molecules are CUT A (a central regulator of MHC class II expression), chaperone proteins such as CLIP, HLA-DO, HLA-DM (enhancers of antigen fragment loading into MHC class II), and / or costimulatory molecules such as CD40, CD80, CD86 (enhancers of T cell antigen recognition and T cell activation).

[0306] The CIITA protein is a transcriptional activator that enhances the activation of transcription of MHC class II genes by interacting with a conserved set of DNA-binding proteins associated with the class II promoter region (Steimle et al., 1993, Cell 75: 135-146). The transcriptional activation function of CIITA has been mapped to the amino-terminal acidic domain (amino acids 26-137). Nucleic acid molecules encoding proteins that interact with CIITA are referred to as CIITA-interacting protein 104 (also referred to herein as CIP104). Both CITTA and CIP104 have been shown to enhance transcription from MHC class II promoters and are therefore useful as APC reprogramming molecules of the present invention. In some embodiments, the APC reprogramming molecule is full-length CIITA, CIP 104, or other related molecules or active fragments thereof, such as amino acids 26-137 of CIITA, or amino acids having at least 80% sequence identity thereto and maintaining the ability to enhance the transcriptional activation of MHC class II genes.

[0307] In some embodiments, the LPMP / mRNA therapeutic composition may include a recall antigen, sometimes referred to as a memory antigen. A recall antigen is an antigen that an individual has previously encountered and for which there are existing memory lymphocytes. In some embodiments, the recall antigen may be an infectious disease antigen that an individual has likely encountered, such as an influenza antigen. Recall antigens help promote a stronger immune response.

[0308] The antigen or neoepitope selected for inclusion in the LPMP / mRNA therapeutic composition is typically a high affinity binding peptide. In some embodiments, the antigen or neoepitope binds to HLA protein with higher affinity than wild-type peptide. In some embodiments, the antigen or neoepitope has an IC50 of at least 5000nM, at least 500nM, at least 250nM, at least 200nM, at least 150nM, at least 100nM, at least 50nM, or less. Typically, peptides with a predicted IC50<50nM are generally considered to be medium to high affinity binding peptides and are selected to test their affinity empirically using biochemical assays of HLA binding.

[0309] The cancer antigen may be an individualized cancer antigen. The LPMP / mRNA therapeutic composition may contain one or more known tumor-specific cancer antigens or RNA encoding each subject-specific cancer antigen, referred to as neoepitopes or subject-specific epitopes or antigens. A "subject-specific cancer antigen" is an antigen that has been identified as being expressed in a particular patient's tumor. Subject-specific cancer antigens may be typically present in tumor samples in general, but may also be absent. A tumor-associated antigen that is not expressed or is barely expressed in non-cancerous cells, or whose expression in non-cancerous cells is sufficiently reduced compared to that in cancerous cells to elicit an immune response that is elicited upon vaccination, is referred to as a neoepitope. Neoepitopes, such as tumor-associated antigens, are completely foreign to the body and therefore do not generate an immune response against healthy tissues or are masked by protective components of the immune system. In some embodiments, a neoepitope-based LPMP / mRNA therapeutic composition is desirable, as such a vaccine formulation will maximize the specificity for a patient's specific tumor. Mutation-derived neoepitopes can arise from point mutations, nonsynonymous mutations resulting in different amino acids in the protein, read-through mutations in which a stop codon is altered or deleted resulting in translation of a longer protein with a novel tumor-specific sequence at the C-terminus, splice site mutations resulting in the inclusion of an intron in the mature mRNA and thus a unique tumor-specific protein sequence, chromosomal rearrangements (i.e., gene fusions) giving rise to chimeric proteins with tumor-specific sequences at the junction of two proteins, frameshift mutations or deletions leading to a new open reading frame with a novel tumor-specific protein sequence, and translocations.

[0310] Thus, in some embodiments, the LPMP / mRNA therapeutic composition comprises at least one cancer antigen comprising a mutation selected from the group consisting of a frameshift mutation and a recombination, or any of the other mutations described herein. In some embodiments, the LPMP / mRNA therapeutic composition comprises at least one immunogenic polypeptide comprising a mutation selected from the group consisting of a frameshift mutation and a recombination, or any of the other mutations described herein. In some embodiments, the LPMP / mRNA therapeutic composition comprises at least one signaling polypeptide comprising a mutation selected from the group consisting of a frameshift mutation and a recombination, or any of the other mutations described herein.

[0311] Nucleic acid sequence In some embodiments, the polynucleotide is a polynucleotide construct that encodes one or more wild-type or engineered antigens (or antibodies to antigens). In some embodiments, the polynucleotide comprises an antigenic polypeptide or immunogenic variant, or an immunogenic fragment thereof. The antigen may be derived from a tumor, e.g., a tumor-specific antigen, a tumor-associated antigen, a tumor neo-antigen, or a combination thereof. In some embodiments, the polynucleotide comprises a signaling protein or an anti-cancer protein, such as a secreted cytokine, a cytokine receptor complex, a hormone, or an immune enhancer.

[0312] In some embodiments, the polynucleotide may be an mRNA, an siRNA or siRNA precursor, a microRNA (miRNA) or miRNA precursor, a plasmid, a dicer substrate small interfering RNA (dsiRNA), a small hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), a peptide nucleic acid (PNA), a morpholino, a locked nucleic acid (LNA), a piwi-interacting RNA (piRNA), a ribozyme, a deoxyribozyme (DNAzyme), an aptamer, a circular RNA (circRNA), a guide RNA (gRNA), or a DNA molecule encoding any of these RNAs. In one embodiment, the polynucleotide is an mRNA.

[0313] In some embodiments, the polynucleotide is selected from the group consisting of p53, ART-4, BAGE, ss-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, CLAUDIN-12, C-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A2. and encoding a polypeptide comprising E-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Plac-1, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or S ART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survivin, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, WT-1, or a combination thereof.

[0314] In some embodiments, the polynucleotide encodes an IL-2 peptide, IL-2-Ra, tdTomato, Cre recombinase, GFP, eGFP, anti-CD19, CD20, CAR-T, anti-HER2, etanercept (Enbrel), Humira, erythropoietin, epogen, filgrastim, Keytruda, rituximab, romiplostim, sargramostim, or variants, fragments, or subunits thereof. Exemplary sequences include those shown in Table 9.

[0315] In some embodiments, the polynucleotide encodes an IL-15 peptide, IL-15-Ra, or a fragment or subunit thereof. In one embodiment, the polypeptide is an IL-15 peptide, or a fragment or subunit thereof. Exemplary sequences include those shown in Table 9.

[0316] In some embodiments, the polynucleotide is IL-1α, IL-1 β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A / B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, TGF-β, GM-CSF, M-CSF, G-CSF, TNF-α, TNF-β, LAF, TCGF, ​​BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β, IFN-γ, Utelog Robin, Foxp3, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, Encodes CXCL16, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, XCL1, XCL2, CX3CL1, or a fragment, subunit, or variant thereof.

[0317] An antigen variant or other polypeptide variant refers to a molecule whose amino acid sequence is different from a wild type, natural or reference sequence. A polypeptide variant may have substitutions, deletions, and / or insertions at certain positions in the amino acid sequence compared to a natural or reference sequence. Usually, a variant has at least 50% identity with a wild type, natural or reference sequence. In some embodiments, a variant shares at least 80% or at least 90% identity with a wild type, natural or reference sequence.

[0318] Variant polypeptides encoded by the nucleic acids of the present disclosure may contain amino acid changes that confer any of a number of desirable properties, for example, enhancing their immunogenicity, enhancing their expression, and / or improving their stability or PK / PD properties in a subject. Variant antigens / polypeptides can be made using routine mutagenesis techniques and assayed as needed to determine whether they have the desired properties. Assays for determining expression levels and immunogenicity are well known in the art, and examples of such assays are described in the Examples section. Similarly, the PK / PD properties of protein variants can be measured using art-recognized techniques, for example, by determining the expression of antigens in vaccinated subjects over time and / or by examining the durability of the induced immune response. The stability of the protein(s) encoded by the variant nucleic acids may be measured by assaying thermal stability or stability upon urea denaturation, or may be measured using in silico predictions. Methods for such experiments and in silico determinations are known in the art.

[0319] The term "identity" refers to the relationship between the sequences of two or more polypeptides (e.g., antigens, anti-cancer proteins, or signaling proteins) or polynucleotides (nucleic acids), as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or among sequences, as determined by the number of matches between a series of two or more amino acid or nucleic acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences, with gap alignment (if any) specified by a particular mathematical model or computer program (e.g., "algorithm"). The identity of related antigens, proteins, or nucleic acids can be readily calculated by known methods. "Percentage of identity" as applied to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid or nucleic acid residues) of a candidate amino acid or nucleic acid sequence that are identical to the residues of the amino acid or nucleic acid sequence of a second sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art. It is understood that identity depends on the calculation of percent identity, but the value may vary depending on the gaps and penalties introduced in the calculation. Generally, a variant of a particular polynucleotide or polypeptide (e.g., an antigen, an anti-cancer protein, or a signaling protein) has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those of skill in the art.Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). Another common local alignment technique is based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147:195-197). A common global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48:443-453). More recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is intended to produce global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

[0320] Thus, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and / or additions, deletions, and covalent modifications with respect to reference sequences, particularly polypeptide (e.g., antigen or signaling protein) sequences disclosed herein, are included within the scope of this disclosure. For example, sequence tags or amino acids such as one or more lysines can be added to the peptide sequence (e.g., at the N-terminus or C-terminus). Sequence tags can be used for peptide detection, purification, or localization. Lysines can be used to increase peptide solubility or to enable biotinylation. Alternatively, amino acid residues located in the carboxy- and amino-terminal regions of the peptide or protein amino acid sequence can be optionally deleted to result in truncated sequences. Certain amino acids (e.g., C- or N-terminal residues) can be alternatively deleted depending on the use of the sequence, for example, expression of the sequence as part of a larger sequence linked to a soluble or solid support. In some embodiments, sequences such as (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (e.g., foldon regions, etc.) can be replaced with alternative sequences that achieve the same or similar functions. In some embodiments, cavities in the core of the protein can be filled to improve stability, for example, by introducing larger amino acids. In other embodiments, buried hydrogen bond networks can be replaced with hydrophobic residues to improve stability. In still other embodiments, glycosylation sites can be removed and replaced with appropriate residues. Such sequences are readily identifiable to those skilled in the art. It should also be understood that some of the sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N-terminus or C-terminus) that can be deleted, for example, before use in preparing an RNA (e.g., mRNA) vaccine.

[0321] As will be appreciated by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of the coronavirus antigen of interest. For example, any protein fragment (meaning a polypeptide sequence that is at least one amino acid residue shorter than the reference antigen sequence but otherwise identical) of a reference protein is provided herein, provided that the fragment is immunogenic and confers a protective immune response against coronavirus. In addition to variants that are identical to the reference protein but are truncated, in some embodiments, the antigen comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein. Antigens / antigenic polypeptides can range in length from about 4, 6, or 8 amino acids to the full-length protein.

[0322] In some embodiments, the polynucleotide is an mRNA. Messenger RNA (mRNA) is any RNA that encodes (at least one) protein (naturally occurring, non-naturally occurring, or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. Those skilled in the art will understand that unless otherwise stated, the nucleic acid sequences described in this application may recite "T" in a representative DNA sequence, but when the sequence represents an RNA (e.g., mRNA), "T" is replaced with "U". Thus, any of the DNAs disclosed and identified by a particular sequence identification number herein also discloses the corresponding RNA (e.g., mRNA) sequence that is complementary to the DNA, and each "T" in the DNA sequence is replaced with "U".

[0323] In some embodiments, a polynucleotide (e.g., mRNA) has an open reading frame (ORF) that encodes an antigen, tumor antigen, or signaling polypeptide. An open reading frame (ORF) is a continuous stretch of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). ORFs typically code for proteins. The sequence may further include additional elements, such as 5'UTR and 3'UTR.

[0324] In some embodiments, the RNA (eg, mRNA) further comprises a 5'UTR, a 3'UTR, a poly(A) tail, and / or a 5' cap analog.

[0325] In some embodiments, the mRNA comprises a 5' untranslated region (UTR) and / or a 3' UTR.

[0326] In some embodiments, the mRNA is derived from (a) a DNA molecule, or (b) an RNA molecule. In the mRNA, T is optionally replaced with U.

[0327] In some embodiments, the mRNA is derived from a DNA molecule. The DNA molecule may further comprise a promoter. In some embodiments, the promoter is a T7 promoter, a T3 promoter, or a SP6 promoter. In some embodiments, the promoter is located in the 5'UTR.

[0328] In some embodiments, the mRNA is an RNA molecule. The RNA molecule may be a self-replicating RNA molecule.

[0329] In some embodiments, the mRNA is an RNA molecule. The RNA molecule may further comprise a 5' cap. The 5' cap may have a cap1 structure, a cap1(m6A) structure, a cap2 structure, a cap3 structure, a cap0 structure, or any combination thereof.

[0330] In some embodiments, the polynucleotide is an mRNA encoding an IL-2 molecule. In one embodiment, the IL-2 molecule comprises a naturally occurring IL-2 molecule, a fragment of a naturally occurring IL-2 molecule, or a variant thereof. In one embodiment, the IL-2 molecule comprises a variant of a naturally occurring IL-2 molecule (e.g., an IL-2 variant described herein), or a fragment thereof.

[0331] In one embodiment, the polynucleotide is an mRNA encoding an IL-2 molecule comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of an IL-2 molecule provided in any one of Tables I-III.

[0332] In some embodiments, the polynucleotide is an mRNA encoding an IL-15 molecule. In one embodiment, the IL-15 molecule comprises a naturally occurring IL-15 molecule, a fragment of a naturally occurring IL-15 molecule, or a variant thereof. In one embodiment, the IL-15 molecule comprises a variant of a naturally occurring IL-15 molecule (e.g., an IL-15 variant described herein), or a fragment thereof.

[0333] In one embodiment, the polynucleotide is an mRNA encoding an IL-15 molecule comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of an IL-15 molecule provided in Table IV.

[0334] In some embodiments, the mRNA comprises a 5' untranslated region (UTR) and / or a 3' UTR.

[0335] In some embodiments, the mRNA comprises a 5'UTR. The 5'UTR may comprise a Kozak sequence.

[0336] In some embodiments, the mRNA comprises a 3'UTR. In some embodiments, the 3'UTR comprises one or more sequences derived from a split amino-terminal enhancer (AES). In some embodiments, the 3'UTR comprises a sequence derived from mitochondrially encoded 12S rRNA (mtRNRl).

[0337] In some embodiments, the mRNA comprises a poly(A) sequence. In one embodiment, the poly(A) sequence is a 110 nucleotide sequence consisting of a sequence of 30 adenosine residues, a linker sequence of 10 nucleotides, and a sequence of 70 adenosine residues. [Table I-1] [Table I-2] [Table I-3] [Table I-4] [Table I-5] [Table I-6] [Table II-1] [Table II-2] [Table II-3] [Table II-4] [Table II-5] [Table II-6] [Table III-1] [Table III-2] [Table III-3] [Table III-4] [Table III-5] [Table III-6] [Table III-7] [Table III-8] [Table III-9] [Table IV]

[0338] Stabilizing Element Naturally occurring eukaryotic mRNA molecules may contain stabilizing elements, including but not limited to untranslated regions (UTRs), at their 5'-ends (5'UTRs) and / or their 3'-ends (3'UTRs), in addition to other structural features, such as a 5'-cap structure or a 3'-poly(A) tail. Both the 5'UTR and the 3'UTR are typically transcribed from genomic DNA and are elements of the immature mRNA. The characteristic structural features of mature mRNAs, such as the 5'-cap and the 3'-poly(A) tail, are usually added to the transcribed (immature) mRNA during mRNA processing.

[0339] In some embodiments, the polynucleotide has an open reading frame encoding an antigenic, tumor antigenic, or signaling polypeptide with at least one modification, at least one 5'-end cap, and is formulated in lipid nanoparticles. 5'-capping of the polynucleotide can be completed simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs to generate a 5'-guanosine cap structure according to the manufacturer's protocol: 3'-O-Me-m7G(5')ppp(5') G [ARCA cap] G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). 5'-capping of the modified RNA can be completed post-transcriptionally using vaccinia virus capping enzyme to generate a "cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). The cap 1 structure can be generated using both vaccinia virus capping enzyme and a 2'-0 methyl-transferase to generate m7G(5')ppp(5')G-2'-O-methyl. The cap 2 structure can be generated from the cap 1 structure, followed by 2'-0-methylation of the 5'-antipinaltimete nucleotide using a 2'-O methyl-transferase. The cap 3 structure can be generated from the cap 2 structure, followed by 2'-O-methylation of the 5'-preantipinaltimete nucleotide using a 2'-O methyl-transferase. The enzymes can be derived from recombinant sources.

[0340] A 3'-poly(A) tail is typically a stretch of adenine nucleotides added to the 3' end of a transcribed mRNA. It may contain up to about 400 adenine nucleotides in some cases. In some embodiments, the length of the 3'-poly(A) tail may be an essential element for the stability of an individual mRNA.

[0341] In some embodiments, the polynucleotide comprises a stabilizing element. The stabilizing element may comprise, for example, a histone stem loop. A 32 kDa protein, stem-loop binding protein (SLBP), has been identified. It associates with histone stem loops at the 3' end of histone messages in both the nucleus and the cytoplasm. Its expression level is regulated by the cell cycle, peaking during S phase when histone mRNA levels also rise. The protein has been shown to be essential for efficient 3' end processing of histone pre-mRNA by U7 snRNP. SLBP remains associated with stem loops after processing and then stimulates translation of mature histone mRNA into histone protein in the cytoplasm. The RNA binding domain of SLBP is conserved through metazoans and protozoans, and its binding to histone stem-loops depends on the structure of the loop. The minimal binding site contains at least three nucleotides 5' and two nucleotides 3' to the stem-loop.

[0342] In some embodiments, the polynucleotide (e.g., mRNA) comprises a coding region, at least one histone stem loop, and, optionally, a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal should generally enhance the expression level of the encoded protein. The encoded protein, in some embodiments, is not a histone protein, a reporter protein (e.g., luciferase, GFP, EGFP, b-galactosidase, EGFP), or a marker or selection protein (e.g., alpha-globin, galactokinase, and xanthine:guanine phosphoribosyltransferase (GPT)).

[0343] In some embodiments, a polynucleotide (e.g., an mRNA) contains a combination of a poly(A) sequence or a polyadenylation signal and at least one histone stem-loop, both of which represent alternative mechanisms in nature, but act synergistically to increase protein expression beyond the levels observed with either of the individual elements. The synergistic effect of the combination of poly(A) and at least one histone stem-loop is not dependent on the order of the elements or the length of the poly(A) sequence. In some embodiments, the RNA (e.g., an mRNA) does not contain a histone downstream element (HDE). A "histone downstream element" (HDE) comprises a purine-rich polynucleotide stretch of approximately 15-20 nucleotides 3' of a naturally occurring stem-loop, which represents a binding site for U7 snRNA involved in processing of histone pre-mRNA to mature histone mRNA. In some embodiments, the nucleic acid does not contain an intron.

[0344] A polynucleotide (e.g., mRNA) may or may not contain enhancer and / or promoter sequences, which may be modified or unmodified, or may be activated or inactivated. In some embodiments, a histone stem-loop generally comprises intramolecular base pairing of two adjacent partially or completely reverse-complementary sequences separated by a spacer, consisting of a short sequence derived from a histone gene and forming a loop of the structure. The unpaired loop region typically cannot base pair with either of the stem-loop elements. This occurs more frequently in RNA, as it is a major component of many RNA secondary structures, but can also occur in single-stranded DNA. The stability of the stem-loop structure generally depends on the length of the paired region, the number of mismatches or bulges, and the base composition. In some embodiments, hobble base pairing (non-Watson-Crick base pairing) can occur. In some embodiments, at least one histone stem-loop sequence comprises 15-45 nucleotides in length.

[0345] In some embodiments, the polynucleotide (e.g., mRNA) has one or more AU-rich sequences removed. These sequences, sometimes called AURES, are destabilizing sequences found in the 3'UTR. AURES may be removed from the RNA vaccine. Alternatively, AURES may remain in the RNA vaccine.

[0346] Signal peptide In some embodiments, the polynucleotide (e.g., mRNA) has an ORF encoding a signal peptide fused to a coronavirus antigen. Signal peptides, which include 15-60 amino acids at the N-terminus of a protein, are typically required for translocation across membranes on the secretory pathway and thus universally control the entry of most proteins into the secretory pathway in both eukaryotes and prokaryotes. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates transport of the growing peptide chain across it for processing. ER processing produces a mature protein, and the signal peptide is typically cleaved from the precursor protein by the host cell's ER-resident signal peptidases, or they are not cleaved and function as a membrane anchor. Signal peptides may also facilitate targeting of proteins to cell membranes. Signal peptides may have a length of 15-60 amino acids. For example, the signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, the signal peptide is 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40 -50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15 to 20 amino acids in length.

[0347] Signal peptides from heterologous genes (which naturally regulate expression of genes other than coronavirus antigens) are known in the art and can be tested for desired properties and then incorporated into the nucleic acids of the present disclosure. In some embodiments, signal peptides can include those described in WO2021 / 154763, which is incorporated by reference in its entirety.

[0348] Fusion proteins In some embodiments, the polynucleotide (e.g., mRNA) encodes an antigenic fusion protein. Thus, the encoded antigen or antigens may comprise two or more proteins (e.g., proteins and / or protein fragments) linked together. Alternatively, the protein to which the protein antigen is fused does not promote a strong immune response against itself, but rather promotes a strong immune response against the coronavirus antigen. The antigen fusion protein, in some embodiments, retains functional properties from each original protein.

[0349] Scaffold part The polynucleotide (e.g., mRNA), in some embodiments, encodes a fusion protein that includes a coronavirus antigen linked to a scaffold moiety. In some embodiments, such a scaffold moiety confers a desired property to the antigen encoded by the nucleic acid of the present disclosure. For example, the scaffold protein may improve the immunogenicity of the antigen, for example, by altering the structure of the antigen, altering the uptake and processing of the antigen, and / or binding the antigen to a binding partner.

[0350] In some embodiments, the scaffold moiety is a protein that can self-assemble into highly symmetric, stable, and structurally organized protein nanoparticles in the 10-150 nm diameter range, a size range highly suitable for optimal interaction with various cells of the immune system.

[0351] In some embodiments, bacterial protein platforms may be used. Non-limiting examples of these self-assembling proteins include ferritin, lumazine and encapsulin.

[0352] Ferritin is a protein whose main function is intracellular iron storage. It consists of 24 subunits, each consisting of four alpha-helical bundles, which self-assemble into a quaternary structure with octahedral symmetry (Cho KJ et al. J Mol Biol. 2009; 390:83-98). Several high-resolution structures of ferritin have been determined, confirming that Helicobacter pylori ferritin consists of 24 identical protomers, whereas in animals there are ferritin light and heavy chains that can assemble alone or combine in different ratios into particles of 24 subunits (Granier T. et al. J Biol Inorg Chem. 2003; 8:105-111; Fawson DM et al. Nature. 1991; 349:541-544). Ferritin self-assembles into nanoparticles with robust thermal and chemical stability. Therefore, ferritin nanoparticles are well suited to carry and expose antigens.

[0353] Fusidine synthase (FS) is also well suited as a nanoparticle platform for antigen display. FS, involved in the penultimate catalytic step in the biosynthesis of riboflavin, is an enzyme present in a wide range of organisms, including archaea, bacteria, fungi, plants, and eubacteria (Weber SE Flavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology. 2014). The FS monomer is 150 amino acids long and consists of a beta sheet with tandem alpha helices flanking its sides. A large number of different quaternary structures have been reported for FS, showing its morphological versatility, from homopentamers to symmetric assemblies of 12 pentamers that form capsids with a diameter of 150A. Even FS cages of more than 100 subunits have been described (Zhang X. et al. J Mol Biol. 2006; 362:753-770).

[0354] Encapsulin, a novel protein cage nanoparticle isolated from Thermophia Thermotoga maritima, can also be used as a platform for presenting antigens on the surface of self-assembled nanoparticles. Encapsulin is assembled from 60 copies of identical 31 kDa monomers with a thin and icosahedral T=1 symmetric cage structure with inner and outer diameters of 20 nm and 24 nm, respectively (Sutter M. et al. Nat Struct Mol Biol. 2008, 15: 939-947). The exact function of encapsulin in T. maritima is not yet clearly understood, but its crystal structure has recently been solved and its function has been hypothesized as a cellular compartment that encapsulates proteins such as DyP (peroxidase decolorizing pigment) and Flp (ferritin-like protein) involved in oxidative stress response (Rahmanpour R. et al. FEBS J. 2013, 280: 2097-2104).

[0355] In some embodiments, the polynucleotide encodes a coronavirus antigen (e.g., SARS-CoV-2 S protein) fused to a foldon domain. The foldon domain may be obtained, for example, from bacteriophage T4 fibritin (see, e.g., Tao Y, et al. Structure. 1997 Jun 15; 5(6):789-98).

[0356] Linkers and cleavable peptides In some embodiments, a polynucleotide (e.g., an mRNA) encodes two or more polypeptides, referred to herein as a fusion protein. In some embodiments, the mRNA further encodes a linker located between at least one or each domain of the fusion protein. The linker can be, for example, a cleavable linker or a protease-sensitive linker. In some embodiments, the linker is selected from the group consisting of an F2A linker, a P2A linker, a T2A linker, an E2A linker, and combinations thereof. This family of self-cleaving peptide linkers, called 2A peptides, has been described in the art (see, e.g., Kim, JH et al. (2011) PLoS ONE 6:el8556). In some embodiments, the linker is an F2A linker. In some embodiments, the fusion protein contains three domains with an intervening linker having the structure: domain-linker-domain-linker-domain.

[0357] Any cleavable linker known in the art may be used in the context of the present disclosure. Examples of such linkers include F2A linkers, T2A linkers, P2A linkers, E2A linkers (see, for example, International Publication No. WO 2017 / 127750, which is incorporated herein by reference in its entirety). Those skilled in the art will understand that other art-recognized linkers may be suitable for use in the constructs of the present disclosure (e.g., encoded by the nucleic acids of the present disclosure). Similarly, those skilled in the art will understand that other polycistronic constructs (mRNAs that encode more than one antigen / polypeptide separately within the same molecule) may be suitable for use as provided herein.

[0358] Array Optimization In some embodiments, the ORF that codes for the antigen, tumor antigen, signal transduction or anti-cancer protein of the present disclosure is codon-optimized.Codon optimization methods are known in the art.For example, any one or more ORFs of the sequences provided herein may be codon-optimized. Codon optimization can be used in some embodiments to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that can impair gene assembly or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove / add post-translational modification sites in the encoded protein (e.g., glycosylation sites), add, remove, or shuffle protein domains, insert or delete restriction enzyme sites, modify ribosome binding sites and mRNA degradation sites, adjust translation rates to allow various domains of a protein to fold properly, or reduce or remove problematic secondary structures in polynucleotides. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or proprietary services. In some embodiments, the open reading frame (ORF) sequence is optimized using an optimization algorithm.

[0359] In some embodiments, the codon-optimized sequence shares less than 95% sequence identity with a naturally occurring or wild-type sequence ORF (e.g., a naturally occurring or wild-type mRNA sequence encoding a coronavirus antigen). In some embodiments, the codon-optimized sequence shares less than 90% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a coronavirus antigen). In some embodiments, the codon-optimized sequence shares less than 85% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a coronavirus antigen). In some embodiments, the codon-optimized sequence shares less than 80% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a coronavirus antigen). In some embodiments, the codon-optimized sequence shares less than 75% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a coronavirus antigen).

[0360] In some embodiments, the codon-optimized sequence shares 65%-85% (e.g., about 67%-85% or about 67%-80%) sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a coronavirus antigen). In some embodiments, the codon-optimized sequence shares 65%-75% or about 80% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a coronavirus antigen).

[0361] In some embodiments, the codon-optimized sequence encodes an antigen that is as immunogenic or more immunogenic (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% or more) than a coronavirus antigen encoded by a non-codon-optimized sequence. When transfected into a mammalian host cell, the modified mRNA has a stability of 12-18 hours, or more than 18 hours, e.g., 24, 36, 48, 60, 72 hours, or more than 72 hours, and can be expressed by the mammalian host cell.

[0362] In some embodiments, the codon-optimized RNA may be an RNA with an enhanced level of G / C. The G / C content of a nucleic acid molecule (e.g., mRNA) may affect the stability of the RNA. An RNA with an increased amount of guanine (G) and / or cytosine (C) residues may be more functionally stable than an RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. As an example, WO 02 / 098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by replacing existing codons with ones that promote higher RNA stability without changing the resulting amino acid. The approach is limited to the coding region of the RNA.

[0363] Chemically unmodified nucleotides In some embodiments, the polynucleotide (e.g., mRNA) is not chemically modified and comprises standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, the nucleotides and nucleosides of the polynucleotide (e.g., mRNA) comprise standard nucleoside residues such as those present in transcribed RNA (e.g., A, G, C, or U). In some embodiments, the nucleotides and nucleosides of the polynucleotide (e.g., mRNA) comprise standard deoxyribonucleosides such as those present in DNA (e.g., dA, dG, dC, or dT).

[0364] chemical modification A polynucleotide (e.g., mRNA) in some embodiments comprises an RNA having an open reading frame encoding a coronavirus antigen, and the nucleic acid comprises nucleotides and / or nucleosides that can be standard (unmodified) or modified as known in the art. In some embodiments, the nucleotides and nucleosides of a polynucleotide (e.g., mRNA) comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include art-recognized modifications in the sugar, backbone, or nucleobase moieties of the nucleotides and / or nucleosides.

[0365] The nucleic acid of a polynucleotide (eg, an mRNA) can contain standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, non-naturally occurring nucleotides and nucleosides, or any combination thereof.

[0366] The nucleic acids of a polynucleotide (e.g., DNA nucleic acids and RNA nucleic acids such as mRNA nucleic acids) in some embodiments comprise a variety (more than two) different types of standard and / or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two, or more (optionally different) types of standard and / or modified nucleotides and nucleosides.

[0367] In some embodiments, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into a cell or organism exhibit reduced degradation in the cell or organism, respectively, compared to unmodified nucleic acids comprising standard nucleotides and nucleosides.

[0368] In some embodiments, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into a cell or organism may exhibit reduced immunogenicity (e.g., reduced innate immune response) in the cell or organism, respectively, compared to unmodified nucleic acids comprising standard nucleotides and nucleosides.

[0369] Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) in some embodiments contain non-naturally occurring modified nucleotides that are introduced during or after synthesis of the nucleic acid to achieve a desired function or property. Modifications may be present on the internucleotide linkage, the purine or pyrimidine base, or the sugar. Modifications may be introduced at the end of the chain or anywhere else within the chain, using chemical synthesis or using polymerase enzymes. Any region of the nucleic acid may be chemically modified.

[0370] The present disclosure provides modified nucleosides and nucleotides of nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids). A "nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). A "nucleotide" refers to a nucleoside that includes a phosphate group. Modified nucleotides can be synthesized by any useful method, e.g., chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. A nucleic acid can include a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the nucleic acid will include a region of nucleotides.

[0371] Modified nucleotide base pairing includes not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides containing non-standard or modified bases and / or modified nucleotides, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between non-standard and standard bases, or between two complementary non-standard base structures, for example, in nucleic acids having at least one chemical modification. One example of such non-standard base pairing is base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker may be incorporated into the nucleic acid of the present disclosure.

[0372] In some embodiments, a polynucleotide (e.g., an mRNA) contains a uridine at one or more or all uridine positions of a nucleic acid. In some embodiments, an mRNA is uniformly modified for a particular modification (e.g., completely modified, modified throughout the entire sequence). For example, a nucleic acid can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by substitution with a modified residue, such as those modified residues described above.

[0373] The nucleic acids of a polynucleotide (e.g., an mRNA) may be partially or completely modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purines or pyrimidines, or any one or more or all of A, G, U, C) may be uniformly modified in the nucleic acids of the present disclosure, or in a given sequence region thereof (e.g., in an mRNA, including or excluding a poly(A) tail). In some embodiments, every nucleotide X in a nucleic acid of the present disclosure (or in a sequence region thereof) is a modified nucleotide, where X may be any one of the nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0374] Nucleic acids may contain from about 1% to about 100% modified nucleotides (with respect to overall nucleotide content or with respect to any one or more types of nucleotides, i.e., A, G, U, or C) or any intervening percentage (e.g., 1%-20%, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-10 ... 0%, 1%~95%, 10%~20%, 10%~25%, 10%~50%, 10%~60%, 10%~70%, 10%~80%, 10%~90%, 10%~95%, 10%~100%, 20%~25%, 20%~50%, 20%~60%, 20%~70%, 20%~80%, 20%~90%, 20%~95%, 20%~100%, 50%~60%, 50%~70%, The amino acid sequence may contain 50%-80%, 50%-90%, 50%-95%, 50%-100%, 70%-80%, 70%-90%, 70%-95%, 70%-100%, 80%-90%, 80%-95%, 80%-100%, 90%-95%, 90%-100%, and 95%-100%). Of course, any remaining percentages are accounted for by the presence of unmodified A, G, U, or C.

[0375] The mRNA may contain a minimum of 1% and a maximum of 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the nucleic acid may contain modified pyrimidines, such as modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracils in the nucleic acid are replaced with modified uracils (e.g., 5-substituted uracils). The modified uracils may be replaced by a compound with a single unique structure, or by multiple compounds with different structures (e.g., two, three, four, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in the nucleic acid are substituted with modified cytosines (e.g., 5-substituted cytosines). The modified cytosines may be replaced by a compound having a single unique structure, or may be replaced by multiple compounds having different structures (e.g., 2, 3, 4, or more unique structures).

[0376] Untranslated Regions (UTRs) A polynucleotide (e.g., mRNA) may contain one or more regions or portions that act or function as untranslated regions. When an mRNA is designed to code at least one protein of interest, the polynucleotide may contain one or more of these untranslated regions (UTRs). Wild-type untranslated regions of a nucleic acid sequence are transcribed but not translated. In an mRNA, the 5'UTR begins at the transcription initiation site and follows but does not include the initiation codon, while the 3'UTR begins immediately after the stop codon and continues until the transcription termination signal. There is growing evidence regarding the regulatory role that UTRs play in the stability and translation of nucleic acid molecules. The regulatory properties of UTRs may be incorporated into the polynucleotides of the present disclosure, inter alia, to enhance the stability of the molecule. Certain properties may be incorporated to ensure controlled downregulation of the transcript when it is misdirected to an undesired organ site. A variety of 5'UTR and 3'UTR sequences are known and available in the art.

[0377] The 5'UTR is the region of an mRNA immediately upstream (5') from the start codon (the first codon of an mRNA transcript that is translated by the ribosome). The 5'UTR does not code for a protein (it is non-coding). Naturally occurring 5'UTRs have properties that play a role in translation initiation. They have properties such as the Kozak sequence, which is commonly known to be involved in the process by which the ribosome initiates the translation of many genes. The Kozak sequence has the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". The 5'UTR is also known to form secondary structures involved in elongation factor binding.

[0378] In some embodiments, the 5'UTR is a heterologous UTR, i.e., a UTR found in nature associated with a different ORF. In another embodiment, the 5'UTR is a synthetic UTR, i.e., does not occur in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties, for example to increase gene expression, but also UTRs that are completely synthetic. Exemplary 5'UTRs include a-globin or b-globin from Xenopus or human (8278063, 9012219), human cytochrome b-245 polypeptide, and hydroxysteroid (17b) dehydrogenase, and tobacco etch virus (U.S. Patent Nos. 8278063, 9012219, incorporated herein by reference in their entirety). The CMV immediate early 1 (IE1) gene (US2014 / 0206753, WO2013 / 185069, incorporated herein by reference in its entirety), the sequence GGGAUCCUACC (WO2014 / 144196) may also be used. In another embodiment, the 5'UTR of the TOP gene is a 5'UTR of the TOP gene lacking the 5'TOP motif (oligopyrimidine tract) (e.g., WO2015 / 101414, WO2015 / 101415, WO2015 / 062738, WO2015 / 024667, WO2015 / 024667), and a 5'UTR element derived from the ribosomal protein large 32 (L32) gene. 5'UTR elements from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (WO 2015 / 024667), or the 5'UTR element from the 5'UTR of ATP5A1 (WO 2015 / 024667) may be used. In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5'UTR.

[0379] The 3'UTR is the region of an mRNA immediately downstream (3') from the stop codon (the codon of the mRNA transcript that signals the end of translation). The 3'UTR does not code for a protein (it is non-coding). Native or wild-type 3'UTRs are known to have stretches of adenosines and uridines embedded in them. These AU-rich features are particularly prevalent in genes with high turnover rates. Based on their sequence and functional properties, AU-rich elements (AREs) can be divided into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules that contain this type of ARE include GM-CSF and TNF-α. Class III AREs are less well defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples of this class.

[0380] Most proteins that bind to AREs are known to destabilize messengers, but members of the ELAV family, particularly HuR, have been reported to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule results in HuR binding and thus stabilization of the message in vivo.

[0381] The introduction, removal or modification of 3'UTR AU-rich elements (AREs) can be used to modulate the stability of polynucleotides (e.g., mRNAs). When engineering a particular nucleic acid, one or more copies of an ARE can be introduced to reduce the stability of the nucleic acid of the present disclosure, thereby reducing translation and reducing the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability and thus increase the translation and production of the resulting protein. Transfection experiments can be performed in relevant cell lines using the nucleic acids of the present disclosure, and protein production can be assayed at various time points after transfection. For example, cells can be transfected with the relevant protein by using an ELISA kit with molecules engineering different AREs, and the protein produced can be assayed 6 hours, 12 hours, 24 hours, 48 ​​hours, and 7 days after transfection.

[0382] The 3'UTR may be heterologous or synthetic.

[0383] Those skilled in the art will appreciate that heterologous or synthetic 5'UTRs can be used with any desired 3'UTR sequence. For example, a heterologous 5'UTR can be used with a synthetic 3'UTR having a heterologous 3'UTR.

[0384] Non-UTR sequences may be used as regions or subregions within nucleic acid. For example, introns or parts of intron sequences may be incorporated into regions of the nucleic acid sequences of the present disclosure. The incorporation of intron sequences may increase protein production as well as nucleic acid levels.

[0385] A combination of features may be included in a flanking region or may be contained within another feature. For example, an ORF may be flanked by a 5'UTR that may contain a strong Kozak translation initiation signal and / or a 3'UTR that may contain an oligo(dT) sequence for templated addition of a polyA tail. The 5'UTR may include a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes, such as the 5'UTRs described in US Patent Application Publication Nos. 2010 / 0293625 and PCT / US2014 / 069155, which are incorporated herein by reference in their entirety. It should be understood that any UTR from any gene may be incorporated into a region of a nucleic acid sequence. Additionally, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs that are not variants of the wild-type region. These UTRs or portions thereof may be placed in the same orientation as the transcript from which they were selected, or may be oriented or positioned differently. Thus, 5' or 3' UTR can be inverted, shortened, extended, and made with one or more other 5' or 3' UTRs. As used herein, the term "altered" with respect to a UTR sequence means that the UTR is changed in some way with respect to a reference sequence. For example, the 3' or 5' UTR can be changed with respect to the wild-type or native UTR by changing the orientation or position as taught above, or by including additional nucleotides, deleting nucleotides, exchanging or rearranging nucleotides. Any of these changes that generate a "modified" UTR (whether 3' or 5') include variant UTRs.

[0386] In some embodiments, double, triple or quadruple UTRs, such as 5'UTR or 3'UTR, can be used. As used herein, a "double" UTR is one in which two copies of the same UTR are encoded, either in tandem or substantially in tandem. For example, double beta-globin 3'UTRs can be used as described in US Patent Publication No. 2010 / 0129877, the contents of which are incorporated herein by reference in their entirety.

[0387] It is also within the scope of the present disclosure to have patterned UTR.As used herein, "patterned UTR" is a UTR that reflects a repeating or alternating pattern, such as ABABAB or AABBABBAABB or ABCABCABC or its variants that are repeated once, twice or more than three times.In these patterns, each letter A, B or C represents a UTR that is different at the nucleotide level.

[0388] In some embodiments, the flanking region is selected from a family of transcripts whose proteins share common functions, structures, properties or characteristics.For example, the subject polypeptide may belong to a family of proteins expressed in a particular cell, tissue or at a certain time during development.The UTR from any of these genes may be exchanged with any other UTR from the same or different protein family to generate a new polynucleotide.As used herein, "family of proteins" is used in the broadest sense and refers to a group of two or more subject polypeptides that share at least one function, structure, property, localization, origin or expression pattern.

[0389] The untranslated region may also include a translation enhancer element (TEE). As a non-limiting example, the TEE may include those described in U.S. Patent Application No. 2009 / 0226470, the entirety of which is incorporated herein by reference, and those known in the art. The in vitro transcription of the RNA cDNA encoding the polynucleotide described herein may be transcribed using an in vitro transcription (IVT) system. In vitro transcription of RNA is known in the art and described in WO 2014 / 152027, the entirety of which is incorporated herein by reference. In some embodiments, the RNA of the present disclosure is prepared according to any one or more of the methods described in WO 2018 / 053209 and WO 2019 / 036682, each of which is incorporated herein by reference.

[0390] In some embodiments, RNA transcripts are generated using a non-amplified linearized DNA template in an in vitro transcription reaction to generate RNA transcripts. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of an RNA polynucleotide, such as, but not limited to, coronavirus mRNA. In some embodiments, cells, such as bacterial cells, such as E. coli, such as DH-1 cells, are transfected with a plasmid DNA template. In some embodiments, the transfected cells are cultured to replicate the plasmid DNA, which is then isolated and purified. In some embodiments, the DNA template comprises an RNA polymerase promoter, such as a T7 promoter operably linked to the 5'-side of a gene of interest.

[0391] In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, encodes a 3' UTR and a poly(A) tail. The particular nucleic acid sequence composition and length of the in vitro transcription template depends on the mRNA encoded by the template.

[0392] "5' untranslated region" (UTR) refers to the region of an mRNA immediately upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript that is translated by a ribosome) that does not code for a polypeptide. When an RNA transcript is generated, the 5'UTR may contain a promoter sequence. Such promoter sequences are known in the art. It should be understood that such promoter sequences are not present in the vaccines of the present disclosure.

[0393] "3' untranslated region" (UTR) refers to the region of an mRNA immediately downstream (i.e., 3') from a termination codon (i.e., a codon in an mRNA transcript that signals the termination of translation) that does not encode a polypeptide.

[0394] An "open reading frame" is a contiguous stretch of DNA beginning with a start codon (eg, methionine (ATG)) and ending with a stop codon (eg, TAA, TAG, or TGA) that encodes a polypeptide.

[0395] A "poly(A) tail" is a region of an mRNA downstream, e.g., immediately downstream (i.e., 3'), from the 3'UTR that contains multiple consecutive adenosine monophosphates. A poly(A) tail can contain 10-300 adenosine monophosphates. For example, a poly(A) tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50-250 adenosine monophosphates. In relevant biological environments (e.g., within a cell, in vivo), the poly(A) tail functions, for example, to protect the mRNA from enzymatic degradation in the cytoplasm, direct transcription termination, and / or aid in the transport of the mRNA from the nucleus and translation.

[0396] In some embodiments, the nucleic acid comprises 200 to 3,000 nucleotides. For example, the nucleic acid may comprise 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.

[0397] In vitro transcription systems typically include a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase.

[0398] NTPs may be produced in-house, selected from a source, or synthesized as described herein. NTPs may be selected from, but are not limited to, those described herein, including natural and non-natural (modified) NTPs.

[0399] Any number of RNA polymerases or variants can be used in the method of the present disclosure.Polymerase can be selected from, but is not limited to, phage RNA polymerase, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and / or mutant polymerase, such as, but not limited to, polymerase that can incorporate modified nucleic acid and / or modified nucleotide, including chemically modified nucleic acid and / or nucleotide.Some embodiments exclude the use of DNase.

[0400] In some embodiments, the RNA transcript is capped via enzymatic capping. In some embodiments, the polynucleotide (e.g., mRNA) comprises a 5' end cap, e.g., 7mG(5')ppp(5')NlmpNp.

[0401] chemical synthesis Solid-phase chemical synthesis. Polynucleotides (e.g., mRNA) can be produced in whole or in part using solid-phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method in which molecules are immobilized on a solid support and synthesized stepwise in a reactant solution. Solid-phase synthesis is useful for site-specific introduction of chemical modifications in nucleic acid sequences.

[0402] Solution Phase Chemical Synthesis Synthesis of polynucleotides (eg, mRNA) by sequential addition of monomer building blocks may be carried out in solution phase.

[0403] Combination of synthetic methods. Each of the synthetic methods discussed above has its own advantages and limitations. Attempts have been made to combine these methods to overcome limitations. Such combinations of methods are within the scope of this disclosure. The use of solid-phase or liquid-phase chemical synthesis combined with enzymatic ligation provides an efficient method for producing long-chain nucleic acids that cannot be obtained by chemical synthesis alone.

[0404] Ligation of nucleic acid regions or subregions Ligase-assisted assembly of nucleic acids may also be used. DNA or RNA ligase promotes intermolecular ligation of the 5' and 3' ends of a polynucleotide chain through the formation of a phosphodiester bond. Nucleic acids such as chimeric polynucleotides and / or circular nucleic acids may be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by ligase-catalyzed reactions to generate recombinant DNAs with different functions. Two oligodeoxynucleotides, one with a 5' phosphoryl group and the other with a free 3' hydroxyl group, serve as substrates for DNA ligase.

[0405] purification Purification of nucleic acids as described herein may include, but is not limited to, nucleic acid cleanup, quality assurance and quality control. Cleanup may be performed by methods known in the art, such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark), or HPLC-based purification methods, such as strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). When used in relation to nucleic acids, such as "purified nucleic acids," the term "purified" refers to something that is separated from at least one contaminant. A "contaminant" is any substance that makes another incompatible, impure, or inferior. Thus, purified nucleic acids (e.g., DNA and RNA) exist in a form or setting different from that found in nature, or in a form or setting different from that which existed before being subjected to a processing or purification method.

[0406] Quality assurance and / or quality control checks may be performed using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.

[0407] In some embodiments, the nucleic acid may be sequenced by methods including, but not limited to, reverse transcriptase-PCR.

[0408] Quantification In some embodiments, polynucleotides (e.g., mRNA) may be quantified when present within exosomes or from one or more bodily fluids, including peripheral blood, serum, plasma, peritoneal fluid, urine, cerebrospinal fluid (CSF), saliva, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, milk, bronchoalveolar lavage fluid, semen, prostatic fluid, bovine fluid or ejaculate, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyle, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretions, fecal fluid, pancreatic juice, lavage fluid of sinus cavities, bronchopulmonary aspirate, blastocyst fluid, and umbilical cord blood. Alternatively, exosomes may be obtained from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

[0409] The assay may be performed using construct-specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or a combination thereof, while exosomes may be isolated using immunohistochemistry methods such as enzyme-linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or a combination thereof.

[0410] These methods allow researchers to monitor the levels of remaining or delivered nucleic acid in real time, which is possible because the nucleic acids of the present disclosure, in some embodiments, differ from endogenous forms by structural or chemical modifications.

[0411] In some embodiments, the nucleic acid may be quantified using methods such as, but not limited to, ultraviolet-visible spectroscopy (UV / Vis). A non-limiting example of a UV / Vis spectrometer is the NANODROP® spectrometer (ThermoFisher, Waltham, MA). The quantified nucleic acid may be analyzed to determine whether the nucleic acid may be of the appropriate size and to ensure that no degradation of the nucleic acid has occurred. Nucleic acid degradation may be checked by methods such as agarose gel electrophoresis, HPLC-based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).

[0412] Treatment method Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention and / or treatment of cancer in humans and other mammals. LPMP / mRNA formulations can be used as therapeutic or prophylactic agents. They can be used in medicines to prevent and / or treat cancer.

[0413] In one embodiment, the LPMP / mRNA therapeutic composition is used to provide prophylactic protection from cancer. Prophylactic protection from cancer can be achieved after administration of the LPMP / mRNA therapeutic composition (as a vaccine). The vaccine can be administered once, twice, three times, four times or more, although it is likely that a single administration of the vaccine (optionally followed by a single booster) will be sufficient. It is more desirable to administer the vaccine to individuals with cancer to achieve a therapeutic response. Administration may need to be adjusted accordingly.

[0414] In some embodiments, the LPMP / mRNA therapeutic composition (as a vaccine) is administered on a schedule of up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 7 months, up to 8 months, up to 9 months, up to 10 months, up to 11 months, up to 1 year, up to 1.5 years, up to 2 years, up to 3 years, or up to 4 years. The schedule may be the same or may vary. In some embodiments, the schedule is weekly for the first 3 weeks and monthly thereafter.

[0415] The LPMP / mRNA therapeutic composition (as a vaccine) may be administered by any route. In some embodiments, the vaccine is administered by EVI or IV route.

[0416] At any point during treatment, the patient may be tested to determine if the vaccine mutations are still appropriate, and based on that analysis, the vaccine may be adjusted or reformulated to include one or more different mutations or to remove one or more mutations.

[0417] Therapeutic and Prophylactic Compositions Provided herein are compositions (eg, pharmaceutical compositions), methods, kits, and reagents for the prevention, treatment, or diagnosis of cancer in humans and other mammals.

[0418] In some embodiments, LPMP / mRNA therapeutic compositions (as vaccines) can be used to prime immune effector cells, for example by activating peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject.

[0419] In an exemplary embodiment, the LPMP / mRNA therapeutic composition can be administered to a subject (e.g., a mammalian subject, such as a human subject) and the RNA polynucleotide is translated in vivo to produce an antigenic polypeptide.

[0420] The LPMP / mRNA therapeutic composition can induce translation of a polypeptide (e.g., an antigen, tumor antigen, or signaling protein) in a cell, tissue, or organism. In exemplary embodiments, such translation occurs in vivo, although embodiments in which such translation occurs ex vivo, in culture, or in vitro can be envisioned. In exemplary embodiments, a cell, tissue, or organism is contacted with an effective amount of a composition containing an LPMP / mRNA therapeutic composition that contains a polynucleotide having at least one translatable region that encodes an antigenic (e.g., tumor antigenic) or signaling polypeptide.

[0421] An "effective amount" of an LPMP / mRNA therapeutic composition is provided based at least in part on the target tissue, the target cell type, the means of administration, the physical characteristics of the polynucleotide (e.g., size and extent of modified nucleosides), and other components of the LPMP / mRNA therapeutic composition, as well as other determinants. In general, an effective amount of an LPMP / mRNA therapeutic composition provides an induced or boosted immune response as a function of intracellular antigen or polypeptide production, preferably more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or polypeptide. Increased antigen production can be indicated by increased cell transfection (percentage of cells transfected with the LPMP / mRNA therapeutic composition), increased protein translation from the polynucleotide, decreased nucleic acid degradation (e.g., indicated by an increased period of protein translation from the modified polynucleotide), or a change in the therapeutic agent or antigen-specific immune response of the host cell.

[0422] In some embodiments, the LPMP / mRNA therapeutic composition may be used to treat cancer.

[0423] LPMP / mRNA therapeutic compositions may be administered prophylactically or therapeutically to healthy individuals, or as part of an active immunization scheme, either at the onset of cancer or during active cancer after the onset of symptoms. In some embodiments, the amount of LPMP / mRNA therapeutic composition provided to a cell, tissue, or subject may be an amount effective for immunoprophylaxis.

[0424] The LPMP / mRNA therapeutic composition may be administered with other prophylactic or therapeutic compounds. As a non-limiting example, the prophylactic or therapeutic compound may be an immune enhancer, adjuvant, or booster. As used herein, when referring to a composition such as a vaccine, the term "booster" refers to an extra administration of a prophylactic (vaccine) composition. A booster (or booster vaccine) may be administered after an earlier administration of the prophylactic composition. The administration time between the first administration of the prophylactic composition and the booster can be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, It may be 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or more than 99 years. In exemplary embodiments, the administration time between the first administration of the prophylactic composition and the booster may be, without limitation, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

[0425] In one embodiment, the LPMP / mRNA therapeutic composition may be administered intramuscularly or intradermally, similar to the administration of vaccines known in the art.

[0426] LPMP / mRNA therapeutic compositions may be utilized in a variety of situations depending on the severity of the cancer or the degree or level of unmet medical need. As a non-limiting example, LPMP / mRNA therapeutic compositions may be utilized to treat cancer at any stage. LPMP / mRNA therapeutic compositions have superior properties in terms of generating much greater antibody titers, T cell responses, and generating early responses than commercially available anti-cancer vaccines. Without intending to be bound by theory, the inventors hypothesize that LPMP / mRNA therapeutic compositions, as mRNAs, are better designed to generate the proper protein conformation upon translation because LPMP / mRNA therapeutic compositions utilize natural cellular machinery. Unlike conventional vaccines that are produced ex vivo and may induce undesirable cellular responses, LPMP / mRNA therapeutic compositions are presented to cell systems in a more natural manner.

[0427] A non-limiting list of cancers that LPMP / mRNA therapeutic compositions can treat is given below. Peptide epitopes or antigens can be derived from any antigen of these cancers or tumors. Such epitopes are called cancer or tumor antigens. Cancer cells can express cell surface molecules specifically during different stages of tumor progression. For example, cancer cells can express a cell surface antigen in a benign state, but downregulate that particular cell surface antigen upon metastasis. It is therefore envisioned that tumor or cancer antigens can encompass antigens produced during the progression of any stage of cancer. The methods of the present invention can be adjusted to accommodate these changes. For example, several different LPMP / mRNA therapeutic compositions can be produced for a particular patient. For example, a first vaccine may be used at the beginning of treatment. At a later time, a new LPMP / mRNA therapeutic composition may be produced and administered to the patient, which causes different antigens to be expressed.

[0428] Provided herein are pharmaceutical compositions comprising the LPMP / mRNA therapeutic composition, optionally in combination with one or more pharma- ceutically acceptable excipients.

[0429] The LPMP / mRNA therapeutic composition may be formulated or administered alone or in combination with one or more other components. For example, the LPMP / mRNA therapeutic composition may include other components, including, but not limited to, immune enhancing agents (e.g., adjuvants). In some embodiments, the LPMP / mRNA therapeutic composition does not include an immune enhancing agent or adjuvant (i.e., is immune enhancing agent or adjuvant free).

[0430] In other embodiments, the LPMP / mRNA therapeutic composition may be combined with any other therapy useful in treating a patient. For example, a patient may be treated with an LPMP / mRNA therapeutic composition and an anti-cancer therapeutic agent. Thus, in one embodiment, the method of the present invention may be used in conjunction with one or more cancer therapeutic agents, for example, in conjunction with anti-cancer therapeutic agents, conventional cancer vaccines, chemotherapy, radiation therapy, etc. (e.g., simultaneously or as part of an overall treatment procedure). Parameters of cancer treatment that may be varied may include, but are not limited to, dosage, timing or duration of administration or therapy, and cancer treatment may vary in dosage, timing, or duration. Another treatment for cancer is surgery, which may be used alone or in combination with any of the conventional therapies. Any agent or therapy known to be useful or that has been or is currently used in the prevention or treatment of cancer (e.g., conventional cancer vaccines, chemotherapy, radiation therapy, surgery, hormonal therapy, and / or biological therapy / immunotherapy) may be used in combination with the composition of the present invention according to the present invention described herein. Those skilled in the medical field can determine the appropriate treatment for a subject.

[0431] Examples of such agents (i.e., anti-cancer therapeutics) include, but are not limited to, DNA interactive agents, such as, but not limited to, alkylating agents (e.g., nitrogen mustards, e.g., chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, uracil mustard; aziridines such as thiotepa; methanesulfonate esters such as busulfan; nitrosoureas such as carmustine, lomustine, streptozocin, platinum complexes such as cisplatin, carboplatin; bioreductive alkylating agents such as mitomycin and procarbazine, dacarbazine, and altretamine); DNA strand breaking agents, such as, bleomycin; intercalating topoisomerase II inhibitors, such as intercalating agents, e.g., amsacrine, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitoxantrone, and non-intercalating agents, e.g., etoposide and teniposide; non-intercalating topoisomerase II inhibitors, e.g., etoposide and teniposide; and DNA minor groove binders, e.g., plicamidin; antimetabolites, e.g., folate antagonists, such as, but not limited to, methotrexate and trimetrexate; pyrimidine antagonists, e.g., fluorouracil, fluorodeoxyuridine, CB3717, azacytidine, and floxiridine; purine antagonists, e.g., mercaptopurine, 6-thioguanine, pentostatin; sugar-modified analogs such as tarabin and fludarabine; and ribonucleotide reductase inhibitors such as hydroxyurea; tubulin interacting agents, including but not limited to colchicine, vincristine and vinblastine, both alkaloids and paclitaxel and cytoxan, hormonal agents, including but not limited to estrogens, conjugated estrogens and ethinyl estradiol and diethylstilbesterol, chlortrianisene and idenestrol; progestins, including hydroxyprogesterone caproate, medroxyprogesterone, and megestrol; and androgens, such as testosterone, testosterone propionate; fluoxymesterone, methyltestosterone;Corticosteroids, such as prednisone, dexamethasone, methylprednisolone, and prednisolone; luteinizing hormone releasing hormone agents or gonadotropin releasing hormone antagonists, such as leuprolide acetate and goserelin acetate; antihormonal antigens, such as, but not limited to, antiestrogens such as tamoxifen, antiandrogens such as flutamide; and antiadrenal agents such as mitotane and aminoglutethimide; cytokines, such as, but not limited to, IL-1.alpha, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-18, TGF-β, GM-CSF, M-CSF, G-CSF, TNF-a, TNF-β, LAF, TCGF, ​​BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-a, IFN-β, IFN-γ, and uteroglobin (U.S. Pat. No. 5,696,092); anti-angiogenic agents, including but not limited to agents that inhibit VEGF (e.g., other neutralizing antibodies), soluble receptor constructs, tyrosine kinase inhibitors, antisense strategies, RNA aptamers, and ribozymes against VEGF or VEGF receptors, immunotoxins and coagulants, tumor vaccines, and antibodies. Further examples of such agents (i.e., anti-cancer therapeutic agents) include cytokines, including, but not limited to, IL-15 or recombinant IL-15;

[0432] Specific examples of anticancer therapeutic agents include, but are not limited to, acivicin, aclarubicin, codazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, and bleoma sulfate. Isin, briquinal sodium, bropirimine, busulfan, cactinomycin, calsterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexorumaplatin, desaguanine, desaguanine mesylate, diaziquone, Docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, zuazomycin, edatrexate, eflomitine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, elbrozole, esorubicin hydrochloride, estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, phen Retinides, floxuridine, fludarabine phosphate, fluorouracil, flurocitabine, foskidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, irmofosine, interleukin II (including recombinant interleukin II or rIL-2), interferon alpha-2a, interferon alpha-2b, interferon alpha-nl, interferon alpha-n3, interferon beta-I a, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocalcin, mitocromine, mitodilline, mitomarcine, mitomarcine Syn, Mitosper, Mitotene, Mitoxantrone hydrochloride, Mycophenolic acid, Nocodazole, Nogalamycin, Ormaplatin, Oxisuran, Paclitaxel, Pegaspargase, Periomycin, Pentamustine, Peplomycin sulfate, Perfosfamide, Pipobroman, Piposulfan, Piroxantrone hydrochloride, Plicamycin, Promestane, Porfimer sodium, Porfiromycin, Prednimustine, Procarbazine hydrochloride, Puromycin, Puromycin hydrochloride, Pyrazofurin, Ribopurin, logretoimide, safingol, safingol hydrochloride, semustine, simtrazene, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, tallysomycin, tecogalan sodium, tegafur, teroxantrone hydrochloride, temoporfin, teniposide, teroxiron, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, trefoil citrate These include miphene, trestrone acetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tuburozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglisinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrocidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, and zorubicin hydrochloride.

[0433] Other anti-cancer drugs include, but are not limited to, 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; angiogenesis inhibitors; anti-dorsal morphogenetic protein; ara-CDP-DL-PTBA; BCR / ABL antagonists; CaRest M3; CARN 700;Casein kinase inhibitor (ICOS);Clotrimazole;Colismycin A;Colismycin B;Combretastatin A4;Crambecidin 816;Cryptophycin 8;Curacine A;Dehydrodidemnin B;Didemnin B;Dihydro-5-azacytidine;Dihydrotaxol, Duocarmycin SA;Kahalalide F;Lamellarin-N triacetate;Leuprolide + estrogen + progesterone;Lysoclinamide 7;Monophosphoryl lipid A + Myobacterium cell wall sk;N-Acetyldinarine;N-Substituted benzamides;O6-Benzylguanine;Placetin A;Placetin B;Platinum complexes;Platinum compounds;Platinum triamine complexes;Rhenium Re 186 etidronate; RII retinamide; rubiginone B1; SarCNU; sarcophytol A; sargramostim; senescence induction inhibitor 1; spicamycin D; talimustine; 5-fluorouracil; thrombopoietin; thymotrin; thyroid-stimulating hormone; variolin B; thalidomide; veraresol; veramine; verudine; verteporfin; vinorelbine; vinxartin; vitaxin; zanoterone; zeniplatin and zilascorub.

[0434] The present invention also encompasses the administration of compositions comprising LPMP / mRNA therapeutic compositions in combination with radiation therapy, including the use of X-rays, gamma rays, and other radiation sources to destroy cancer cells. In some embodiments, radiation therapy is administered as external beam radiation or teletherapy, where radiation is directed from a distant source. In other embodiments, radiation therapy is administered as internal therapy or brachytherapy, where a radioactive source is placed in the body near the cancer cells or tumor mass.

[0435] In certain embodiments, suitable anti-cancer regimen is selected according to the type of cancer.For example, patients with ovarian cancer can be administered a prophylactically or therapeutically effective amount of a composition comprising LPMP / mRNA therapeutic composition in combination with a prophylactically or therapeutically effective amount of one or more other agents useful for ovarian cancer therapy, including but not limited to intraperitoneal radiation therapy, such as P32 therapy, total abdominal and pelvic radiation therapy, combinations of cisplatin, paclitaxel (taxol) or docetaxel (taxotere) with cisplatin or carboplatin, combinations of cyclophosphamide with cisplatin, combinations of cyclophosphamide with carboplatin, combinations of 5-FU with leucovorin, etoposide, liposomal doxorubicin, gemcitabine or topotecan.Cancer treatments and their dosages, routes of administration and recommended methods of use are known in the art and described in references such as Physician's Desk Reference (56th ed., 2002).

[0436] In some embodiments, the LPMP / mRNA therapeutic composition is administered with a T cell activator, such as an immune checkpoint regulator. Immune checkpoint regulators include both stimulatory and inhibitory checkpoint molecules, i.e., anti-CTLA4 and anti-PD1 antibodies. Stimulatory checkpoint inhibitors function by promoting checkpoint processes. Some stimulatory checkpoint molecules are members of the tumor necrosis factor (TF) receptor superfamily-CD27, CD40, OX40, GITR, and CD137, while others belong to the B7-CD28 superfamily-CD28 and ICOS. OX40 (CD134) is involved in the proliferation of effector and memory T cells. Anti-OX40 monoclonal antibodies have been shown to be effective in treating advanced cancers. MEDI0562 is a humanized OX40 agonist. GITR, a glucocorticoid-induced T FR family-related gene, is involved in T cell proliferation. Some antibodies against GITR have been shown to promote anti-tumor responses. ICOS, inducible T cell costimulator, is important in T cell effector function. CD27 supports antigen-specific proliferation of naive T cells and participates in the generation of T cell and B cell memory. Several antagonistic anti-CD27 antibodies are in development. CD122 is the interleukin-2 receptor beta subunit. KTR-214 is a CD122-biased immunostimulatory cytokine.

[0437] Inhibitory checkpoint molecules include, but are not limited to, PD-1, TEVI-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligands, or combinations thereof. Ligands for checkpoint proteins include, but are not limited to, CTLA-4PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands. In some embodiments, the anti-PD-1 antibody is BMS-936558 (nivolumab). In other embodiments, the anti-CTLA-4 antibody is ipilimumab (brand name Yervoy, formerly known as MDX-010 and MDX-101).

[0438] The LPMP / mRNA therapeutic composition and the anti-cancer therapeutic agent can be combined to further enhance the immune therapeutic response. The LPMP / mRNA therapeutic composition and the other therapeutic agent may be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously, they can be administered in the same formulation or in separate formulations, but are administered simultaneously. When the administration of the other therapeutic agent and the LPMP / mRNA therapeutic composition is temporally separated, the other therapeutic agents are administered sequentially with each other and with the LPMP / mRNA therapeutic composition. The time separation between the administration of these compounds may be a few minutes or may be longer, for example, hours, days, weeks, or months. For example, in some embodiments, the time separation between the administration of these compounds is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 24 hours, or more. In some embodiments, the time separation between the administration of these compounds is 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or more. In some embodiments, the LPMP / mRNA therapeutic composition is administered before the anti-cancer therapeutic agent. In some embodiments, the LPMP / mRNA therapeutic composition is administered after the anti-cancer therapeutic agent.

[0439] Other therapeutic agents include, but are not limited to, anti-cancer therapeutic agents, adjuvants, cytokines, antibodies, antigens, and the like.

[0440] Therapeutic agents may also include those capable of treating and / or preventing chronic pain and / or symptoms of chronic pain, including, but not limited to: (i) an opioid analgesic, such as morphine, heroin, hydromorphone, oxymorphone, levorphanol, levallorphan, methadone, meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine or pentazocine; (ii) nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, diclofenac, diflucinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tolmetin or zomepirac, Mobic (meloxicam SR), or a pharma- ceutically acceptable salt thereof; (iii) barbiturate analgesics such as amobarbital, aprobarbital, butabarbital, butalbital, methobarbital, metharbital, methohexital, pentobarbital, phenobarbital, secobarbital, talbutal, thiamaryl or thiopental, or a pharma- ceutically acceptable salt thereof; (iv) benzodiazepines having analgesic properties, such as chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, or triazolam, or a pharma- ceutically acceptable salt thereof; (v) H1 antagonists having analgesic properties, such as diphenhydramine, pyrilamine, promethazine, chlorpheniramine or chlorcyclizine, or a pharma- ceutically acceptable salt thereof; (vi) Analgesics, such as glutethimide, meprobamate, methaqualone or dichloralphenazone or a pharma- ceutically acceptable salt thereof; (vii) a skeletal muscle relaxant, such as baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol or orphrenazine, or a pharma- ceutically acceptable salt thereof; (viii) NMDA receptor antagonists such as dextromethorphan ((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine, pyrroloquinoline quinone, or cis-4-(phosphonomethyl)-2-piperidine carboxylic acid, or a pharma- ceutically acceptable salt thereof; (ix) α-adrenergic agonists such as doxazosin, tamsulosin, clonidine or 4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl)quinazoline; (x) tricyclic antidepressants, such as desipramine, imipramine, amitriptyline, or nortriptyline; (xi) anticonvulsants such as carbamazepine or valproate, (xii) tachykinin (NK) antagonists, in particular NK-3, NK-2 or NK-1 antagonists, such as (αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]naphtholidine-6-13-dione (TAK-637), 5-[[(2R,3S)-2 -[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophenyl)-4-)morpholinyl]methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one (MK-869), lanepitant, dapitant or 3-[[2-methoxy-5-(trifluoromethoxy)phenyl]methylami]-2-phenyl-piperidine (2S, 3S), (xiii) muscarinic antagonists, such as oxybutin, tolterodine, propiverine, trospium chloride, or darifenacin; (xiv) COX-2 inhibitors, such as celecoxib, rofecoxib, or valdecoxib, (xv) non-selective COX inhibitors, preferably those with GI protection such as nitroflurbiprofen (HCT-1026); (xvi) coal tar analgesics, especially paracetamol; (xvii) neuroleptics such as droperidol, (xviii) vanilloid receptor agonists (e.g., resiniferatoxin) or antagonists (e.g., capsazepine), (xix) beta-adrenergic agonists, such as propranolol; (xx) Local anesthetics such as mexiletine, (xxi) Corticosteroids, such as dexamethasone, (xxii) serotonin receptor agonists or antagonists, (xxiii) cholinergic (nicotinic) analgesics, (xxiv) Tramadol TM, (xxv) PDEV inhibitors such as sildenafil, vardenafil, or tadalafil.

[0441] In some embodiments, the methods provided include administering an LPMP / mRNA therapeutic composition in combination with an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator, e.g., a checkpoint inhibitor such as an anti-PD-1 antibody, is administered at a dosage level sufficient to deliver 100-300 mg to the subject. In some embodiments, the immune checkpoint modulator, e.g., a checkpoint inhibitor such as an anti-PD-1 antibody, is administered at a dosage level sufficient to deliver 200 mg to the subject. In some embodiments, the immune checkpoint modulator, e.g., a checkpoint inhibitor such as an anti-PD-1 antibody, is administered by intravenous infusion. In some embodiments, the immune checkpoint modulator is administered to the subject two, three, four, or more times. In some embodiments, the immune checkpoint modulator is administered to the subject on the same day as administration of the LPMP / mRNA therapeutic composition.

[0442] The LPMP / mRNA therapeutic composition may be formulated or administered in combination with one or more pharma- ceutically acceptable excipients. In some embodiments, the LPMP / mRNA therapeutic composition includes at least one additional active agent, such as, for example, a therapeutic active agent, a prophylactic active agent, or a combination of both. The LPMP / mRNA therapeutic composition may be sterile, pyrogen-free, or both sterile and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceuticals, such as vaccine compositions, can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005, which is incorporated herein by reference in its entirety.

[0443] In some embodiments, the LPMP / mRNA therapeutic composition is administered to a human, human patient, or subject. The phrase "active ingredient" generally refers to the LPMP / mRNA therapeutic composition or a polynucleotide contained therein, such as an RNA polynucleotide (e.g., an mRNA polynucleotide) that encodes an antigenic or therapeutic polypeptide.

[0444] Formulations of the LPMP / mRNA therapeutic compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods of preparation include the step of bringing into association an active ingredient (e.g., an mRNA polynucleotide) with an excipient and / or one or more other accessory ingredients and then dividing, shaping, and / or packaging the product into the desired single or multiple dosage units as necessary and / or desired.

[0445] LPMP / mRNA therapeutic compositions can be formulated with one or more excipients to (1) increase stability, (2) increase cell transfection, (3) allow sustained or delayed release (e.g., from a depot formulation), (4) modify biodistribution (e.g., targeting to a specific tissue or cell type), (5) increase translation of the encoded protein in vivo, and / or (6) modify the release profile of the encoded protein (antigen) in vivo. In addition to conventional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, etc., excipients can include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells into which a cancer RNA vaccine has been introduced (e.g., for implantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0446] kit The present invention also provides a kit comprising a container with the mRNA therapeutic composition described herein. The kit may further comprise instruction material for applying or delivering the mRNA therapeutic composition to a subject according to the method of the present invention. Those skilled in the art will understand that the instruction for applying the mRNA therapeutic composition in the method of the present invention can be any form of instruction. Such instruction includes, but is not limited to, written instruction material (such as label, booklet, pamphlet), oral instruction material (such as audio cassette or CD), or video instruction (such as video tape or DVD).

[0447] Embodiment 1. An mRNA therapeutic composition comprising: one or more polynucleotides encoding one or more antigenic (e.g., tumor antigenic) polypeptides or signaling polypeptides; The lipid reconstituted plant messenger pack (LPMP) is formulated with natural lipids and ionizable lipids, the ionizable lipids having the following characteristics: (i) at least two ionizable amines; (ii) at least three lipid tails, each of said lipid tails being at least 6 carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) ionizable amine and heteroorganic groups separated by a chain of at least two atoms; and (v) an N:P ratio of at least 10;

[0448] Embodiment 2. The mRNA therapeutic composition of embodiment 1, wherein the natural lipids are extracted from lemon or algae.

[0449] Embodiment 3. The mRNA therapeutic composition of embodiment 1, wherein the LPMP further comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate.

[0450] Embodiment 4. The mRNA therapeutic composition of embodiment 3, wherein the LPMP comprises a molar ratio of ionizable lipids:natural lipids:sterols:PEG lipids of about 35:50:12.5:2.5.

[0451] Embodiment 5. The mRNA therapeutic composition of embodiment 3, wherein the LPMP comprises a molar ratio of ionizable lipids:natural lipids:sterols:PEG lipids of about 35:20:42.5:2.5.

[0452] Embodiment 6. The mRNA therapeutic composition of embodiment 1, wherein the ionizable lipid is selected from the group consisting of 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315.

[0453] Embodiment 7. The ionizable lipid comprises: [ka] wherein R is a C8-C14 alkyl group.

[0454] Embodiment 8. The mRNA therapeutic composition of embodiment 1, wherein the polypeptide comprises a tumor-specific antigen, a tumor-associated antigen, a tumor neoantigen, or a combination thereof.

[0455] 9. The polypeptide is selected from the group consisting of p53, ART-4, BAGE, ss-catenin / m, Bcr-abL, CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, CLAUDIN-12, C-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Plac-1, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survivin, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, WT-1, or combinations thereof.

[0456] 10. The polypeptide is selected from the group consisting of CD2, CD3, CD4, CD8, CD11b, CD14, CD16, CD19, CD20, CD22, CD25, CD27, CD33, CD37, CD38, CD40, CD44, CD45, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5, AGS-16, angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, Carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbBl, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, HLA, HLA-DR, ICOS, IGF1R, integrin αν, integrin ανβ, LAG-3, Lewis The mRNA therapeutic composition of embodiment 1, comprising Y, mesothelin, c-MET, MN carbonic anhydrase IX, MUC1, MUC16, Nectin-4, KGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, syndecan-1, TACI, TAG-72, tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and variants thereof.

[0457] Embodiment 11. The mRNA therapeutic composition of embodiment 1, wherein the polypeptide is IL-2 peptide, IL-2-Ra, tdTomato, Cre recombinase, GFP, eGFP, anti-CD19, CD20, CAR-T, anti-HER2, etanercept (Enbrel), Humira, erythropoietin, Epogen, filgrastim, Keytruda, rituximab, romiplostim, sargramostim, or a fragment or subunit thereof.

[0458] Embodiment 12. The mRNA therapeutic composition of embodiment 1, wherein the polynucleotide is an mRNA encoding an IL-2 molecule comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of an IL-2 molecule provided in any one of Tables I-III.

[0459] Embodiment 13. The mRNA therapeutic composition of embodiment 1, wherein the tumor antigenic polypeptide comprises a tumor antigen selected from the group consisting of carcinoma, sarcoma, melanoma, lymphoma, leukemia, and combinations thereof.

[0460] Embodiment 14 The mRNA therapeutic composition of embodiment 13, wherein the tumor antigenic polypeptide comprises a lung cancer antigen.

[0461] Embodiment 15. The polynucleotide is an mRNA, the mRNA being (a) a DNA molecule, or (b) the RNA molecule in which T is replaced with U;

[0462] Embodiment 16 The mRNA therapeutic composition of embodiment 15, wherein the DNA molecule further comprises a promoter.

[0463] Embodiment 17 The mRNA therapeutic composition of embodiment 16, wherein the promoter is located in the 5'UTR.

[0464] Embodiment 18. The mRNA therapeutic composition of embodiment 15, wherein the promoter is a T7 promoter, a T3 promoter, or an SP6 promoter.

[0465] Embodiment 19 The mRNA therapeutic composition of embodiment 15, wherein the RNA molecule is a self-replicating RNA molecule.

[0466] Embodiment 20 The mRNA therapeutic composition of embodiment 15 or embodiment 19, wherein the RNA molecule further comprises a 5' cap.

[0467] Embodiment 21. The mRNA therapeutic composition of embodiment 20, wherein the 5' cap has a cap1 structure, a cap1(m6A) structure, a cap2 structure, a cap3 structure, a cap0 structure, or any combination thereof.

[0468] Embodiment 22 The mRNA therapeutic composition of embodiment 12, wherein the IL-2 molecule comprises a naturally occurring IL-2 molecule, a fragment of a naturally occurring IL-2 molecule, or a variant thereof.

[0469] Embodiment 23 The mRNA therapeutic composition of embodiment 22, wherein the IL-2 molecule comprises a variant of a naturally occurring IL-2 molecule, or a fragment thereof.

[0470] Embodiment 24. The mRNA therapeutic composition of embodiment 15, wherein the mRNA comprises a 5' untranslated region (UTR) and / or a 3'UTR.

[0471] Embodiment 25. The mRNA therapeutic composition of embodiment 24, wherein the 5'UTR comprises a Kozak sequence.

[0472] Embodiment 26 The mRNA therapeutic composition of embodiment 24, wherein the 3'UTR comprises a sequence derived from an amino-terminal enhancer (AES) of splitting.

[0473] Embodiment 27. The mRNA therapeutic composition of embodiment 24, wherein the 3'UTR comprises a sequence derived from mitochondrially encoded 12S rRNA (mtRNRl).

[0474] Embodiment 28 The mRNA therapeutic composition of embodiment 15, wherein the mRNA comprises a poly(A) sequence.

[0475] Embodiment 29. The mRNA therapeutic composition of embodiment 28, wherein the poly(A) sequence is a 110 nucleotide sequence consisting of a sequence of 30 adenosine residues, a linker sequence of 10 nucleotides, and a sequence of 70 adenosine residues.

[0476] Embodiment 30. The mRNA therapeutic composition of embodiment 1, wherein the LPMP is a lipophilic moiety selected from the group consisting of lipoplexes, liposomes, lipid nanoparticles, polymeric carriers, exosomes, lamellar bodies, micelles, and emulsions.

[0477] Embodiment 31. The mRNA therapeutic composition of embodiment 1, wherein the LPMP is a liposome selected from the group consisting of cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes.

[0478] Embodiment 32 The mRNA therapeutic composition of embodiment 1, wherein the LPMP is a lipid nanoparticle.

[0479] Embodiment 33 The mRNA therapeutic composition of embodiment 1, wherein the LPMPs have a size of less than about 200 nm.

[0480] Embodiment 34 The mRNA therapeutic composition of embodiment 33, wherein the LPMPs have a size of less than about 150 nm.

[0481] Embodiment 35 The mRNA therapeutic composition of embodiment 33, wherein the LPMPs have a size of less than about 100 nm.

[0482] Embodiment 36. The mRNA therapeutic composition of embodiment 32, wherein the lipid nanoparticles have a size of about 55 nm to about 80 nm.

[0483] Embodiment 37 The mRNA therapeutic composition of embodiment 3, wherein the PEG-lipid conjugate is PEG-DMG or PEG-PE.

[0484] Embodiment 38. The mRNA therapeutic composition of embodiment 37, wherein the PEG-DMG is PEG2000-DMG or PEG2000-PE.

[0485] Embodiment 39. The LPMP comprises: about 20 mol % to about 50 mol % of an ionizable lipid; about 20 mol % to about 60 mol % of natural lipids; about 7 mol % to about 20 mol % of a sterol, and The mRNA therapeutic composition of any one of the preceding embodiments, comprising about 0.5 mol % to about 3 mol % of a polyethylene glycol (PEG)-lipid conjugate.

[0486] Embodiment 40. The LPMP comprises: about 35 mole % ionizable lipids; Approximately 50 mol % natural lipids, about 12.5 mole % sterols, and The mRNA therapeutic composition of embodiment 39, comprising about 2.5 mol % of a polyethylene glycol (PEG)-lipid conjugate.

[0487] Embodiment 41. The mRNA therapy composition of any one of the preceding embodiments, wherein the mRNA therapy composition has a total lipid:polynucleotide weight ratio of about 50:1 to about 10:1.

[0488] Embodiment 42. The mRNA therapeutic composition of embodiment 41, wherein the mRNA therapeutic composition has a total lipid:polynucleotide weight ratio of about 44:1 to about 24:1.

[0489] Embodiment 43. The mRNA therapeutic composition of embodiment 41, wherein the mRNA therapeutic composition has a total lipid:polynucleotide weight ratio of about 40:1 to about 28:1.

[0490] Embodiment 44. The mRNA therapeutic composition of embodiment 41, wherein the mRNA therapeutic composition has a total lipid:polynucleotide weight ratio of about 38:1 to about 30:1.

[0491] Embodiment 45. The mRNA therapeutic composition of embodiment 41, wherein the mRNA therapeutic composition has a total lipid:polynucleotide weight ratio of about 37:1 to about 33:1.

[0492] Embodiment 46. The mRNA therapeutic composition of embodiment 1, further comprising a HEPES or TRIS buffer at a pH of about 7.0 to about 8.5.

[0493] Embodiment 47. The mRNA therapeutic composition of embodiment 46, wherein the HEPES or TRIS buffer is at a concentration of about 7 mg / mL to about 15 mg / mL.

[0494] Embodiment 48. The mRNA therapeutic composition of embodiment 46 or 47, further comprising about 2.0 mg / mL to about 4.0 mg / mL NaCl.

[0495] Embodiment 49 The mRNA therapeutic composition of embodiment 1, further comprising one or more cryoprotectants.

[0496] Embodiment 50. The mRNA therapeutic composition of embodiment 49, wherein the one or more cryoprotectants are selected from the group consisting of sucrose, glycerol, and combinations thereof.

[0497] Embodiment 51. The mRNA therapeutic composition of embodiment 50, wherein the mRNA therapeutic composition comprises a combination of sucrose at a concentration of about 70 mg / mL to about 110 mg / mL and glycerol at a concentration of about 50 mg / mL to about 70 mg / mL.

[0498] Embodiment 52 The mRNA therapeutic composition of embodiment 1, wherein the mRNA therapeutic is a lyophilized composition.

[0499] Embodiment 53 The mRNA therapeutic composition of embodiment 52, wherein the lyophilized mRNA therapeutic composition comprises one or more lyoprotectants.

[0500] Embodiment 54 The mRNA therapeutic composition of embodiment 53, wherein the lyophilized mRNA therapeutic composition comprises poloxamer, potassium sorbate, sucrose, or any combination thereof.

[0501] Embodiment 55 The mRNA therapeutic composition of embodiment 54, wherein the poloxamer is poloxamer 188.

[0502] Embodiment 56. The mRNA therapeutic composition of any one of embodiments 52 to 55, wherein the lyophilized mRNA therapeutic composition comprises from about 0.01 to about 1.0% w / w polynucleotide.

[0503] Embodiment 57. The mRNA therapeutic composition of any one of embodiments 52 to 55, wherein the lyophilized mRNA therapeutic composition comprises about 1.0 to about 5.0% w / w lipid.

[0504] Embodiment 58. The mRNA therapeutic composition of any one of embodiments 52 to 55, wherein the lyophilized mRNA therapeutic composition comprises about 0.5 to about 2.5% w / w TRIS buffer.

[0505] Embodiment 59. The mRNA therapeutic composition of any one of embodiments 52 to 55, wherein the lyophilized mRNA therapeutic composition comprises about 0.75 to about 2.75% w / w NaCl.

[0506] Embodiment 60. The mRNA therapeutic composition of any one of embodiments 52 to 55, wherein the lyophilized mRNA therapeutic composition comprises about 85 to about 95% w / w sugar.

[0507] Embodiment 61 The mRNA therapeutic composition of embodiment 60, wherein the sugar is sucrose.

[0508] Embodiment 62. The mRNA therapeutic composition of any one of embodiments 52 to 55, wherein the lyophilized mRNA therapeutic composition comprises about 0.01 to about 1.0% w / w poloxamer.

[0509] Embodiment 63 The mRNA therapeutic composition of embodiment 62, wherein the poloxamer is poloxamer 188.

[0510] Embodiment 64. The mRNA therapeutic composition of any one of embodiments 52 to 55, wherein the lyophilized mRNA therapeutic composition comprises about 1.0 to about 5.0% w / w potassium sorbate.

[0511] Embodiment 65. A method of making an mRNA therapeutic composition, comprising: Reconstituting a membrane comprising the purified PMP lipids in the presence of an ionizable lipid to produce a lipid reconstituted plant messenger pack (LPMP) comprising said ionizable lipid, said ionizable lipid having the following characteristics: (i) at least two ionizable amines; (ii) at least three lipid tails, each of said lipid tails being at least 6 carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) ionizable amine and heteroorganic groups separated by a chain of at least two atoms; and (v) an N:P ratio of at least 10; and and loading said LPMP with one or more polynucleotides encoding one or more antigenic (e.g., tumor antigenic) or signaling polypeptides.

[0512] Embodiment 66. A method of delivering an mRNA therapeutic in a subject, comprising: A method comprising administering to a subject the mRNA therapeutic composition of any one of embodiments 1-64.

[0513] Embodiment 67. A method of inducing an immune response in a subject, comprising: A method comprising administering to a subject the mRNA therapeutic composition of any one of embodiments 1-64.

[0514] Embodiment 68. A method of treating or preventing cancer in a subject, comprising: A method comprising administering to a subject the mRNA therapeutic composition of any one of embodiments 1-64.

[0515] Embodiment 69. The method of any one of embodiments 66-68, wherein the mRNA therapy composition is administered by oral, intravenous, intradermal, intramuscular, intranasal, intraocular, rectal, intrajejunal, intratumoral, and / or subcutaneous administration.

[0516] Embodiment 70 The method of embodiment 69, wherein the mRNA therapeutic composition is administered by oral administration, intravenous administration, intramuscular administration, and / or subcutaneous administration.

[0517] Embodiment 71. The method of any one of embodiments 66-68, wherein the mRNA therapeutic composition is administered at a dosage level sufficient to deliver from about 0.006 mg / kg to about 0.5 mg / kg of polynucleotide (e.g., mRNA) to the subject.

[0518] Embodiment 72. The method of embodiment 71, wherein the mRNA therapeutic composition is administered at a dosage level sufficient to deliver about 0.01 mg / kg, about 0.05 mg / kg, or about 0.1 mg / kg of polynucleotide (e.g., mRNA) to the subject.

[0519] Embodiment 72 The method of embodiment 67 or 68, wherein the mRNA therapeutic composition is administered to the subject once, twice, three times, four times, or more.

[0520] Embodiment 73 The method of embodiment 72, wherein the mRNA therapeutic composition is administered once or twice to the subject.

[0521] Embodiment 74 The method of any one of embodiments 66-68, further comprising administering to the subject an additional therapeutic agent.

[0522] Embodiment 75 The method of embodiment 74, wherein the additional therapeutic agent is an anti-cancer therapeutic agent.

[0523] Embodiment 76 The method of embodiment 75, wherein the additional therapeutic agent is a therapeutic agent that treats and / or prevents chronic pain.

[0524] Embodiment 77 The method of embodiment 76, wherein the additional therapeutic agent is buprenorphine, meloxicam SR, or a combination thereof.

[0525] Embodiment 78 The method of embodiment 74, wherein an additional therapeutic agent is administered prior to, simultaneously with, or following administration of the mRNA therapeutic composition.

[0526] Embodiment 79 The method of embodiment 72, wherein the mRNA therapeutic composition is administered to the subject three or four times.

[0527] Embodiment 80. The method of embodiment 71, wherein the mRNA therapeutic composition is administered at a dosage level sufficient to deliver about 0.006 mg / kg, about 0.02 mg / kg, about 0.2 mg / kg, about 0.4 mg / kg, or about 0.5 mg / kg of polynucleotide (e.g., mRNA) to the subject.

[0528] Embodiment 81. The method of embodiment 71, wherein the mRNA therapeutic composition is administered at a dosage level sufficient to deliver about 0.006 mg / kg, about 0.01 mg / kg, about 0.02 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, or about 0.4 mg / kg of polynucleotide (e.g., mRNA) to the subject.

[0529] Embodiment 82. The method of embodiments 66-68, wherein the mRNA therapeutic composition is administered at a dosage level sufficient to deliver from about 0.0003 mg / kg to about 0.002 mg / kg of polynucleotide (e.g., mRNA) to the subject.

[0530] Embodiment 83. The method of embodiment 82, wherein the mRNA therapeutic composition is administered to the subject once, twice, three times, four times, or more at a dosage level sufficient to deliver from about 0.0003 mg / kg to about 0.5 mg / kg of the polynucleotide to the subject.

[0531] Embodiment 84. The mRNA therapeutic composition of embodiments 66-68, wherein the polynucleotide is an mRNA encoding an IL-15 and / or IL-15Ra molecule comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of an IL-15 molecule provided in Table 9 or any one of Examples 1-9.

[0532] Embodiment 85. The mRNA therapeutic composition of embodiment 84, wherein the IL-15 molecule comprises a naturally occurring IL-15 molecule, a fragment of a naturally occurring IL-15 molecule, or a variant thereof.

[0533] Embodiment 86 The mRNA therapeutic composition of embodiment 84, wherein the IL-15Ra molecule comprises a naturally occurring IL-15Ra molecule, a fragment of a naturally occurring IL-15Ra molecule, or a variant thereof.

[0534] Embodiment 87. The method of embodiments 66-68, wherein the mRNA therapeutic composition induces proliferation or activation of T cells and / or NK cells.

[0535] Embodiment 88 The method of embodiments 66-68, wherein the mRNA therapeutic composition modulates the proliferation or activation of T cells and / or NK cells.

[0536] Embodiment 89 The method of embodiment 88, wherein the mRNA therapeutic composition modulates the proliferation or activation of CD4 T cells and / or CD8 T cells.

[0537] Embodiment 90 The method of embodiment 88, wherein the mRNA therapeutic composition modulates the proliferation or activation of T cells and / or NK cells in the blood, lymph nodes, spleen, or any combination thereof.

[0538] Embodiment 91 The method of embodiment 88, wherein the mRNA therapeutic composition regulates the proliferation or activation of T cells and / or NK cells in a specific organ, such as the lung, intestine, or skin.

[0539] Embodiment 92. The method of embodiment 88, wherein the mRNA therapeutic composition modulates T cell and / or NK cell proliferation or activation for 6 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 13 days, or more after administration.

[0540] Embodiment 93. The method of embodiments 66-68, wherein the mRNA therapeutic composition modulates the activity or expression of granzyme B / perforin, CD25 (IL-2Ra), TNF-alpha, IL-15, IL-6, IL-8, IL-12, GM-CSF, G-CSF, IL-1, IL-2, IL-4, IL-5, IL-8, IL-9, IL-10, IL-13, IL-17, IL-33, PD-L1, CCL2, CCL3, CCL4, CXCL1, CXCL2, CXCL10 (IP-10), CCL20, CD40, TNF-β, LAF, TCGF, ​​BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-a, IFN-β, IFN-γ, or any combination thereof.

[0541] Embodiment 94. The method of embodiment 93, wherein the mRNA therapeutic composition modulates an immune response for 6 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 13 days, 15 days, 20 days, or more after administration...

Claims

1. 1. An mRNA therapeutic composition comprising: comprising one or more polynucleotides encoding one or more tumor antigenic or signaling polypeptides; Formulated into a lipid reconstituted plant messenger pack (LPMP) comprising natural lipids and ionizable lipids, wherein the ionizable lipids have the following characteristics: (i) at least two ionizable amines; (ii) at least three lipid tails, each of said lipid tails being at least six carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) an ionizable amine and heteroorganic group separated by a chain of at least two atoms; and (v) an N:P ratio of at least 10;

2. 2. The mRNA therapeutic composition of claim 1, wherein the natural lipid is extracted from lemon or algae.

3. The mRNA therapeutic composition of claim 1, wherein the LPMP further comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate.

4. The mRNA therapeutic composition of claim 3, wherein the LPMP comprises an ionizable lipid:natural lipid:sterol:PEG lipid molar ratio of about 35:50:12.5:2.5 or about 35:20:42.5:2.

5.

5. 2. The mRNA therapeutic composition of claim 1, wherein the ionizable lipid is selected from the group consisting of 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, lipid 5, SM-102 (lipid H), and ALC-315.

6. 2. The mRNA therapeutic composition of claim 1, wherein the polypeptide is IL-2 peptide, IL-2-Ra, IL-15 peptide, IL-15-Ra, anti-CD19, CD20, CAR-T, anti-HER2, etanercept (Enbrel), Humira, erythropoietin, Epogen, filgrastim, Keytruda, rituximab, romiplostim, sargramostim, or a fragment or subunit thereof.

7. 2. The mRNA therapeutic composition of claim 1, wherein the polynucleotide is an mRNA encoding an IL-2 molecule comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of an IL-2 molecule provided in any one of Tables I-III.

8. The mRNA therapeutic composition of claim 1, wherein the LPMP has a size of less than about 200 nm.

9. The LPMP is about 20 mol % to about 50 mol % of said ionizable lipid; about 20 mol % to about 60 mol % naturally occurring lipids; about 7 mol % to about 20 mol % of a sterol, and 4. The mRNA therapeutic composition of claim 3, comprising about 0.5 mol% to about 3 mol% of a polyethylene glycol (PEG)-lipid conjugate.

10. 10. The mRNA therapeutic composition of claim 1, wherein the mRNA therapeutic composition has a total lipid:polynucleotide weight ratio of about 50:1 to about 10:

1.

11. 10. The mRNA therapeutic composition of claim 1, further comprising a HEPES or TRIS buffer at a pH of about 7.0 to about 8.

5.

12. The mRNA therapeutic composition of claim 1, further comprising one or more cryoprotectants.

13. The mRNA therapeutic composition of claim 1, wherein the mRNA therapeutic is a lyophilized composition comprising one or more lyoprotectants.

14. The mRNA therapeutic composition of any one of claims 1 to 13 for delivering an mRNA therapeutic agent in a subject.

15. The mRNA therapeutic composition of claim 14 for inducing an immune response in a subject.

16. The mRNA therapeutic composition of claim 14 for treating or preventing cancer in a subject.

17. 15. The mRNA therapeutic composition of claim 14, which is administered by oral, intravenous, intradermal, intramuscular, intranasal, intraocular, rectal, intrajejunal, intratumoral, and / or subcutaneous administration.

18. 15. The mRNA therapeutic composition of claim 14, administered at a dosage level sufficient to deliver about 0.006 mg / kg to about 0.5 mg / kg of the polynucleotide to the subject.

19. The mRNA therapeutic composition of claim 14, which is administered to a subject once, twice, three times, four times, or more.

20. The mRNA therapeutic composition of claim 14, wherein an additional therapeutic agent is delivered to the subject, and the additional therapeutic agent is an anti-cancer therapeutic agent or a therapeutic agent for treating and / or preventing chronic pain.

21. 15. The mRNA therapeutic composition of claim 14, wherein the mRNA therapeutic composition is administered at a dosage level sufficient to deliver about 0.0003 mg / kg to about 0.002 mg / kg of the polynucleotide to the subject.

22. The mRNA therapeutic composition of claim 1, wherein the polynucleotide is an mRNA encoding an IL-15 and / or IL-15Ra molecule comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of an IL-15 molecule provided in Table 9 or any one of Examples 1-9.

23. The mRNA therapeutic composition of claim 14, which regulates the proliferation or activation of T cells and / or NK cells.

24. The mRNA therapeutic composition of claim 23, which regulates the proliferation or activation of CD4 T cells and / or CD8 T cells.

25. 24. The mRNA therapeutic composition of claim 23, which provides modulation in the blood, lymph nodes, spleen, or any combination thereof.

26. 15. The mRNA therapeutic composition of claim 14, which modulates the activity or expression of granzyme B / perforin, CD25 (IL-2Ra), TNF-alpha, IL-15, IL-6, IL-8, IL-12, GM-CSF, G-CSF, IL-1, IL-2, IL-4, IL-5, IL-8, IL-9, IL-10, IL-13, IL-17, IL-33, PD-L1, CCL2, CCL3, CCL4, CXCL1, CXCL2, CXCL10 (IP-10), CCL20, CD40, TNF-β, LAF, TCGF, ​​BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β, IFN-γ, or any combination thereof.

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