mRNA encoding constitutively active cyclic GMP-AMP synthase and a lipid delivery vehicle therefor

Abstract

The present disclosure relates to a composition for the constitutive expression of cyclic GMP-AMP synthase in cells of a mammalian subject and its use for enhancing the immunogenicity of an mRNA vaccine. The mRNA can be encapsulated in lipid nanoparticles (LNPs) or complexed with lipids (RNA-Lipoplex). The present disclosure also relates to a composition further comprising a pathogen recognition receptor agonist. In some embodiments, the compositions of the present disclosure do not contain lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 341,987, filed on May 13, 2022, which is hereby incorporated by reference in its entirety.

[0002] Reference to Electronic Sequence Listing The content of the electronic sequence listing (165532000540SEQLIST.xml; size: 22,326 bytes; and creation date: May 10, 2023) is hereby incorporated by reference in its entirety.

[0003] Field The present disclosure relates to compositions for the constitutive expression of cyclic GMP - AMP synthase in cells of mammalian subjects and its use for enhancing the immunogenicity of mRNA vaccines. The above - mentioned mRNA can be encapsulated in lipid nanoparticles (LNPs) or complexed with lipids (RNA - Lipoplex). The present disclosure also relates to compositions further comprising pathogen - recognition receptor agonists.

Background Art

[0004] Background Advances in mRNA chemistry and delivery systems have enabled the rapid generation of several effective mRNA COVID-19 vaccines (Hou et al., Nature Review Materials, 6:1078-1094, 2021). However, recent reports have found that the T cell reactivity against the SARS-CoV-2 spike protein is considerably lower in vaccinated non-infected individuals than in individuals previously infected with SARS-CoV-2 (Naranbhai et al., Cell, 185:1-11, 2022). This is a problem considering that higher levels of SARS-CoV-2-reactive CD4+ and CD8+ T cell immune responses have been found to be associated with milder COVID-19 disease (Rydyznski Moderbacher et al., Cell, 183(4):996-1012, 2020). Therefore, there is a need in the art for formulations that enhance the cellular immune response elicited by mRNA vaccines. In particular, an adjuvant suitable for increasing the adaptive immune response against antigens encoded by nucleic acids is desired.

Prior Art Documents

Non-Patent Documents

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Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Means for Solving the Problems

[0006] Brief Summary The present disclosure relates to a composition for the constitutive expression of cyclic GMP-AMP synthase in cells of a mammalian subject and its use for enhancing the immunogenicity of mRNA vaccines. The mRNA can be encapsulated in lipid nanoparticles (LNP) or complexed with lipids (RNA-Lipoplex). The present disclosure also relates to a composition further comprising a pathogen recognition receptor agonist. BRIEF DESCRIPTION OF THE DRAWINGS

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[0020] Detailed Description The present disclosure relates to compositions for the constitutive expression of cyclic GMP-AMP synthase in cells of a mammalian subject and its use for enhancing the immunogenicity of mRNA vaccines. The mRNA can be encapsulated in lipid nanoparticles (LNPs) or complexed with lipids (RNA-Lipoplex). The present disclosure also relates to compositions further comprising a pathogen recognition receptor agonist. In some embodiments, the compositions of the present disclosure do not contain lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain.

[0021] The induction of an inflammatory response may be desirable, for example, for immunotherapy or vaccination. COVID-19 mRNA vaccines (including LNPs filled with mRNA encoding SARS-CoV-2 antigens) have been found to be effective in reducing the frequency and severity of infection. As described herein, inducing the cGAS-STING innate immune pathway aids compositions comprising LNPs filled with mRNA encoding a protein antigen (which, in an exemplary embodiment, is ovalbumin (OVA)). STING signaling is particularly important among all possible innate immune signaling pathways. This is because, unlike some other signaling pathways, cGAS-STING activation does not induce translational inhibition.

[0022] To induce cGAS-STING signaling, a cGAS mutant lacking a portion of its N-terminus was designed to be constitutively active (cGASΔN). The mRNA encoding the cGAS mutant was packaged into LNPs for cell uptake and protein expression. The LNP-packaged mRNA encoding cGASΔN has the ability to assist (adjuvant) the immune response via the cGAS-STING pathway. Importantly, including mRNA encoding constitutively active cGAS (e.g., cGASΔN) increases the potency of LNPs filled with mRNA encoding an antigen by increasing inflammatory signals.

[0023] General Techniques and Definitions The practice of the present disclosure, unless otherwise indicated, uses conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

[0024] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless otherwise indicated. For example, "an additive" includes one or more additives.

[0025] The term "comprising" as used herein indicates that there is no limitation and that such embodiments may include additional elements. In contrast, the term "consisting of" is exclusive and indicates that such embodiments do not include additional elements (except for trace impurities). The term "consisting essentially of" is partially exclusive and indicates that such embodiments may further include elements that do not substantially change the basic characteristics of such embodiments.

[0026] As used herein in connection with a value, the term "about" encompasses from 90% to 110% of that value (e.g., about 900 Daltons of molecular weight refers to a molecular weight of from 810 Daltons to 990 Daltons).

[0027] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to produce a beneficial or desired result, including clinical results, and thus the "effective amount" depends on the context in which it is applied. For example, in the context of administering an immunogenic composition comprising an antigen and one or more mRNAs encoding constitutively active cGAS, the effective amount comprises sufficient mRNA to stimulate an immune response against the antigen (e.g., an antigen-reactive antibody and / or a cellular immune response).

[0028] The terms "individual" and "subject" refer to a mammal. Mammals include, but are not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats). In some embodiments, the subject is a human patient (e.g., a human patient suffering from cancer and / or an infectious disease).

[0029] As used herein in connection with an immunological composition, the term "dosage" refers to the measured portion of the immunogenic composition that is taken (administered to, or received by) a subject at any given time.

[0030] As used herein, the terms "isolated" and "purified" refer to a substance that has been removed from at least one of the naturally associated components (e.g., removed from its natural environment). By way of example, when used in connection with a phospholipid, an isolated phospholipid is at least 90%, 95%, 96%, 97%, 98% or 99% pure as determined by thin layer chromatography or gas chromatography. As a further example, when used in connection with a recombinant protein, an isolated protein refers to a protein that has been removed from the culture medium of the host cell that produced the protein.

[0031] As used herein, the terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in a form that allows for the biological activity of the active ingredient to be effective and that does not contain additional components that are toxic to an unacceptable degree to the individual to whom the formulation or composition is administered. Such formulations or compositions are intended to be sterile.

[0032] As used herein, "excipient" includes a pharmaceutically acceptable excipient, carrier, vehicle or stabilizer that is non-toxic to the cells or mammals being exposed thereto at the dosages and concentrations used. Often, a physiologically acceptable excipient is an aqueous pH buffer solution.

[0033] As used herein, the term "antigen" refers to a substance that is recognized and specifically bound by an antibody or by a T cell antigen receptor. Antigens can include peptides, polypeptides, proteins, glycoproteins, polysaccharides, glycoconjugates, sugars, gangliosides, lipids and phospholipids; portions of these and combinations thereof. In the context of the present disclosure, the term "antigen" typically refers to a polypeptide encoded by a nucleic acid sequence of mRNA or DNA. The polypeptide antigen is preferably at least 8 amino acid residues in length and may include one or more post-translational modifications.

[0034] The term "agonist" is used in the broadest sense and includes any molecule that activates signal transduction through a receptor. In some embodiments, the agonist binds to the receptor. For example, a TLR8 agonist binds to TLR8 and activates the TLR8 signal transduction pathway.

[0035] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group. Cx alkyl refers to an alkyl group having x carbon atoms. Cx-Cy alkyl or Cx-y alkyl refers to an alkyl group having from x to y carbon atoms (including both ends).

[0036] "Alkylene" refers to a divalent saturated aliphatic hydrocarbyl group.

[0037] "Alkenyl" refers to a monovalent hydrocarbyl group having at least one double bond (>C=C<). Cx alkenyl refers to an alkenyl group having x carbon atoms. Cx-Cy alkenyl or Cx-y alkenyl refers to an alkenyl group having from x to y carbon atoms (including both ends).

[0038] A "stimulation" of a response or parameter includes inducing and / or enhancing that response or parameter when compared to the same conditions except for the parameter of interest, or alternatively when compared to another condition (e.g., an increase in TLR signal transduction in the presence of a TLR agonist compared to the absence of the TLR agonist). For example, a "stimulation" of an immune response means an increase in that response. Depending on the parameter being measured, that increase can be from 2-fold to 2,000-fold, or 5-fold to 500-fold, or more, or from 2-fold, 5-fold, 10-fold, 50-fold, or 100-fold to 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold.

[0039] Conversely, "inhibition" of a response or parameter includes reducing and / or suppressing that response or parameter when compared to the same conditions in other respects except for the parameter of interest, or alternatively when compared to another condition (e.g., a decrease in abnormal cell proliferation after administration of a composition of the present disclosure compared to no administration or treatment with a placebo composition). For example, "inhibition" of an immune response means a decrease in the response. Depending on the parameter being measured, that decrease can be from 2-fold to 2,000-fold, or 5-fold to 500-fold, or more, or from 2-fold, 5-fold, 10-fold, 50-fold, or 100-fold to 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold.

[0040] The relative terms "higher" and "lower" refer to a measurable increase or decrease, respectively, in a response or parameter when compared to the same conditions in other respects except for the parameter of interest, or alternatively when compared to another condition. For example, "a higher level of DC overactivation" refers to the level of DC overactivation as a result of a treatment condition that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the level of DC overactivation as a result of a control condition. Similarly, "a lower level of DC overactivation" refers to the level of DC overactivation as a result of a treatment condition that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold lower than the level of DC overactivation as a result of a control condition.

[0041] As used herein, the term "immunization" refers to a process that increases the immune response of a mammalian subject to an antigen and thus improves its ability to resist or overcome an infection and / or resist a disease.

[0042] The term "vaccination" as used herein refers to the introduction of a vaccine into the body of a mammalian subject.

[0043] As used herein, the term "adjuvant" refers to a substance that, when added to a composition containing an antigen or a nucleic acid encoding an antigen, enhances or potentiates an immune response to the antigen in the mammalian recipient upon exposure.

[0044] As used herein, the terms "treating" or "treatment" refer to the practice of administering to an individual (human or otherwise) one or more therapeutic agents in an attempt to obtain a beneficial or desired result (including clinical results). Beneficial or desired clinical results include, but are not limited to, alleviation or improvement of one or more symptoms or signs of a disease, diminishment of the extent of a disease, stabilization (i.e., non-worsening) of a disease state, prevention of the spread of a disease, delay or slowing of disease progression, amelioration or palliation of a disease state, and remission (whether partial or total). "Treatment" may also mean prolonging survival as compared to the expected survival of an untreated individual. Further, "treating" and "treatment" can occur by administration of a single dose of one or more therapeutic agents or can occur upon administration of a series of doses of one or more therapeutic agents. "Treating" or "treatment" does not require complete alleviation of symptoms or signs, does not require cure, and specifically includes protocols having only a palliative effect on an individual. "Palliating" a disease or disorder means that the degree and / or undesirable clinical manifestations of the disease or disorder are reduced, and / or the time course of progression of the disease or disorder is slowed, as compared to the predicted course of non-treatment.

[0045] As used herein in the context of cGAS, the term "constitutively-active" refers to a cGAS variant that binds to DNA in the cytoplasm and catalyzes cGAMP synthesis even under some conditions where native cGAS has little or no enzymatic activity. In some preferred embodiments, said "constitutively-active cGAS" is a "truncated cGAS" such as cGASΔN that contains an enzyme domain that binds DNA at the C-terminus in the absence of an N-terminal disordered domain. That is, cGASΔN is a constitutively-active cGAS lacking the regulation of enzymatic activity impaired by the N-terminal disordered domain of full-length cGAS.

[0046] I. Constitutively-Active Cyclic GMP-AMP Synthase Cyclic GMP-AMP synthase, also called cGAMP synthase or cGAS, is an enzymatic sensor of cytosolic DNA. cGAS recognizes double-stranded DNA independent of its sequence, leading to dimerization, formation of liquid-like droplets, and production of the second messenger 2’3’ cyclic GMP-AMP (cGAMP). Said cGAMP binds to STING, activates it, and results in the expression of interferon and other inflammatory mediators. Specifically, STING activation results in a signaling cascade that ultimately activates the IRF3 and NF-kB transcription factors. IRF3 activation leads to the expression of genes such as IP-10 and type I interferons that induce an antiviral immune response. NF-kB activation induces the expression of inflammatory cytokines (e.g., IL-6 that enhances the inflammatory immune response).

[0047] Human cGAS is 522 amino acids in length and contains an N-terminal phosphoinositide-binding domain (residues 1-59) and a C-terminal DNA-binding and enzyme domain (residues 160-522) (Barnett et al., Cell, 176:1432-1446, 2019). Importantly, it has been found that the expression of cGASΔN in human leukemia monocytic cell lines results in higher levels of interferon and the expression of genes stimulated by interferon (Barnett, supra, 2019).

[0048] The amino acid sequence of human cGAS (GenBank No. NP_612450.2) is as follows:

Chem.

[0049] The amino acid sequence of the N-terminal domain of cGAS is as follows:

Chem.

[0050] The amino acid sequence of the C-terminal domain (cGASΔN) of cGAS is as follows:

Chem.

[0051] The nucleotide sequence encoding human cGASΔN (which was codon-optimized for expression in mouse cells) is shown as SEQ ID NO: 17. Using a DNA template having this nucleotide sequence, mRNA encoding human cGASΔN was prepared, filled into LNPs, and tested as described in Examples 1, 2, and 3.

[0052] The compositions and methods of the present disclosure include nucleic acids encoding catalytically active cGAS as a constitutive adjuvant for improving the adaptive immune response induced by mRNA vaccines. In some preferred embodiments, the constitutively active cGAS is a truncated cGAS (cGASΔN) lacking the N-terminal phosphoinositide-binding domain. In some preferred embodiments, the constitutively active cGAS is a truncated cGAS (cGASΔN) containing the C-terminal DNA-binding and enzyme domains.

[0053] Homologs of cGAS are expressed in species across the animal kingdom, and the cGAS amino acid sequence is conserved in higher primates. An alignment of the amino acid sequence of the C-terminal domain of human cGAS and the amino acid sequences of the C-terminal domains of multiple non-human primate cGAS proteins is shown in FIG. 1.

Table I

[0054] In some preferred embodiments, the constitutively active cGAS described above is a truncated human cGAS lacking the N-terminal domain (SEQ ID NO: 11). In some preferred embodiments, the constitutively active cGAS described above is a truncated human (SEQ ID NO: 1) containing the C-terminal domain. In some preferred embodiments, cGASΔN comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. In some preferred embodiments, cGASΔN comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. In some preferred embodiments, cGASΔN comprises the consensus amino acid sequence of SEQ ID NO: 9. For expression in transfected cells, the nucleic acid encoding cGASΔN is in operable combination with the start codon (ATG).

[0055] "Percent (%) sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software (such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software). One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment over the entire length of the sequences being compared. For example, the % sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or including a particular % sequence identity to, with, or against a given amino acid sequence B) can be calculated as follows: 100 × fraction X / Y. In this case, X is the number of amino acid residues that are matched and scored as identical by the sequences in the alignment of the programs for A and B, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % sequence identity of A to B will not be equal to the % sequence identity of B to A.

[0056] II. mRNA Encoding the Antigen The compositions and methods of the present disclosure may contain mRNA encoding an antigen or, alternatively, are suitable for use with a formulation containing mRNA encoding an antigen. In some embodiments, the antigen is a proteinaceous antigen. The terms "polypeptide" and "protein" are used interchangeably herein in relation to an antigen comprising a peptide chain at least 8 amino acids in length. In some embodiments, the antigen is 8 to 1800 amino acids, 9 to 1000 amino acids, or 10 to 100 amino acids in length. The polypeptide may be post-translationally modified such as by phosphorylation, hydroxylation, sulfonation, palmitoylation, and / or glycosylation.

[0057] In some embodiments, the antigen is a tumor antigen comprising the amino acid sequence of at least one full-length protein or a fragment thereof. In some embodiments, the tumor antigen comprises an amino acid sequence derived from a tumor protein or a fragment thereof. In some embodiments, the mammalian antigen is a neoantigen or is encoded by a gene containing a mutation as compared to a gene in normal cells derived from a mammalian subject. Neoantigens are thought to be particularly useful in enabling T cells to distinguish between cancer cells and non-cancer cells (see, e.g., Schumacher and Schreiber, Science, 348:69-74, 2015). In other embodiments, the tumor antigen comprises a viral antigen (e.g., an antigen of a virus that causes cancer).

[0058] In some embodiments, the tumor antigen is a fusion protein comprising two or more polypeptides, where each protein comprises an amino acid sequence from a different tumor antigen or non-contiguous amino acid sequences from the same tumor antigen. In some of these embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, where each polypeptide comprises non-contiguous amino acid sequences from the same tumor antigen.

[0059] In some embodiments, the antigen is a microbial antigen. In some embodiments, the microbial antigen includes a viral antigen, a bacterial antigen, a protozoal antigen, a fungal antigen, or a combination thereof. In some embodiments, the microbial antigen includes a surface protein or other antigenic subunit of the microorganism.

[0060] In some preferred embodiments, the mRNA includes a 5'untranslated region (5'UTR) at the 5'end of the coding region and a 3'untranslated region (3'UTR) at the 3'end of the coding region. In some preferred embodiments, the mRNA includes one or both of a 5'cap structure and a polyA tail.

[0061] In some embodiments, the mRNA further encodes a ribosome skipping sequence (e.g., a 2A-like sequence shown as SEQ ID NO: 12). Additional 2AL sequences are shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In some embodiments where the mRNA includes two or more coding regions, the coding regions can be separated by a 2AL sequence. For example, the 2AL sequence can be located between the coding region of constitutively active cyclic GMP-AMP synthase (cGAS) and the coding region of the antigen (e.g., cGASΔNs-2AL-antigen or antigen-2AL-cGASΔN). Similarly, in some embodiments where the mRNA includes two or more coding regions for antigens, the coding regions can be separated by a 2AL sequence (e.g., antigen1-2AL-antigen2). Additional 2AL sequences for use in the mRNA of the present disclosure are known in the art (see, e.g., Luke et al., J. Gen. Virol, 89:1036-1042, 2008. The 2AL sequence in FIG. 2 is incorporated herein by reference).

[0062] III. Lipid-based delivery vehicle The compositions and methods of the present disclosure may include lipid-based delivery vehicles for mRNA vaccines. In some embodiments, the vehicle is a lipid nanoparticle (LNP). In other embodiments, the vehicle is a lipid that forms a complex (RNA-Lipoplex) with the mRNA.

[0063] In some embodiments, the LNP comprises at least one lipid selected from the group consisting of ionizable lipids, cationic lipids, phospholipids, pegylated lipids, structural lipids, and mixtures thereof. In some embodiments, the at least one lipid comprises an ionizable lipid. In some embodiments, the at least one lipid comprises a cationic lipid. In some embodiments, the at least one lipid comprises a phospholipid. In some embodiments, the at least one lipid comprises a pegylated lipid. In some embodiments, the at least one lipid comprises a structural lipid. In some embodiments, the at least one lipid comprises an ionizable lipid, a phospholipid, a pegylated lipid, and a structural lipid.

[0064] In some embodiments, the lipid components of the RNA-Lipoplex include one or more lipids. In some preferred embodiments, the one or more lipids include a first lipid and a second lipid, where the first lipid is different from the second lipid. In some embodiments, the first lipid is a cationic lipid and the second lipid is a neutral or anionic lipid.

[0065] The structures of lipids suitable for use in the lipid-based mRNA delivery vehicles of the present disclosure are shown in FIGS. 13A and 13B, which are adopted from FIG. 2 of Hou et al., Nature Review Materials, 6:1078-1094, 2021.

[0066] IV. Pathogen Recognition Receptor Agonist The compositions and methods of the present disclosure may further comprise additional pathogen recognition receptor (PRR) agonists. In some embodiments, the PRR agonists include agonists of toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), or C-type lectin receptors (CLRs). In some embodiments, the PRR agonists include TLR7 / 8 agonists.

[0067] A. TLR7 / 8 Agonist As used herein, the term “TLR7 / 8 agonist” refers to an agonist of TLR7 and / or TLR8. In one aspect, the TLR7 / 8 agonist is a TLR7 agonist. In another aspect, the TLR7 / 8 agonist is a TLR8 agonist. In a further aspect, the TLR7 / 8 agonist is an agonist of both TLR7 and TLR8. The TLR7 / 8 agonists of the present disclosure are suitable for hyperactivating human dendritic cells in response to DAMPs such as oxPAPC or PGPC.

[0068] In some aspects, the TLR7 / 8 agonist is a small molecule. In some embodiments, the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less, or a salt thereof. That is, the small molecule TLR7 / 8 agonist is not a large molecule such as a recombinant protein or synthetic oligonucleotide that can be regulated by the U.S. FDA’s Center for Biologics Evaluation and Research. Rather, the small molecule TLR7 / 8 agonist can be regulated by the FDA’s Center for Drug Evaluation and Research. In some embodiments, the small molecule has a molecular weight of about 90 to about 900 Daltons. In some embodiments, the TLR7 / 8 agonist includes imidazoquinoline compounds. In some preferred embodiments, the TLR7 / 8 agonist includes resiquimod (R848).

[0069] B. Other PRR agonists In some situations, the pathogen recognition receptor (PRR) agonist includes a toll-like receptor (TLR) agonist, provided that the TLR agonist does not include a TLR7 / 8 agonist. In some embodiments, the TLR agonist includes an agonist of one or more of TLR2, TLR3, TLR4, TLR5, TLR9, and TLR13. In some embodiments, the PRR agonist is a TLR2 / 6 agonist (e.g., Pam2CSK4). In other embodiments, the TLR agonist is a TLR4 agonist (e.g., monophosphoryl lipid A (MPLA)). However, in preferred embodiments, the TLR agonist is not an agonist of TLR2, TLR4, and / or TLR9. For example, in a preferred embodiment, the TLR9 agonist is not a TLR4 ligand (e.g., LPS (endotoxin)).

[0070] In other situations, the PRR agonist includes a NOD-like receptor (NLR) agonist. In a further situation, the PRR agonist includes a RIG-I-like receptor (RLR) agonist. In an additional situation, the PRR agonist includes a C-type lectin receptor (CLR) agonist.

[0071] V. Pharmaceutical formulations Some compositions of the present disclosure are pharmaceutical formulations containing pharmaceutically acceptable additives. The pharmaceutical formulations of the present disclosure can be in the form of a solution or a suspension. Alternatively, the pharmaceutical formulation can be a dehydrated solid (e.g., a lyophilized or spray-dried solid). The pharmaceutical formulations of the present disclosure are preferably sterile and preferably essentially free of endotoxin. The term "pharmaceutical formulation" is used interchangeably herein with the terms "pharmaceutical product" and "medicine". In some embodiments, the pharmaceutical formulation contains various components in a specific ratio based on the intended purpose of the formulation.

[0072] Examples of pharmaceutically acceptable additives of the present disclosure include, for example, solvents, buffers, tonicity modifiers, diluents, and preservatives (see, for example, Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the pharmaceutical formulation may include an additive that functions as one or more of a solvent, buffer, tonicity modifier, and diluent (for example, sodium chloride in a salt solution can serve as both an aqueous vehicle and a tonicity modifier).

[0073] In some embodiments, the pharmaceutical formulation includes an aqueous vehicle as a solvent. Suitable vehicles include, for example, sterile water, salt solutions, phosphate buffered salt solutions, and Ringer's solution. In some embodiments, the composition is isotonic.

[0074] The pharmaceutical formulation may include a buffer. The buffer controls the pH to inhibit the degradation of the active agent during processing, storage, and, if necessary, reconstitution. Suitable buffers include, for example, salts containing acetate, citrate, phosphate, or sulfate. Other suitable buffers include, for example, those containing amino acids (for example, arginine, glycine, histidine, and lysine). The buffer may further include hydrochloric acid or sodium hydroxide. In some embodiments, the buffer maintains the pH of the composition within the range of 6 to 9. In some embodiments, the pH is higher than 6, 7, or 8 (lower limit). In some embodiments, the pH is lower than 9, 8, or 7 (upper limit). That is, the pH is in the range of about 6 to 9 where the lower limit is lower than the upper limit.

[0075] The pharmaceutical composition may include a tonicity modifier. Suitable tonicity modifiers include, for example, dextrose, glycerol, sodium chloride, glycerin, and mannitol.

[0076] The pharmaceutical preparation may contain a bulking agent. The bulking agent is particularly useful when the pharmaceutical composition is to be lyophilized before administration. In some embodiments, the bulking agent is a protective agent that aids in the stabilization of the active agent and prevention of its degradation during freezing or spray drying and / or during storage. Suitable bulking agents are sugars (monosaccharides, disaccharides and polysaccharides) (e.g., sucrose, lactose, trehalose, mannitol, sorbitol, glucose and raffinose).

[0077] The pharmaceutical preparation may contain a preservative. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in a preferred embodiment, the pharmaceutical preparation is prepared under aseptic conditions and is in a single-use container and thus does not require the inclusion of a preservative.

[0078] The pharmaceutical preparation of the present disclosure is suitable for parenteral administration. That is, the pharmaceutical preparation of the present disclosure is not intended for enteral administration (e.g., not by oral, gastric, or rectal means).

[0079] VI. Method of Use In some aspects, the present disclosure relates to methods of use of any one of the compositions or formulations described herein. The methods of use are suitable for a plurality of uses including stimulating an immune response. In some embodiments, the methods of use include a method of treating cancer. In some embodiments, the methods of use include a method of inhibiting abnormal cell proliferation. In some embodiments, the methods of use include a method of treating or preventing an infectious disease. The methods include administering to an individual in need thereof an effective amount of a formulation or composition described herein to achieve a particular outcome. The individual is a mammalian subject (e.g., a human patient). In other embodiments, the individual is a non-human patient. In some embodiments, the individual is a canine patient. That is, in some embodiments, the methods of use include clinical use, but in other embodiments, the methods of use include pre-clinical and / or veterinary use. With respect to pre-clinical use, the mammalian subject can be a non-human primate (e.g., a monkey or an ape) or a rodent (e.g., a mouse or a rat). With respect to veterinary use, the mammalian subject can be a farm animal (e.g., a cow), a sport animal (e.g., a horse), or a pet (e.g., a companion animal (e.g., a dog or a cat)).

[0080] A. Stimulation of the immune response Briefly, the present disclosure provides a method of stimulating an immune response in an individual, the method comprising administering to the individual a composition or formulation described herein in an amount sufficient to stimulate an immune response in the individual. "Stimulating" an immune response (used interchangeably with "eliciting" an immune response) means increasing the immune response, which can result from eliciting a new immune response (e.g., as a result of a primary vaccination regimen) or enhancing an existing immune response (e.g., as a result of a booster vaccination regimen). In some embodiments, stimulating an immune response includes one or more of the following: stimulating cytokine production; stimulating B lymphocyte proliferation; stimulating interferon pathway-related gene expression; stimulating chemokine-related gene expression; and stimulating dendritic cell (DC) maturation. Methods for measuring stimulation of an immune response are known in the art.

[0081] For example, the present disclosure provides a method of inducing an antigen-specific immune response in an individual by administering to the individual a composition or formulation described herein in an amount sufficient to induce an antigen-specific immune response in the individual. In preferred embodiments, the composition or formulation comprises an antigen. In some embodiments, the composition or formulation is administered to the tissue of the individual, including the antigen. The immune response can include one or more of an antigen-specific antibody response, an antigen-specific cytotoxic T lymphocyte (CTL) response, and an antigen-specific helper T (Th) cell response. "Inducing" an antigen-specific antibody response means an increase in the titer of antigen-specific antibodies above a threshold level such as a baseline titer or serum protection level prior to administration. "Inducing" an antigen-specific CTL response means an increase in the frequency of antigen-specific CTLs found in peripheral blood above a baseline frequency prior to administration. "Inducing" an antigen-specific Th cell response means an increase in the frequency of antigen-specific Th cells found in peripheral blood above a baseline frequency prior to administration.

[0082] The analysis of the immune response (both qualitative and quantitative) can be by any method known in the art and can include antigen-specific antibody production (including measurement of specific antibody subclasses), activation of specific populations of lymphocytes such as B cells and helper T cells, production of cytokines such as IFN-α, IFN-γ, IL-6, IL-12, and / or release of histamine, but is not limited thereto. Methods for measuring the antigen-specific antibody response include enzyme-linked immunosorbent assay (ELISA). Activation of specific populations of lymphocytes can be measured by proliferation assays and fluorescence-activated cell sorting (FACS). Cytokine production can also be measured by ELISA. In some embodiments, the method of stimulating the immune response includes stimulation of interleukin-1β (IL-1β) secretion, interferon-γ (IFN-γ) secretion, and / or tumor necrosis factor α (TNF-α) secretion by monocytes-derived dendritic cells or peripheral blood mononuclear cells. In some preferred embodiments, at least 50%, 55%, 60%, 65%, 70% or 75% of the cells that have contacted the composition of the present disclosure remain viable 40 to 56 hours (or about 48 hours) after contact.

[0083] In some embodiments, the above method is suitable for stimulating an anti-tumor immune response. In other embodiments, the above method is suitable for stimulating an anti-microbial immune response. In some embodiments, the above anti-microbial response is an anti-bacterial immune response. In some embodiments, the above anti-microbial response is an anti-fungal immune response. In some embodiments, the above anti-microbial response is an anti-viral immune response. In some embodiments, the above anti-microbial response is an anti-parasitic immune response

[0084] B. Treatment or prevention of diseases The present disclosure further provides a method for treating or preventing a disease in an individual, the method comprising administering to the individual a composition or formulation described herein in an amount sufficient to treat or prevent the disease in the individual. In some embodiments, the disease is cancer. In some embodiments, the disease is abnormal cell proliferation. In other embodiments, the disease is an infectious disease.

[0085] In some embodiments, the method comprises treating cancer in an individual or treating a mammalian subject otherwise having cancer. In some embodiments, the cancer is a blood cancer (e.g., lymphoma, leukemia, or myeloma). In other embodiments, the cancer is a non-blood cancer (e.g., sarcoma, carcinoma, or melanoma). In some embodiments, the cancer is malignant.

[0086] In some embodiments, the method comprises inhibiting abnormal cell proliferation in an individual. "Abnormal cell proliferation" refers to the growth of a benign or malignant tumor. Malignant tumors can be metastatic tumors.

[0087] In some embodiments, the method comprises treating or preventing an infectious disease in an individual. In some embodiments, the infectious disease is caused by a viral infection. In other embodiments, the infectious disease is caused by a bacterial infection. In further embodiments, the infectious disease is caused by a fungal infection. In still further embodiments, the infectious disease is caused by a protozoan infection. Infectious diseases caused by zoonotic pathogens that infect humans and other animals (e.g., mammals or birds) are of particular importance. In some embodiments, the zoonotic pathogen is transmitted to humans via an intermediate species (vector). Numbered embodiments In this section, Embodiment 1 refers to both Embodiment 1' and Embodiment 1". Embodiment 1'. A composition comprising a first mRNA encapsulated in a first lipid nanoparticle (LNP) and a second mRNA encapsulated in a second LNP, wherein the first mRNA comprises a coding region of constitutively active cyclic GMP-AMP synthase (cGAS), the second mRNA comprises a coding region of an antigen, and the first LNP and the second LNP both comprise at least one lipid selected from the group consisting of phospholipids and at least one of ionizable lipids, pegylated lipids, structural lipids, and mixtures thereof. Embodiment 1”. A composition comprising an mRNA encapsulated in a lipid nanoparticle (LNP), wherein the mRNA comprises a coding region of constitutively active cyclic GMP-AMP synthase (cGAS), the LNP comprises at least one lipid selected from the group consisting of phospholipids and at least one of ionizable lipids, pegylated lipids, structural lipids, and mixtures thereof, and optionally the composition further comprises a further mRNA encapsulated in a further lipid nanoparticle, the further mRNA comprises a coding region of an antigen of interest, and the LNP comprises at least one lipid selected from the group consisting of phospholipids and at least one of ionizable lipids, pegylated lipids, structural lipids, and mixtures thereof. Embodiment 2. A composition comprising a first mRNA and a second mRNA encapsulated in a lipid nanoparticle (LNP), wherein the first mRNA comprises a coding region of constitutively active cyclic GMP-AMP synthase (cGAS), the second mRNA comprises a coding region of an antigen; and the LNP comprises at least one lipid selected from the group consisting of phospholipids and at least one of ionizable lipids, pegylated lipids, structural lipids, and mixtures thereof. Embodiment 3. A composition comprising mRNA encapsulated in lipid nanoparticles (LNP), wherein the mRNA comprises a first coding region and a second coding region separated by a 2A-like sequence, wherein the first coding region is the coding region of constitutively active cyclic GMP-AMP synthase (cGAS), and the second coding region is the coding region of an antigen, or the first coding region is the coding region of an antigen, and the second coding region is the coding region of constitutively active cyclic GMP-AMP synthase (cGAS), and the LNP comprises at least one lipid selected from the group consisting of phospholipids, and ionizable lipids, pegylated lipids, structural lipids, and mixtures thereof. Embodiment 4. The composition according to any one of Embodiments 1 to 3, wherein the at least one lipid comprises an ionizable lipid, a pegylated lipid, and a structural lipid. Embodiment 5. The ionizable lipid is i) 8-[(2-Hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102) or an analog or derivative thereof; and / or ii) 6-((2-Hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexane-1-aminium (ALC-0315) or an analog or derivative thereof; and / or iii) (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) ALC-0315 or an analog or derivative thereof, The composition according to any one of Embodiments 1 to 4. Embodiment 6. The pegylated lipid is the composition according to any one of Embodiments 1 to 5, selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and combinations thereof. Embodiment 7. The pegylated lipid is the composition according to any one of Embodiments 1 to 5, comprising polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG]. Embodiment 8. The structural lipid is the composition according to any one of Embodiments 1 to 7, selected from the group consisting of cholesterol, fucosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and combinations thereof. Embodiment 9. The structural lipid is the composition according to any one of Embodiments 1 to 7, comprising cholesterol. Embodiment 10. The phospholipid is i) a hydrophilic head selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lyso-phosphatidylcholine, and sphingomyelin; and ii) one or more fatty acid tails selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid, The composition according to any one of Embodiments 1 to 9. Embodiment 11. The phospholipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-doundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and combinations thereof, A composition according to any one of Embodiments 1 to 9, selected from the group consisting of. Embodiment 12. The composition according to Embodiment 11, wherein the phospholipid contains 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Embodiment 13. The composition according to any one of Embodiments 1 to 12, wherein the composition further contains at least one additive, and optionally, the additive contains sucrose here. Embodiment 14. The composition according to any one of Embodiments 1 to 13, wherein the composition does not contain lysophosphatidylcholine (LPC), and optionally, the LPC has a single C13-C24 acyl chain here. Embodiment 15. A composition containing mRNA complexed with one or more lipids (RNA-Lipoplex), wherein the mRNA contains a coding region of constitutively active cyclic GMP-AMP synthase (cGAS), and the one or more lipids contain a first lipid and a second lipid. Embodiment 16. A composition containing a first mRNA and a second mRNA complexed with one or more lipids (RNA-Lipoplex), wherein the first mRNA contains a coding region of constitutively active cyclic GMP-AMP synthase (cGAS), the second mRNA contains a coding region of an antigen, and the one or more lipids contain a first lipid and a second lipid. Embodiment 17. A composition containing mRNA complexed with one or more lipids (RNA-Lipoplex), wherein the mRNA contains a first coding region and a second coding region separated by a 2A-like sequence, the first coding region is a coding region of constitutively active cyclic GMP-AMP synthase (cGAS), and the second coding region is a coding region of an antigen, or the first coding region is a coding region of an antigen, and the second coding region is a coding region of constitutively active cyclic GMP-AMP synthase (cGAS), and the one or more lipids contain a first lipid and a second lipid. Embodiment 18. The composition according to any one of Embodiments 15 to 17, wherein the first lipid is a cationic lipid and the second lipid is a neutral or anionic lipid. Embodiment 19. The cationic lipid is i) 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) or an analog or derivative thereof; and ii) 1,2-dioleoyl-3-triethylammonium propane (DOTAP) or an analog or derivative thereof, The composition according to Embodiment 18, comprising one or both of the above. Embodiment 20. The neutral or anionic lipid is i) 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or an analog or derivative thereof; and / or ii) cholesterol or an analog or derivative thereof; and / or iii) 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or an analog or derivative thereof, The composition according to Embodiment 18 or Embodiment 19, comprising the above. Embodiment 21. The composition according to any one of Embodiments 1 to 20, further comprising a TLR7 / 8 agonist. Embodiment 22. The composition according to Embodiment 21, wherein the TLR7 / 8 agonist is a low molecular weight compound having a molecular weight of 900 daltons or less. Embodiment 23. The composition according to Embodiment 22, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound. Embodiment 24. The composition according to Embodiment 23, wherein the TLR7 / 8 agonist comprises resiquimod (R848). Embodiment 25. The composition according to any one of Embodiments 1 to 24, wherein the mRNA or the first mRNA and the second mRNA comprise a 5'untranslated region (5'UTR) and a 3'untranslated region (3'UTR). Embodiment 26. The mRNA is the composition according to any one of Embodiments 1 to 25, which contains a 5' cap structure. Embodiment 27. The mRNA is the composition according to any one of Embodiments 1 to 26, which contains a polyA tail. Embodiment 28. The mRNA is the composition according to any one of Embodiments 1 to 27, which is nucleoside-modified mRNA. Embodiment 29. The constitutively active cGAS has a tendency to be more self-DNA reactive than its wild-type counterpart, and is the composition according to any one of Embodiments 1 to 28. Embodiment 30. The cGAS is a truncated cGAS (cGASΔN) lacking an amino-terminal phosphoinositide-binding domain, and is the composition according to any one of Embodiments 1 to 29. Embodiment 31. The cGASΔN contains the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and is the composition according to Embodiment 30. Embodiment 32. The cGASΔN is (i) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; or (ii) the consensus amino acid sequence of SEQ ID NO: 9, and is the composition according to Embodiment 31. Embodiment 33. The cGASΔN is encoded by the nucleotide sequence of SEQ ID NO: 17, and is the composition according to Embodiment 31. Embodiment 34. The coding region of the cGASΔN is in an operable combination with the start codon (ATG), and is the composition according to any one of Embodiments 30 to 33. Embodiment 35. The antigen is a tumor antigen, and is the composition according to any one of Embodiments 1 to 34. Embodiment 36. The composition according to Embodiment 35, wherein the tumor antigen is a tumor-associated antigen. Embodiment 37. The composition according to Embodiment 35, wherein the tumor antigen is a neoantigen. Embodiment 38. The composition according to any one of Embodiments 1 to 34, wherein the antigen comprises a microbial antigen. Embodiment 39. The composition according to Embodiment 38, wherein the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoal antigen, or a fungal antigen. Embodiment 40. The composition according to any one of Embodiments 1 to 34, wherein the antigen comprises a surface protein of a pathogen or a fragment thereof. Embodiment 41. The composition according to Embodiment 40, wherein the pathogen can cause a disease in a human subject. Embodiment 42. The composition according to Embodiment 40 or Embodiment 41, wherein the pathogen is a virus. Embodiment 43. The composition according to Embodiment 42, wherein the virus is SARS-CoV-2. Embodiment 44. The composition according to Embodiment 43, wherein the antigen is the spike (S) glycoprotein of the SARS-CoV-2, and optionally, the spike glycoprotein is a prefusion-stabilized variant here. Embodiment 45. The composition according to any one of Embodiments 1 to 44, which does not contain lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA). Embodiment 46. The composition according to any one of Embodiments 1 to 45, which does not contain oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC) or a species of oxPAPC. Embodiment 47. The composition according to Embodiment 46, which does not contain 2-[[(2R)-2-[(E)-7-carboxy-5-hydroxyhepta-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (HOdiA-PC), [(2R)-2-[(E)-7-carboxy-5-oxohepta-6-enoyl]oxy-3-hexadecanoyloxypropyl] 2-(trimethylazaniumyl)ethyl phosphate (KOdiA-PC), 1-palmitoyl-2-(5-hydroxy-8-oxo-octenoyl)-sn-glycero-3-phosphocholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxoocta-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (KOOA-PC), [(2R)-3-hexadecanoyloxy-2-(5-oxopentanoyloxy)propyl] 2-(trimethylazaniumyl)ethyl phosphate (POVPC), [(2R)-2-(4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl] 2-(trimethylazaniumyl)ethyl phosphate (PGPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopenta-3-en-1-ylidene]methyl]oxirane-2-yl]butanoyloxy]propyl] 2-(trimethylazaniumyl)ethyl phosphate (PECPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxirane-2-yl]butanoyloxy]propyl] 2-(trimethylazaniumyl)ethyl phosphate (PEIPC) and / or 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PAzePC). Embodiment 48. A pharmaceutical preparation comprising the composition according to any one of Embodiments 1 to 47 and a pharmaceutically acceptable additive. Embodiment 49. A method for generating hyperactivated dendritic cells, the method comprising contacting the dendritic cells with an effective amount of the composition according to any one of Embodiments 1 to 47, any one of Embodiments 21 to 47, or the formulation according to Embodiment 48 to generate hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing cell death upon exposure within about 48 hours. Embodiment 50. The dendritic cells are (i) contacted with the composition in vivo; or (ii) contacted with the composition ex vivo, The method according to Embodiment 49. Embodiment 51. The hyperactivated dendritic cells (i) secrete one or more of RANTES, IP-10, and IFNα at a higher level than unstimulated dendritic cells or dendritic cells contacted with empty LNP; and / or (ii) express at least one cell surface marker selected from the group consisting of CD40, CD86, CD69, MHC class II, MHC class I, CCR7, and combinations thereof, The method according to Embodiment 49 or Embodiment 50. Embodiment 52. At least 10 3 , 10 4 , 10 5 or 10 6 of the hyperactivated dendritic cells generated by the method according to any one of Embodiments 49 to 51, and a pharmaceutically acceptable additive, a pharmaceutical formulation. Embodiment 53. A method for stimulating an immune response against an antigen, the method comprising administering an effective amount of the pharmaceutical formulation according to Embodiment 48 or Embodiment 52 to an individual in need of stimulating an immune response against the antigen to stimulate the immune response against the antigen. Embodiment 54. A method for treating cancer, the method comprising administering to an individual in need of treating cancer an effective amount of the pharmaceutical preparation according to Embodiment 48 or Embodiment 52 to treat the cancer. Embodiment 55. A method for inhibiting abnormal cell growth, the method comprising administering to an individual in need of inhibiting abnormal cell growth an effective amount of the pharmaceutical preparation according to Embodiment 48 or Embodiment 52 to inhibit abnormal cell growth. Embodiment 56. A method for treating or preventing an infectious disease, the method comprising administering to an individual in need of treating or preventing an infectious disease an effective amount of the pharmaceutical preparation according to Embodiment 48 to treat or prevent the infectious disease. Embodiment 57. The method according to Embodiment 56, wherein the infectious disease is a viral disease. Embodiment 58. The method according to Embodiment 56, wherein the infectious disease is a bacterial disease. Embodiment 59. The method or pharmaceutical preparation according to any one of Embodiments 49 to 55, wherein the dendritic cell is a mammalian cell. Embodiment 60. The method or pharmaceutical preparation according to Embodiment 59, wherein the mammalian cell is a human cell. Embodiment 61. The method according to any one of Embodiments 53 to 59, wherein the individual is a mammal. Embodiment 62. The method according to Embodiment 61, wherein the mammal is a human. Embodiment 63. The method according to Embodiment 61, wherein the mammal is a dog or a cat. Embodiment 64. The composition, preparation, or method according to any one of Embodiments 1 to 63, wherein the phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and the at least one lipid comprises 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octyl nonyl ester (SM-102) or an analog or derivative thereof, and cholesterol. Embodiment 65. The at least one lipid further comprises a pegylated lipid, and optionally here the pegylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG], the composition, formulation, or method according to Embodiment 64. Embodiment 66. The LNP has an effective diameter of less than about 250 nanometers, optionally about 25 to about 250 nanometers, optionally about 50 to about 200 nanometers, or optionally about 75 to about 175 nanometers, the composition, formulation, or method according to any one of Embodiments 1 to 65. Embodiment 67. The LNP has an effective diameter of less than about 200 nanometers, the composition, formulation, or method according to Embodiment 66. Embodiment 68. The LNP has an effective diameter of less than about 150 nanometers, the composition, formulation, or method according to Claim 67.

Examples

[0088] Abbreviations: BMDC (bone marrow-derived dendritic cell); CDS (cytosolic DNA sensor); cyclic GMP-AMP synthase (cGAS); CLR (C-type lectin receptor); DAMP (damage-associated molecular pattern); DC (dendritic cell); dLN (draining lymph node); DLS (dynamic light scattering); DMG-PEG-2000 (polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG]; DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine); ELSD (evaporative light scattering detector); FLT3L (Fms-related tyrosine kinase 3 ligand); GFP (green fluorescent protein); GV (GenVoy ILM TMFormulation); (HOdiA-PC (1-palmitoyl-2-(5-hydroxy-8-oxo-6-octenedioyl)-sn-glycero-3-phosphatidylcholine); HOOA-PC (1-palmitoyl-2-(5-hydroxy-8-oxoocta-6-enoyl)-sn-glycero-3-phosphocholine); IFNγ (Interferon-gamma); IL-1b / IL1-beta / IL-1β (Interleukin-1 beta); KOdiA-PC (1-(palmitoyl)-2-(5-keto-6-octenedioyl) phosphatidylcholine); KOOA-PC (1-palmitoyl-(5-keto-8-oxo-6-octenoyl)-sn-glycero-3-phosphocholine); LNP (Lipid nanoparticles); LPC / Lyso PC (Lysophosphatidylcholine); Lyso PC(22:0) (1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine); LPS (Lipopolysaccharide); MC3 ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (also known as DLin-MC3-DMA); mcg or μg (Microgram); moDC (Monocyte-derived dendritic cells); MPLA (Monophosphoryl lipid A); NLR (NOD-like receptor); OVA (Ovalbumin); oxPAPC (Oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine); PAMP (Pathogen-associated molecular pattern); PBMC (Peripheral blood mononuclear cells); PGPC (1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine); POVPC (1-palmitoyl-2-(5’-oxo-valeroyl)-sn-glycero-3-phosphocholine); PRR (Pathogen recognition receptor); RLR (RIG-I-like receptor); R848 (Resiquimod); SC (Subcutaneously); STING (Stimulator of interferon genes); TNFα (Tumor necrosis factor-alpha); and TLR (Toll-like receptor).

[0089] For the purposes of clarity and understanding, the present disclosure has been described in some detail by way of illustration and example, but it will be apparent to those skilled in the art that certain changes and modifications can be made. Accordingly, the following examples should not be construed as limiting the scope of the present disclosure, which is clearly set forth by the appended claims.

[0090] Example 1: Mouse DCs can be activated in vitro by LNP containing cGASΔN mRNA. Materials and Methods Materials. GenVoy ILM TM The ionizable lipid mixture was purchased from Precision Nanosystems. CleanCap OVA and GFP mRNAs were purchased as off-the-shelf products from Trilink (with 120-residue polyA tail, 5-methoxyuridine base modification, and codon optimization for expression in mammalian systems). cGASΔN mRNA was custom ordered from Trilink and synthesized via in vitro transcription from linearized template DNA. The nucleotide sequence of the human cGASΔN sequence was codon-optimized for expression in mouse cells. The synthetic nucleotide sequence is shown as SEQ ID NO: 17. The above mRNA sequences were capped using Trilink's proprietary Clean Cap mRNA technology (with N1-methylpseudouridine base modification and 120-residue polyA tail). The above sequences contain Bbsl restriction enzyme sites. The mRNA was treated with phosphatase after synthesis.

[0091] LNP synthesis. Lipid nanoparticles (LNPs) were prepared using the GenVoy ILM TM ionizable lipid mixture (Precision Nanosytems). OVA mRNA, GFP mRNA, and cGASΔN mRNA (Trilink) were each prepared at 0.17 mg / mL in sodium citrate buffer (pH 4). GenVoy ILM TMwas used at 12.5 mM. LNPs were synthesized using a NanoAssemblr Ignite instrument (Precision Nanosystems). Lipids in ethanol were combined with the mRNA solution at a 1:3 volume ratio individually using a flow rate of 12 mL / min. The LNPs were washed in 10 volumes of phosphate buffered saline (PBS) (pH 7.4) to remove residual ethanol and then concentrated using an Amicon 10K MWCO centrifugal filter. The LNPs were filtered through a 0.2 μm filter before use.

[0092] LNP Characterization. Encapsulation of mRNA into LNPs was quantified using a RiboGreen assay (ThermoFisher) according to the manufacturer's protocol. Samples were diluted to fall within the range of the standard curve. LNPs were lysed using Triton® X-100 to evaluate encapsulation of mRNA into LNPs. Both total mRNA and encapsulated mRNA were quantified. The size of the LNPs was evaluated using dynamic light scattering (DLS) on a NanoBrook Omni (Brookhaven). The LNPs were diluted 1:10 in PBS before performing DLS. Three 90-second measurements were recorded for each sample.

[0093] Generation of Mouse Bone Marrow-Derived FLT3L-DCs. The femurs and tibias of the legs were removed from the mice, cut with scissors, and flushed into sterile tubes. The bone marrow suspension was treated with ACK lysis buffer for 1 minute and then passed through a 40 μm cell strainer. Cells were counted and resuspended in medium consisting of complete IMDM (I10) supplemented with 10% FBS, penicillin and streptomycin, as well as L-glutamine and sodium pyruvate. The cells were then seeded at 8×10 6 bone marrow cells per well of a P12 plate. Recombinant mouse FLT3L (Miltenyi) was added to the cultures at 200 ng / mL. Differentiated cells were used for subsequent assays on day 8. The efficiency of differentiation was monitored by flow cytometry using a BD Symphony A3, where CD11c + MHC-II +The cells always exceeded 80% of the living cells. For each experiment, 5 - 15 mice were used to generate DCs from the bone marrow.

[0094] Mouse FLT3L-DC stimulation. FLT3L-DCs were harvested 8 days after differentiation, counted, and treated with lipid nanoparticles (LNP) based on mRNA content. The cells were treated with 1 μg / mL mRNA in 200 μl of complete medium / well. Approximately 24 hours after incubation, the cells and culture supernatant were used for downstream readouts. 150 mL of cell supernatant was collected from each well to measure cytokine secretion from DCs. LEGENDplex TM Mouse Anti-Virus Response Panel (Biolegend) was used according to the manufacturer's protocol. Data were collected using a BD FACS Symphony and analyzed using the cloud-based software provided by Biolegend. To evaluate DC activation after LNP exposure, FLT3L DCs were collected 24 hours after LNP addition, stained, and the expression of the following cell surface markers: CD11c, MHC-II, CD24, SIRP1a, CD40, CD86, CD69, and H2kb (MHC-I) was measured. Graphs show the mean and SD of triplicates. Data are representative of two experiments.

[0095] Results Lipid nanoparticles filled with mRNA encoding cGASΔN (cGASΔN LNP) showed a sizing and filling profile similar to that of lipid nanoparticles filled with mRNA encoding a model antigen. cGASΔN mRNA-filled LNP showed a size and mRNA filling profile similar to that of LNP filled with model antigen mRNA - OVA and GFP. All mRNA-filled LNP had an average effective diameter of less than 150 nm (Figure 3A) and a relatively uniform size profile indicated by a polydispersity index of less than 0.3 (Figure 3B). All mRNA was filled into GenVoy ILM LNP, and the measured mRNA filling showed 90 - 110% of the theoretically filled amount of mRNA (Figure 3C).

[0096] cGASΔN LNPs induce IFN-I-dependent responses in mouse DCs. FLT3L DCs were stimulated with empty LNPs (without mRNA), or LNPs containing either GFP mRNA or OVA mRNA or cGASΔN mRNA alone. Alternatively, DCs were stimulated with LNPs containing OVA mRNA + empty LNPs, or LNPs containing OVA mRNA + cGASΔN, or LNPs containing cGASΔN + LNPs containing GFP mRNA. LNPs were added to cell cultures based on the concentration of mRNA at 1 μg / mL. Twenty-four hours after stimulation, cell supernatants were measured for the presence of cytokines using a multiplex cytokine bead array Legendplex assay. Interestingly, the inventors found that upon cGASΔN LNP treatment, BMDCs secrete RANTES, IP-10, and IFNα at significantly higher levels than when treated with mRNA antigen-loaded LNPs alone (Figures 4A, 4B, 4C). These data demonstrated that constitutively active cGASΔN was successfully expressed and functional in mouse DCs. Pro-inflammatory cytokines dependent on NFkB, including IL-6 and TNFα, were not secreted. This suggests that constitutively active cGASΔN induces NFkB activation in specific cell types. The inventors compared the induction of these cytokines upon LNP treatment with stimulation with LPS and R848 PAMPs. Interestingly, IP-10, RANTES, and IFNα were expressed at either stronger or similar levels by cGASΔN LNP treatment compared to higher doses of LPS or R848. This suggests that the IRF3 pathway can be strongly induced by the expression of constitutively active cGASΔN.

[0097] cGASΔN LNPs induce mouse DC activation. To evaluate DC activation, LNP-treated DCs were stained with activation markers on total cDCs and the cDC1 and cDC2 subsets of DCs 24 hours after treatment. The expression of the T cell co-stimulatory surface molecules CD40, CD86, and CD69 increased when cells were treated with cGASΔN LNPs, as evaluated by median fluorescence intensity (MFI), but not when treated with empty LNPs or GFP LNPs or OVA-containing LNPs (Figures 5A, 5B, 5C). This trend also held for the cDC1 subset. Furthermore, MHCII and H2kB antigen presentation molecules increased on cDCs when treated with cGASΔN LNPs compared to standard mRNA antigen LNPs (Figures 5D, 5E). This also held for the cDC1 subset. Additionally, CCR7 surface expression, which indicates the potential for DCs to migrate to LNs, increased on cDCs when treated with cGASΔN LNPs compared to standard mRNA antigen LNPs (Figure 5F). This trend also held for the cDC1 subset. Overall, these data indicate that cGASΔN LNPs induce cGAS-STING pathway activation within DCs, leading to the DC activation state and the production of type I IFN.

[0098] Example 2: Human cells can be activated in vitro to an inflammatory state by LNPs containing cGASΔN mRNA. Materials and methods Materials. Lipids for LNP were purchased from Cayman Chemicals (SM102) or Avanti (22:0 LPC, DSPC, DMG-PEG2000). Cholesterol was purchased from Sigma. GenVoy ILM lipids were purchased from Precision Nanosystems. CleanCap OVA and GFP mRNA were purchased as off-the-shelf products from Trilink (with 120 polyA tails and base-modified 5-methoxyuridine, optimized for mammalian systems). cGASΔN mRNA was custom-ordered and synthesized via in vitro transcription from linearized template DNA (Trilink). The human cGASΔN sequence was codon-optimized for expression in mice. The above sequence was capped using Trilink's proprietary Clean Cap mRNA technology (with base-modified N1-methylpseudouridine and 120 polyA tails). The above sequence contains Bbsl restriction enzyme sites. The sequence information was used to construct a plasmid from which mRNA was synthesized synthetically. The mRNA was phosphatase-treated after synthesis.

[0099] LNP synthesis. Lipid nanoparticles (LNP) were prepared using GenVoy ILM LNP lipid mix (Precision Nanosytems) or a custom-ordered LNP lipid mix (Table 2-1). OVA mRNA, GFP mRNA, and cGASΔN mRNA (Trilink) were each prepared at 0.17 mg / mL in sodium citrate buffer (pH 4). GenVoy ILM and custom-ordered LNP lipid mixes were used at 12.5 mM. LNP were synthesized using a NanoAssemblr Ignite instrument (Precision Nanosystems). Lipids in ethanol were combined with the mRNA solution individually at a volume ratio of 1:3 using a flow rate of 12 mL / min. The LNP were washed in 10 volumes of phosphate buffered saline (PBS) (pH 7.4) to remove residual ethanol and then concentrated using an Amicon 10K MWCO centrifugal filter. The LNP were filtered through a 0.2 μm filter before use.

Table 2-1

[0100] LNP Characterization. Encapsulation of mRNA into LNP was quantified using the RiboGreen assay (ThermoFisher) according to the manufacturer's protocol. Samples were diluted to fall within the range of the standard curve. LNP was lysed using Triton® X-100 to evaluate encapsulation of mRNA into LNP. Both total mRNA and encapsulated mRNA were quantified. The size of LNP was evaluated using dynamic light scattering (DLS) on a NanoBrook Omni (Brookhaven). LNP was diluted 1:10 in PBS before performing DLS. Three 90-second measurements were recorded for each sample.

[0101] Generation of Human Monocyte-Derived Dendritic Cells (moDC). Human monocytes were isolated from Leukopaks purchased from Miltenyi using the StraightFrom Leukopak CD14 MicroBead Kit (Miltenyi) according to the manufacturer's instructions. Monocytes were then aliquoted and frozen in fetal bovine serum containing 10% dimethyl sulfoxide. For studies in monocyte-derived dendritic cell (moDC) cultures, monocytes were thawed and cultured in RPMI medium (R10 medium) containing 10% FBS, 50 units / mL penicillin, 50 mg / mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 mM β-mercaptoethanol, 10 mM HEPES, and Gibco MEM non-essential amino acids. Recombinant human GM-CSF (50 ng / mL) and IL-4 (25 ng / mL) were added to the R10 medium to differentiate monocytes into moDC. Cells were cultured with GM-CSF and IL-4 for 6 days and replenished with additional nutrients in R10 medium containing GM-CSF and IL-4 on day 3. Six days after differentiation, moDC were collected and counted. Cells were seeded at 1E5 cells / well in a 96-well flat-bottom plate in R10 medium.

[0102] THP-1 cell generation. THP1-Null2 (Invivogen) cells were thawed into RPMI medium containing 10% heat-inactivated FBS, 25 mM HEPES, 100 U / mL penicillin / streptomycin, and 100 μg / mL normocin and maintained therein, with 100 μg / mL zeocin replenished every 2 passages. Cells were passaged twice a week at a density of 0.4 - 0.6×10 6 cells / mL and maintained up to 20 passages. Thereafter, new vials were thawed. Prior to treatment with LNP, THP1-Null2 cells were collected from the flask and seeded at 100,000 cells / well in a 96-well flat-bottom tissue culture plate in RPMI medium containing 10% heat-inactivated FBS, 25 mM HEPES, and 100 U / mL penicillin / streptomycin.

[0103] Human cell activation. THP-1 cells and moDCs were treated with lipid nanoparticles (LNPs) based on the concentration of the mRNA content. The cells were treated with 0.2 μg / mL mRNA delivered in LNPs at a total stimulant volume of 200 μL / well. After an overnight incubation, the cells and culture supernatant were used for downstream readouts. 150 microliters of the cell supernatant was collected. One day after LNP addition, cells and supernatant were collected from the THP-1 culture and the cytokines IL-6 (Lumit assay by Promega) and IP-10 (ELISA kit from Biolegend) were measured. THP-1 cells were used to stain activation markers for flow cytometry analysis. To measure cytokine secretion from moDCs, the LegendPlex Anti-virus Response Panel (Biolegend) was used according to the manufacturer's protocol. Data were collected using a BD FACS Symphony and analyzed using cloud-based software provided by Biolegend. Both moDCs and THP-1 cells were collected one day after LNP addition and stained to measure the expression of the following cell surface activation markers: CD11c, CD209, CD40, CD80, CD83, CD86, HLA-ABC, and HLA-DR. Studies were performed on two different human donor samples and THP-1 cells, and each biological condition was tested in triplicate. The graphs show data from two human samples or THP-1 cells. The data are representative of two experiments.

[0104] HEK-Blue IL-1β reporter assay. moDCs were treated with lipid nanoparticles (LNP) based on the concentration of the mRNA content. Cells were treated with 1 μg / mL OVA mRNA, 1 μg / mL cGASΔN mRNA, or 0.2 μg / mL mRNA delivered in LNP at a total stimulator volume of 200 μL / well. LNP was delivered with medium, R848, or R848 + MCC950 (an inhibitor of the NLRP3 inflammasome). In the presence of MCC950, IL-1β should not be secreted in an inflammasome-dependent manner. HEK-Blue IL-1β reporter cells (Invivogen) were used to evaluate the functional IL-1β secreted by human moDCs in response to treatment with cGASΔN LNP over 48 hours. HEK-Blue IL-1β reporter cells are derived from HEK293 cells and express an NK-kB / AP-1-inducible SEAP (secreted embryonic alkaline phosphatase) reporter that induces SEAP production once the IL-1β receptor encounters mature IL-1β. SEAP production is monitored by a color change using the QUANTI-Blue solution. HEK-Blue IL-1β reporter cells were thawed into DMEM medium containing 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 100 μg / mL normocin, maintained therein, and supplemented with 100 μg / mL zeocin every 2 passages. Cells were passaged twice a week and maintained in T75 culture flasks at 0.4 - 0.6×10 6Cells were passaged between 1×10⁵ and 1×10⁶ cells / mL. Cells were maintained up to 20 passages. Thereafter, new vials were thawed. Before treatment with LNP, HEK-Blue IL-1β reporter cells were collected from the flask and seeded at 50,000 cells / well in a 96-well flat-bottom tissue culture plate in DMEM medium containing 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin. 50 μL of the medium from cGASΔN-activated moDC or 50 μL of the medium containing recombinant human IL-1β at a known concentration was added to the wells containing HEK-Blue IL-1β reporter cells to create a standard curve. Cells were incubated overnight, and then 20 μL of the SEAP-containing supernatant from the incubated samples was mixed with 180 μL of the QUANTI-Blue solution. This reaction was carried out at 37 °C for 1 hour, and then the absorbance at 630 nm was measured by spectrophotometry.

[0105] Results To determine whether cGASΔN LNP enables greater cell activation than standard LNP, LNP was synthesized to contain mRNA encoding the Ova antigen or mRNA encoding constitutively active cGASΔN. As an experimental control, LNP was made with mRNA encoding GFP. These LNP were tested individually or in combination in THP-1 cells (a human myeloid cell line) and primary human moDC. Two lipid formulations: GenVoy (Precision Nanosystems) and a custom lipid mix were included to determine whether the activity is LNP formulation-dependent. LNP was added to the cell culture based on a concentration of 0.2 μg / mL of mRNA.

[0106] The cGASΔN LNPs exhibited sizing and encapsulation profiles similar to those of the model antigen-loaded LNPs. The cGASΔN mRNA-loaded LNPs showed similar size and mRNA encapsulation profiles to those of the LNPs loaded with model antigen mRNAs (OVA and GFP). All the mRNA-loaded LNPs had an average effective diameter of less than 150 nm (Figure 6A) and a relatively uniform size profile indicated by a polydispersity index of less than 0.3 (Figure 6B). All the mRNAs were encapsulated into GenVoy ILM LNPs, and 70 - 90% of the theoretically encapsulable amount of mRNA was encapsulated in the LNPs (Figure 6C). Both GenVoy and custom lipid mixes enabled LNP formation with similar sizing, but the optimized GenVoy formulation showed an increase in size uniformity, as indicated by a lower polydispersity index (Figure 6B). Both GenVoy and custom lipid mixes enabled similar encapsulation levels of all the mRNAs, and the GenVoy formulation encapsulated the mRNA slightly more efficiently.

[0107] cGASΔN LNPs activate inflammatory pathways in human moDCs. Frozen monocytes from two donors were thawed and differentiated into moDCs using GM-CSF and IL-4. Cells were treated with 0.2 μg / mL of mRNA-loaded LNPs. Using a multiplex cytokine bead array assay, multiple inflammatory cytokines were found to be upregulated. Upon cGASΔN LNP treatment, IL-6 (Figure 7A), TNFα (Figure 7B), IP-10 (Figure 7C), IFNλ1 (Figure 7D), IFNβ (Figure 7E), and IFNα2 (Figure 7F) were detectable at elevated levels within 1 day compared to treatment with LNPs loaded with a model antigen. The expression of IL-6 suggested that constitutively active cGAS can induce NF-κB activation and is likely cell-type dependent. Interestingly, IP-10, IFNλ1, and IFNβ were more strongly expressed by cGASΔN LNP treatment than when cells were treated with 10 μg / mL LPS (which served as a positive control). This suggests that the IRF3 pathway can be strongly induced by the expression of constitutively active cGAS.

[0108] LNP-treated moDCs were stained 1 day after treatment to quantify the expression of activation markers. By measuring the median fluorescence intensity (MFI), CD83 expression was increased when cells were treated with cGASΔN LNPs compared to standard LNPs (Figure 8A). The expression of T cell co-stimulatory molecules was also measured. CD40 and CD80 expression increased in the overall population of moDCs and at the single-cell level when they were treated with cGASΔN LNPs (Figure 8B, Figure 8C). HLA-DR was expressed by almost all moDCs, but MFI measurements revealed that MHC class II molecules increased in individual cells when moDCs were treated with cGASΔN LNPs (Figure 8D). In the case of MHC class I molecules, HLA-ABC staining did not reveal any cGASΔN LNP-dependent changes.

[0109] cGASΔN LNPs activate the inflammatory pathway in THP-1. One day after incubation, THP-1 cell supernatants were measured for the presence of cytokines dependent on NF-kB or IRF3 activation, IL-6 and IP-10, respectively. At both time points, IL-6 was not differentially expressed by THP-1 cells treated with cGAS LNPs. This suggests that NF-kB was not activated. In contrast, IP-10 (a gene controlled by IRF3) was expressed within one day of cGAS LNP treatment (Figure 9A). cGAS LNPs induced IP-10 expression, whereas other control LNPs induced little or no IP-10. Detectable IP-10 indicated that the packaging of constitutively active cGAS transcripts into LNPs could be a way to activate the innate immune system.

[0110] To further verify that cGAS LNPs activate innate immune signaling, THP-1 cells were collected one day after starting incubation to stain for cell surface expression of activation markers. THP-1 samples were gated on live single-cell events. CD40 expression was observed to increase after cell treatment with cGAS LNPs, whereas other control LNPs with GFP or Ova transcripts were not observed (Figure 9B). CD40 is a receptor that enhances the immune response through cell-cell interactions with its cognate ligand.

[0111] Treatment with cGASΔN LNPs + R848 results in moDC hyperactivation. moDCs were stimulated with R848 and cGASΔN to determine whether the combination of NF-kB stimulation and cGAS-STING pathway activation enables more potent inflammatory activity. Specifically, the ability of moDCs to be hyperactivated by this combination was tested. Hyperactivation was represented by the ability of DCs to secrete IL-1β while remaining viable. moDCs were either left unstimulated (PBS-treated) or treated with cGASΔN LNP or OVA LNP in the presence of medium, R848, or R848 and MCC950. Since MCC950 is an NLRP3 inflammasome inhibitor, cells treated with MCC950 cannot generate IL-1β in an inflammasome-dependent manner. Importantly, all cells treated with LNP showed a survival rate similar to that of moDCs treated with PBS alone. This indicates that none of the above cells underwent pyroptosis in response to LNP treatment (Figure 10A). To determine whether any active IL-1β was generated by these cells in response to treatment with R848 and cGASΔN, the supernatant from the moDC culture was added to HEK-Blue IL-1β reporter cells that generate SEAP in response to IL-1β. As predicted, moDCs treated with PBS, R848, or R848 + MCC950 did not generate IL-1β (Figure 10B). To determine whether hyperactivation was dependent on the LNP lipid composition, LNPs containing OVA were cultured with R848 (conditions that do not permit IL-1β production). Interestingly, LNPs loaded with cGASΔN stimulated IL-1β secretion in moDCs when delivered in combination with R848 (Figure 10B). This response was NLRP3 inflammasome-dependent since treatment with MCC950 eliminated IL-1β production.

[0112] Overall, the data collected from the treatment of THP-1 and human moDCs with LNP led to several conclusions. First, the LNP was successfully synthesized. Furthermore, the expression of constitutively active cGAS resulted in the upregulation of genes associated with the cGAS-STING pathway in a formulation-independent manner. Inflammatory cytokines were expressed, and upregulation of activation markers was observed on the cell surface. The constitutively active cGAS encoded as mRNA and packaged in the LNP can be successfully utilized to initiate innate immune signaling in target cells and promote an inflammatory response. Finally, constitutively active cGAS combined with R848 enabled moDC overactivation and occurred in live DCs with IL-1β secretion.

[0113] Example 3: Mouse cells can be activated in vivo by LNP containing cGASΔN mRNA Materials and Methods Materials. Lipids for LNP were purchased from Cayman Chemicals (SM102) or Avanti (22:0 LPC, DSPC, DMG-PEG2000). Cholesterol was purchased from Sigma. GenVoy ILM lipid was purchased from Precision Nanosystems. CleanCap OVA and GFP mRNA were purchased as off-the-shelf products from Trilink (with 120 polyA tails and base-modified 5-methoxyuridine, optimized for mammalian systems). cGASΔN mRNA was custom synthesized via in vitro transcription from a linearized template DNA (Trilink). The human cGASΔN sequence was codon-optimized for expression in mice. The above sequence was capped using Trilink's proprietary Clean Cap mRNA technology (with base-modified N1-methylpseudouridine and 120 polyA tails). The above sequence contains a Bbsl restriction enzyme site. The sequence information was used to construct a plasmid, from which mRNA was synthetically synthesized. The mRNA was treated with phosphatase after synthesis.

[0114] LNP synthesis. Lipid nanoparticles (LNPs) were prepared as in Example 2 using GenVoy ILM LNP lipid mix (Precision Nanosytems) or a representative LNP lipid mix (Table 2-1, Example 2). OVA mRNA, GFP mRNA, and cGASΔN mRNA (Trilink) were each prepared at 0.17 mg / mL in sodium citrate buffer (pH 4). GenVoy ILM and the representative LNP lipid mix were used at 12.5 mM. LNPs were synthesized using a NanoAssemblr Ignite instrument (Precision Nanosystems). Lipids in ethanol were combined with the mRNA solution individually at a 1:3 volume ratio using a flow rate of 12 mL / min. LNPs were washed in 10 volumes of phosphate buffered saline (PBS) (pH 7.4) to remove residual ethanol and then concentrated using an Amicon 10K MWCO centrifugal filter. LNPs were filtered through a 0.2 μm filter prior to use.

[0115] LNP characterization. The loading of mRNA into LNPs was quantified according to the manufacturer's protocol using a RiboGreen assay (ThermoFisher) as in Example 2. Samples were diluted to fall within the range of the standard curve. LNPs were lysed using Triton® X-100 to evaluate the encapsulation of mRNA into LNPs. Both total mRNA and encapsulated mRNA were quantified. The size of LNPs was evaluated using dynamic light scattering (DLS) on a NanoBrook Omni (Brookhaven). LNPs were diluted 1:10 in PBS prior to performing DLS. Three 90-second measurements were recorded for each sample.

[0116] In vivo immunization with LNP containing cGASΔN mRNA and OVA antigen mRNA. BL6 mice were immunized subcutaneously with LNP according to the following table (Table 3-1). Antigen was delivered using LNP loaded with OVA mRNA, and LNP containing either cGASΔN mRNA as an adjuvant or GFP mRNA without adjuvant activity. Mice were administered the mRNA doses shown in the table. The OVA mRNA dose was fixed at 5 μg / mouse, while the adjuvant mRNA was varied from 1 μg / mouse to 10 μg / mouse. Mice (n = 5 / group) were primed on day 0 and boosted with the same dose on day 7. Seven days after the boost, blood was collected for antibody and T cell responses. Serum was collected from the blood using serum separation tubes, while blood for cell analysis was collected using K2EDTA tubes. After blood collection, the mice were euthanized and the draining regional lymph nodes and spleens were collected and processed into single cell suspensions.

Table 3-1

[0117] OVA-specific T cell tetramer evaluation. OVA-specific T cells in the blood of mice immunized with OVA LNP were evaluated 7 days after the boost. CD8+ T cells specific for SIINFEKL (SEQ ID NO: 16) (OVA MHC-I epitope) were quantified in the blood using tetramer analysis. Briefly, red blood cells in the blood were lysed twice using RBC lysis buffer to completely remove any RBCs in the blood. The cells were washed and then stained for viability (live / dead), SIINFEKL-tetramer binding (MBL), and CD3, CD4, and CD8 expression. The cells were fixed with 4% paraformaldehyde and counting beads were added prior to running to enable total cell counting. Data were collected using a BD FACS Symphony and analyzed using Flowjo (BD).

[0118] OVA-specific antibody evaluation. OVA-specific antibodies in the sera of mice immunized with OVA LNP were evaluated 7 days after the boost. Total OVA-specific IgG, IgG1, and IgG2b were evaluated using ELISA. Briefly, ELISA plates were coated overnight with 10 μg / mL Endofit Ovalbumin (Invivogen), then washed and blocked with 2% bovine serum albumin. The plates were washed again, and then serum was added to the plates at a 1:500 dilution, followed by a total of 7 serial dilutions of the serum at a 1:5 dilution. The samples were washed and then incubated with a detection antibody (Southern Biotech) specific for IgG, IgG1, or IgG2b conjugated to HRP to detect total, Th2-biased, or Th1-biased OVA-specific antibodies, respectively. The plates were washed and then incubated with TMB, and a stop solution was added once the color development was complete.

[0119] Results To determine whether cGASΔN LNP induces a greater antigen-specific T cell and B cell response in vivo, mice were immunized with OVA LNP in combination with cGASΔN or GFP mRNA at different doses of cGASΔN or GFP LNP (Table 3-1). Mice were immunized and then boosted 1 week later. Blood was collected 1 week after the boost for antibody and T cell tetramer analysis. Two lipid formulations, GenVoy (Precision Nanosystems) and a custom lipid mix, were included to determine whether the activity is LNP formulation-dependent.

[0120] cGASΔN LNPs exhibit sizing and loading profiles similar to those of model antigen-loaded LNPs. When cGASΔN mRNA-loaded LNPs were prepared according to Example 2, characterization followed the trends shown in FIGS. 6A-6C. All LNPs exhibited size and mRNA loading profiles similar to those of LNPs loaded with model antigen mRNAs (OVA and GFP). All mRNA-loaded LNPs had an average effective diameter of less than 150 nm and a relatively uniform size profile indicated by a polydispersity index of less than 0.3. All mRNAs were loaded into GenVoy ILM LNPs, and 70-90% of the theoretically loaded amount of mRNA was encapsulated by the LNPs.

[0121] Treatment with OVA LNP and cGASΔN LNP increases OVA-specific CD8+ T cells. The presence of OVA-specific T cells in the blood was analyzed after two immunizations with OVA LNP (5 μg mRNA / mouse) delivered together with cGASΔN LNP (1 or 5 μg mRNA / mouse) or control GFP LNP (1 or 5 μg mRNA / mouse) (Table 3-1). Blood was collected 7 days after the boost, treated to remove red blood cells, and then stained for viability, CD3, CD8, CD4, and SIINFEKL-tetramer reactivity (OVA-specific CD8+ T cell epitope shown as SEQ ID NO: 16). The frequency of SIINFEKL-specific cells among live CD8+ T cells was determined, and the absolute total number of SIINFEKL-specific CD8+ T cells collected in the blood was also determined. When mice were treated with GenVoy LNP-OVA LNP (5 μg mRNA) together with cGASΔN or GFP LNP (1 or 5 μg mRNA), the frequency of SIINFEKL-specific (OVA-specific) CD8+ T cells was significantly increased when cGASΔN mRNA was delivered together with OVA compared to when GFP mRNA was delivered together with OVA (Figure 11A). Mice that did not receive OVA LNP did not have SIINFEKL-specific CD8+ T cells. This trend was not formulation-specific. Because mice that received OVA LNP (5 μg mRNA) and cGASΔN (5 μg mRNA) prepared with a custom lipid mix had a significantly higher frequency of SIINFEKL-specific T cells in the blood than mice that received OVA LNP + GFP LNP (5 μg mRNA) (Figure 11B). This trend also applied to the absolute number of SIINFEKL-specific CD8+ T cells when prepared with either GenVoy (Figure 11C) or a custom lipid mix (Figure 11D). Interestingly, decreasing the dose of cGASΔN mRNA did not seem to have a major impact on the frequency of OVA-specific T cells. This suggests that a small amount of this cGASΔN mRNA may be sufficient to significantly boost the antigen-specific T cell response when combined with a standard lipid preparation containing antigen mRNA.

[0122] Treatment with cGASΔN LNP reduces Th2-specific OVA-specific antibodies without affecting the Th1-specific IgG response. OVA-specific antibodies in the blood were quantified after two immunizations with OVA LNP (5 μg mRNA / mouse) delivered with cGASΔN LNP (1 or 5 μg mRNA / mouse) or control GFP LNP (1 or 5 μg mRNA / mouse) (Table 3-1). Blood was collected 7 days after the boost, serum was isolated, and then total IgG, IgG1 (associated with Th2 response), and IgG2a (associated with Th1 response) were analyzed. Historically, immunization with mRNA antigen-loaded LNP generates a strong antibody response, so the antibody response was evaluated to determine whether the addition of cGASΔN LNP in the treatment reduces the expected response. Interestingly, mice treated with OVA and cGASΔN LNP prepared using the GenVoy formulation showed no significant change in the measured total OVA-specific IgG compared to OVA + GFP LNP treatment (Figure 12A). Mice that did not receive OVA LNP did not generate OVA-specific antibodies. This was not a formulation-specific response: the measured total OVA-specific IgG was not significantly affected by the presence of cGASΔN in LNP prepared using a custom lipid mix (Figure 12B). Since IgG1 is typically associated with the Th2 immune response, OVA-specific IgG1 was also evaluated. OVA-specific IgG1 was significantly reduced by the presence of cGASΔN in LNP prepared using the GenVoy formulation (p < 0.05 for both 1 μg and 5 μg cGASΔN compared to GFP LNP, for dilutions 1:500 and 1:2500) (Figure 12C). This reduced OVA-specific IgG1 response was also measured in mice treated with cGASΔN LNP prepared using a custom lipid mix (p < 0.05 for 5 μg cGASΔN compared to GFP LNP, for dilutions 1:500, 1:2500, and 1:12500) (Figure 12D). Since IgG2a is associated with a stronger inflammatory Th1 response, IgG2a levels were also evaluated.Importantly, mice treated with OVA and cGASΔN LNPs prepared using the GenVoy formulation (Figure 12E) or a custom lipid mix (Figure 12F) did not show a significant change in OVA-specific IgG2a compared to OVA + GFP-treated mice.

[0123] Collectively, the data gathered from in vivo immunization experiments indicate that treatment of mice with cGASΔN LNPs in combination with antigen LNPs significantly improves the antigen-specific adaptive immune response. Mice receiving cGASΔN LNPs showed a significant increase in the number of antigen-specific T cells without sacrificing the antibody response. Indeed, a decrease in antigen-specific IgG1 that did not affect total antigen-specific IgG levels indicates a bias towards a Th1-related immune response. This is beneficial in inducing a protective immune response, which is required for immunotherapy. Collectively, these data suggest that the addition of low doses of cGASΔN LNPs to standard antigen LNP preparations can result in a much more potent immune response against LNP vaccines.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Claims

1. A composition comprising mRNA encapsulated in lipid nanoparticles (LNPs), wherein the mRNA comprises a constitutively active cyclic GMP-AMP synthase (cGAS) coding region, and the LNP comprises phospholipids and at least one lipid selected from the group consisting of ionizable lipids, PEGylated lipids, structural lipids, and mixtures thereof.

2. The composition according to claim 1, comprising a further mRNA, wherein the further mRNA comprises an antigen coding region, wherein: The composition also comprises the aforementioned further mRNA, which is encapsulated within the LNP.

3. The composition according to claim 1, comprising further mRNA, wherein the further mRNA comprises an antigen coding region, and wherein the composition comprises further LNPs in which the further mRNA is encapsulated.

4. The composition according to claim 1, wherein the mRNA comprises a first coding region and a second coding region separated by a 2A-like sequence, wherein the first coding region is the coding region of the constitutively active cyclic GMP-AMP synthase (cGAS) and the second coding region is the coding region of an antigen, or the first coding region is the coding region of an antigen and the second coding region is the coding region of the constitutively active cyclic GMP-AMP synthase (cGAS).

5. The composition according to claim 1, wherein the at least one lipid includes an ionizable lipid, a PEGylated lipid, and a structural lipid.

6. The ionizable lipids are, i) 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid, 1-octyrnonyl ester (SM-102) or its analogs or derivatives; and / or ii) 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexane-1-aminium (ALC-0315) or its analogs or derivatives; and / or iii) (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) or its analog or derivative, The composition according to claim 5, comprising:

7. The composition according to claim 5, wherein the PEG-modified lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and combinations thereof.

8. The composition according to claim 5, wherein the PEGylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG].

9. The composition according to claim 5, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and combinations thereof.

10. The composition according to claim 9, wherein the structural lipid comprises cholesterol.

11. The phospholipids mentioned above are i) Hydrophilic heads selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin; and ii) One or more fatty acid tails selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid, The composition according to claim 5, comprising:

12. The phospholipids mentioned above are 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-Dimiristoyl-sn-glycerophosphocholine (DMPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Diundecanoyl-sn-glycerophosphocholine (DUPC), 1-Palmitoyl-2-Oleoyl-sn-Glycerol-3-Phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-Oleoyl-2-Cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine, 1,2-Dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), Sphingomyelin, and These combinations, A composition according to claim 5, selected from the group consisting of the following.

13. The composition according to claim 5, wherein the composition does not contain lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain.

14. A composition comprising mRNA (RNA-Lipoplex) complexed with one or more lipids, wherein the mRNA comprises a constitutively active cyclic GMP-AMP synthase (cGAS) coding region, and the one or more lipids comprise a first lipid and a second lipid.

15. The composition according to claim 14, further comprising a second mRNA (RNA-Lipoplex) complexed with one or more lipids, wherein the second mRNA comprises an antigen coding region.

16. The composition according to claim 14, wherein the mRNA comprises a first coding region and a second coding region separated by a 2A-like sequence, the first coding region being the coding region of the constitutively active cyclic GMP-AMP synthase (cGAS), and the second coding region being the coding region of an antigen, or the first coding region being the coding region of an antigen, and the second coding region being the coding region of the constitutively active cyclic GMP-AMP synthase (cGAS).

17. The composition according to claim 14, wherein the first lipid is a cationic lipid and the second lipid is a neutral or anionic lipid.

18. The composition according to claim 1, wherein the composition does not contain a TLR7 / 8 agonist.

19. The composition according to claim 1, wherein the cGAS is a shortened cGAS (cGASΔN) lacking an amino-terminal phosphoinositide binding domain.

20. The composition according to claim 19, wherein the cGASΔN comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

1.

21. The aforementioned cGASΔN is, (i) The amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8; or (ii) Consensus amino acid sequence of Sequence ID No. 9, The composition according to claim 20, comprising:

22. The composition according to claim 2, wherein the antigen is a tumor antigen.

23. The composition according to claim 2, wherein the antigen comprises a microbial antigen.

24. The composition according to claim 3, wherein the antigen is a tumor antigen.

25. The composition according to claim 3, wherein the antigen comprises a microbial antigen.

26. A pharmaceutical formulation comprising the composition according to any one of claims 1 to 25, and a pharmaceutically acceptable additive.

27. An in vitro method for generating hyperactivated dendritic cells, the method comprising contacting the dendritic cells with an effective amount of the formulation described in claim 26 to generate hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing cell death within approximately 48 hours of exposure.

28. The aforementioned hyperactivated dendritic cells (i) secreting one or more of RANTES, IP-10, and IFNα at higher levels than unstimulated dendritic cells that have not been in contact with the composition; and / or (ii) Expressing at least one cell surface marker selected from the group consisting of CD40, CD86, CD69, MHC class II, MHC class I, CCR7, and combinations thereof, at higher levels than unstimulated dendritic cells, The method according to claim 27.

29. At least 10 produced by the method of claim 28 ３ , 10 ４ , 10 ５ or 10 ６ A pharmaceutical formulation comprising the aforementioned hyperactivated dendritic cells and a pharmaceutically acceptable additive.

30. Use of an effective amount of the pharmaceutical formulation according to claim 26 for the manufacture of a pharmaceutical for stimulating an immune response to an antigen in an individual that requires stimulation of an immune response to an antigen.

31. Use of an effective amount of the pharmaceutical formulation according to claim 26 for the manufacture of a pharmaceutical for treating cancer in an individual that requires treatment for cancer.

32. The use of an effective amount of the pharmaceutical preparation according to claim 26, characterized in that it is for the manufacture of a pharmaceutical for inhibiting abnormal cell proliferation in an individual that requires the inhibition of abnormal cell proliferation.

33. Use of the pharmaceutical preparation according to claim 26 in an effective amount, characterized in that it is for the manufacture of a pharmaceutical for treating or preventing an infectious disease in an individual who requires treatment or prevention of the infectious disease.

34. The pharmaceutical formulation according to claim 26, for stimulating an immune response to an antigen in an individual that requires stimulation of an immune response to an antigen.

35. The pharmaceutical preparation according to claim 26 for treating cancer in an individual that requires treatment for cancer.

36. A pharmaceutical preparation according to claim 23 for inhibiting abnormal cell proliferation in an individual that requires the inhibition of abnormal cell proliferation.

37. The pharmaceutical preparation according to claim 26 for treating or preventing an infectious disease in an individual who requires treatment or prevention of the infectious disease.

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