Nucleic acids encoding constitutively active cyclic GMP-AMP synthase and immunogenic delivery vehicles therefor

Abstract

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 one or both of a lysophosphatidylcholine (LPC) compound and a pathogen recognition receptor agonist.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 341,984, filed on May 13, 2022, which is hereby incorporated by reference in its entirety.

[0002] Submission of Sequence Listing as an ASCII Text File The content of the electronic sequence listing (165532000240SEQLIST.xml; size: 22,341 bytes; and creation date: May 11, 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 their use for enhancing the immunogenicity of mRNA vaccines. The above - mentioned mRNA can be encapsulated in lipid nanoparticles (LNP) or complexed with lipids (RNA - Lipoplex). The present disclosure also relates to compositions further comprising one or both of lysophosphatidylcholine (LPC) compounds and 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 to 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

[0005]

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 an mRNA vaccine. 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 one or both of a lysophosphatidylcholine (LPC) compound and a pathogen recognition receptor agonist.

Brief Description of the Drawings

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DETAILED DESCRIPTION

[0016] 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 one or both of lysophosphatidylcholine (LPC) compounds and pathogen recognition receptor agonists.

[0017] 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.

[0018] 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 efficacy of LNPs loaded with mRNA encoding an antigen by increasing inflammatory signals.

[0019] 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.

[0020] 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.

[0021] The term "comprising", as used herein, indicates without limitation 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.

[0022] 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).

[0023] An "effective amount" or "sufficient amount" of a substance is that 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 being 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).

[0024] 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).

[0025] 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.

[0026] As used herein, the terms “isolated” and “purified” refer to a substance that has been removed from at least one of the components with which it is naturally associated (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.

[0027] As used herein, the terms “pharmaceutical formulation” and “pharmaceutical composition” refer to a preparation that is in such a form as to be effective for the biological activity of the active ingredient and that contains no additional components that are toxic to an unacceptable degree to the individual to whom the formulation or composition is administered. Such a formulation or composition is intended to be sterile.

[0028] As used herein, “excipient” includes a pharmaceutically acceptable excipient, carrier, vehicle or stabilizer that is non-toxic to the cells or mammals to which it is exposed at the dosages and concentrations used. Often, a physiologically acceptable excipient is an aqueous pH buffer solution.

[0029] 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 include peptides, polypeptides, proteins, glycoproteins, polysaccharides, glycoconjugates, sugars, gangliosides, lipids and phospholipids; portions thereof and combinations thereof may be included. 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 contain one or more post-translational modifications.

[0030] 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.

[0031] "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).

[0032] "Alkylene" refers to a divalent saturated aliphatic hydrocarbyl group.

[0033] "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).

[0034] 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.

[0035] 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 different conditions (e.g., a decrease in abnormal cell growth 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.

[0036] The relative terms "higher" and "lower" refer to a measurable increase or decrease in a response or parameter, respectively, when compared to the same conditions in other respects except for the parameter of interest, or alternatively when compared to different conditions. For example, "a higher level of DC overactivation" refers to the level of DC overactivation as a result of a treatment state that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater 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 state that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold less than the level of DC overactivation as a result of a control condition.

[0037] 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 infection and / or resist disease.

[0038] The term "vaccination" as used herein refers to the introduction of a vaccine into the body of a mammalian subject.

[0039] As used herein, an "adjuvant" refers to a substance that, when added to a composition containing an antigen or a nucleic acid encoding an antigen, enhances or potentiates the immune response to the antigen in a mammalian recipient upon exposure.

[0040] As used herein, the term "treating" or "treatment" refers 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" can 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 lessened and / or the time course of progression of the disease or disorder is slowed as compared to the predicted course of non-treatment.

[0041] As used herein in connection with 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 includes an enzymatic 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 that lacks the regulation of enzymatic activity impaired by the N-terminal disordered domain of full-length cGAS.

[0042] 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. Human cGAS is 522 amino acids in length and includes an N-terminal phosphoinositide-binding domain (residues 1-59) and a C-terminal DNA-binding and enzymatic domain (residues 160-522) (Barnett et al., Cell, 176:1432-1446, 2019). Importantly, expression of cGASΔN in a human leukemia monocytic cell line has been found to result in higher levels of interferon and expression of genes stimulated by interferon (Barnett, supra, 2019).

[0043] The amino acid sequence of human cGAS (GenBank No. NP_612450.2) is as follows:

Chemical formula

[0044] The amino acid sequence of the N-terminal domain of cGAS is as follows:

Chem.

[0045] The amino acid sequence of the C-terminal domain of cGAS (cGASΔN) is as follows:

Chem.

[0046] The nucleotide sequence encoding human cGASΔN (which is 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 the examples.

[0047] The compositions and methods of the present disclosure include nucleic acids encoding constitutively active cGAS as a catalytic adjuvant to improve 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.

[0048] 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 with the amino acid sequences of the C-terminal domains of multiple non-human primate cGAS proteins is shown in FIG. 1.

Table I

[0049] In some preferred embodiments, the constitutively active cGAS is a truncated human cGAS lacking the N-terminal domain (SEQ ID NO: 11). In some preferred embodiments, the constitutively active cGAS 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 a start codon (ATG).

[0050] "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 (e.g., 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 necessary 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 containing 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.

[0051] 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 antigens comprising peptide chains that are 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.

[0052] 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, for example, 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).

[0053] 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.

[0054] 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.

[0055] 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.

[0056] In some embodiments, the mRNA further encodes a ribosome skipping sequence (e.g., a 2A-like (2AL) 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 the 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 the 2AL sequence (e.g., antigen1-2AL-antigen2). Additional 2AL sequences for use with 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 of FIG. 2 is incorporated herein by reference).

[0057] 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.

[0058] In some embodiments, the LNP includes 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 includes an ionizable lipid. In some embodiments, the at least one lipid includes a cationic lipid. In some embodiments, the at least one lipid includes a phospholipid. In some embodiments, the at least one lipid includes a pegylated lipid. In some embodiments, the at least one lipid includes a structural lipid. In some embodiments, the at least one lipid includes an ionizable lipid, a phospholipid, a pegylated lipid, and a structural lipid.

[0059] 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.

[0060] The structures of lipids suitable for use in the lipid-based mRNA delivery vehicles of the present disclosure are shown in FIGS. 9A and 9B, which are adopted from FIG. 2 of Hou et al., Nature Review Materials, 6:1078-1094, 2021.

[0061] IV. Lysophosphatidylcholine Compounds The compositions and methods of the present disclosure may include phospholipids, preferably lysophosphatidylcholine. "Lysophosphatidylcholine" (LPC) or "lysophosphatidylcholine molecule" refers to a glycerol molecule having one phosphocholine group on a hydroxyl group of glycerol and one acyl group on one of the other two hydroxyl groups of the glycerol. The remaining hydroxyl group is unsubstituted.

[0062] In some embodiments, an isolated lysophosphatidylcholine (LPC) having a single acyl chain is of the following form:

Chemical formula

Chemical formula

[0063] In some embodiments, an isolated lysophosphatidylcholine (LPC) having a single acyl chain is of the following form:

Chemical formula

Chemical formula

[0064] The above alkyl or alkenyl chain, together with the carbonyl carbon, forms an acyl chain that is one carbon atom longer than the above alkyl chain or alkenyl chain. For example, the (C23 alkyl)-C(=O)- group forms a C24 acyl chain. Thus, when the group “(alkyl or alkylene)” is a C12-C23 alkyl group (e.g., a C12-C19 alkyl group or a C20-C23 alkyl group), the above (C12-C23 alkyl-C(=O)- group forms a C13-C24 acyl chain (e.g., a C13-C20 acyl chain or a C21-C24 acyl chain). When the group “(alkyl or alkylene)” is a C12-C23 alkenyl group (e.g., a C12-C19 alkenyl group or a C20-C23 alkenyl group), the above (C12-C23 alkenyl-C(=O)- group forms a C13-C24 acyl chain (e.g., a C13-C20 acyl chain or a C21-C24 acyl chain). The acyl chain may also be referred to as a saturated acyl or an unsaturated acyl to distinguish between an alkyl-containing acyl group and an alkenyl-containing acyl group. Standard delta notation or omega notation may be used to indicate the position of one or more double bonds in an unsaturated acyl chain.

[0065] The lysophosphatidylcholine (LPC) compounds of the present disclosure have a single acyl chain where the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain. In some embodiments, the acyl chain is a C18-C22 acyl chain or a C21-C24 acyl chain. In some preferred embodiments, the acyl chain is a C22 acyl chain.

[0066] V. Pathogen Recognition Receptor Agonists The compositions and methods of the present disclosure may further comprise an additional pathogen recognition receptor (PRR) agonist. In some embodiments, the PRR agonist comprises an agonist of a toll-like receptor (TLR), a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), or a C-type lectin receptor (CLR). In some embodiments, the PRR agonist comprises a TLR7 / 8 agonist.

[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 overactivating human dendritic cells in response to LPC.

[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 a 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 comprises an imidazoquinoline compound. In some preferred embodiments, the TLR7 / 8 agonist comprises resiquimod (R848).

[0069] B. Other PRR Agonists In some aspects, 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 a preferred embodiment, 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 aspects, the PRR agonist includes a NOD-like receptor (NLR) agonist. In a further aspect, the PRR agonist includes a RIG-I-like receptor (RLR) agonist. In an additional aspect, the PRR agonist includes a C-type lectin receptor (CLR) agonist.

[0071] VI. Pharmaceutical Formulations Some compositions of the present disclosure are pharmaceutical formulations comprising 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 includes various components in a specific ratio based on the intended purpose of the formulation.

[0072] Pharmaceutically acceptable additives of the present disclosure include, for example, solvents, buffers, tonicity modifiers, bulking agents, and preservatives (see, for example, Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the pharmaceutical formulation may include additives that function as one or more of a solvent, buffer, tonicity modifier, and bulking agent (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 solutions. 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 salts containing, for example, 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 helps stabilize the active agent and prevent 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] VII. 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 methods of treating cancer. In some embodiments, the methods of use include methods of inhibiting abnormal cell growth. In some embodiments, the methods of use include methods 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 the 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 the baseline frequency prior to administration.

[0082] 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 antigen-specific antibody responses 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 an 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 monocyte-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 of 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 otherwise treating a mammalian subject 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 includes 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 includes 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 A composition comprising mRNA encapsulated in lipid nanoparticles (LNP), wherein the mRNA comprises a coding region of constitutively active cyclic GMP-AMP synthase (cGAS), and the LNP comprises a first phospholipid and at least one lipid selected from the group consisting of ionizable lipids, pegylated lipids, structural lipids, a second phospholipid, and mixtures thereof, wherein the first phospholipid comprises lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain. A composition comprising a first mRNA and a second mRNA encapsulated in lipid nanoparticles (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; the LNP comprises a first phospholipid and at least one lipid selected from the group consisting of ionizable lipids, pegylated lipids, structural lipids, a second phospholipid, and mixtures thereof, wherein the first phospholipid comprises lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain. 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 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 LNP comprises a first phospholipid and at least one lipid selected from the group consisting of ionizable lipids, pegylated lipids, structural lipids, a second phospholipid, and mixtures thereof, wherein the first phospholipid comprises lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain. The composition according to any one of Embodiments 1 to 3, wherein the at least one lipid comprises an ionizable lipid, a second phospholipid, 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) or an analog or derivative thereof, The composition according to any one of Embodiments 1 to 4, comprising Embodiment 6. The pegylated 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, the composition according to any one of Embodiments 1 to 5. Embodiment 7. The pegylated lipid comprises polyethylene glycol [PEG]2000 dimyristoyl glycerol [DMG], the composition according to any one of Embodiments 1 to 5. Embodiment 8. The structural lipid is selected from the group consisting of cholesterol, fucosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and combinations thereof, the composition according to any one of Embodiments 1 to 7. Embodiment 9. The structural lipid comprises cholesterol, the composition according to any one of Embodiments 1 to 7. Embodiment 10. The second 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, comprising. Embodiment 11. The second 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-diundecanoyl-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-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 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 second phospholipid comprises 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 comprises at least one additive. Embodiment 14. The composition according to Embodiment 13, wherein the additive comprises sucrose. Embodiment 15. i) mRNA complexed with one or more lipids (RNA-Lipoplex); and ii) Lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain, A composition comprising, wherein the mRNA comprises a coding region of constitutively active cyclic GMP-AMP synthase (cGAS), and the one or more lipids comprise a first lipid and a second lipid. Embodiment 16. i) A first mRNA and a second mRNA complexed with one or more lipids (RNA-Lipoplex); and ii) Lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain, A composition comprising; 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 one or more lipids comprise a first lipid and a second lipid. Embodiment 17. i) mRNA complexed with one or more lipids (RNA-Lipoplex); and ii) Lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain, A composition comprising, wherein the mRNA comprises 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), the second coding region is a coding region of an antigen, or the first coding region is a coding region of an antigen, the second coding region is a coding region of constitutively active cyclic GMP-AMP synthase (cGAS), and the one or more lipids comprise 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 them. 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 Embodiment 21. The composition according to any one of embodiments 1 to 20, wherein the acyl chain of the LPC is a C21-C24 acyl chain. Embodiment 22. The composition according to any one of embodiments 1 to 20, wherein the acyl chain of the LPC is a C22 acyl chain. Embodiment 23. The composition according to any one of embodiments 1 to 22, wherein the acyl chain of the LPC is fully saturated. Embodiment 24. The composition according to embodiment 23, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)]. Embodiment 25. The composition according to any one of embodiments 1 to 24, further comprising a TLR7 / 8 agonist. Embodiment 26. The composition according to embodiment 25, wherein the TLR7 / 8 agonist is a low molecular weight compound having a molecular weight of 900 daltons or less. Embodiment 27. The composition according to embodiment 26, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound. Embodiment 28. The composition according to embodiment 27, wherein the TLR7 / 8 agonist comprises resiquimod (R848). Embodiment 29. The composition according to any one of embodiments 25 to 28, wherein the LPC comprises LPC(22:0) and the TLR7 / 8 agonist comprises resiquimod (R848). Embodiment 30. The composition according to any one of embodiments 1 to 29, 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 31. The mRNA is the composition according to any one of Embodiments 1 to 30, which contains a 5' cap structure. Embodiment 32. The mRNA is the composition according to any one of Embodiments 1 to 31, which contains a polyA tail. Embodiment 33. The mRNA is the composition according to any one of Embodiments 1 to 32, which is a nucleoside-modified mRNA. Embodiment 34. A nucleic acid comprising (i) a coding region of an antigen and (ii) a coding region of constitutively active cyclic GMP-AMP synthase (cGAS), where optionally the nucleic acid is mRNA here and optionally the nucleic acid is DNA here. Embodiment 35. An expression vector containing the nucleic acid according to Embodiment 34. Embodiment 36. The composition according to any one of Embodiments 1 to 35, wherein the constitutively active cGAS tends to have higher self-DNA reactivity than its wild-type counterpart. Embodiment 37. The composition according to any one of Embodiments 1 to 36, wherein the cGAS is a truncated cGAS (cGASΔN) lacking an amino-terminal phosphoinositide-binding domain. Embodiment 38. The composition according to Embodiment 37, wherein 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. Embodiment 39. 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, The composition according to Embodiment 38, which contains. Embodiment 40. The cGASΔN is the composition according to Embodiment 38, which is encoded by the nucleotide sequence of SEQ ID NO: 17. Embodiment 41. The coding region of the cGASΔN is in an operable combination with the start codon (ATG), and the composition according to any one of Embodiments 37 to 40. Embodiment 42. The antigen is a tumor antigen, and the composition according to any one of Embodiments 1 to 41. Embodiment 43. The tumor antigen is a tumor-associated antigen, and the composition according to Embodiment 42. Embodiment 44. The tumor antigen is a neoantigen, and the composition according to Embodiment 42. Embodiment 45. The antigen includes a microbial antigen, and the composition according to any one of Embodiments 1 to 41. Embodiment 46. The microbial antigen includes a viral antigen, a bacterial antigen, a protozoan antigen, or a fungal antigen, and the composition according to Embodiment 44. Embodiment 47. The antigen includes a surface protein of a pathogen or a fragment thereof, and the composition according to any one of Embodiments 1 to 41. Embodiment 48. The pathogen can cause a disease in a human subject, and the composition according to Embodiment 47. Embodiment 49. The pathogen is a virus, and the composition according to Embodiment 47 or Embodiment 48. Embodiment 50. The virus is SARS-CoV-2, and the composition according to Embodiment 49. Embodiment 51. The antigen is the spike (S) glycoprotein of the SARS-CoV-2, and optionally here the spike glycoprotein is a prefusion-stabilized variant, and the composition according to Embodiment 50. Embodiment 52. The composition does not contain lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA), and the composition according to any one of Embodiments 1 to 51. Embodiment 53. The composition does not contain oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC) or a species of oxPAPC, and the composition according to any one of Embodiments 1 to 52. Embodiment 54. The composition 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-oxohept-6-enoyl]oxy-3-hexadecanoyloxypropyl] 2-(trimethylazaniumyl)ethyl phosphate; KOdiA-PC), l-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), the composition according to Embodiment 53. Embodiment 55. A pharmaceutical preparation comprising the composition according to any one of Embodiments 1 to 54 and a pharmaceutically acceptable additive. Embodiment 56. 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 54, any one of Embodiments 25 to 54, or the preparation according to Embodiment 55 to generate hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing cell death upon exposure within about 48 hours. Embodiment 57. The dendritic cells are (i) contacted with the composition in vivo; or (ii) contacted with the composition ex vivo. The method according to Embodiment 56. Embodiment 58. The hyperactivated dendritic cells (i) secrete one or more of IL-6, TNFα, RANTES, and IP-10 at a higher level than unstimulated dendritic cells or dendritic cells contacted with empty LNPs; and / or (ii) express at least one cell surface marker selected from the group consisting of CCR7, CD40, CD80, CD83, CD86, MHC class II, MHC class I, and combinations thereof at a higher level. The method according to Embodiment 56 or Embodiment 57. Embodiment 59. 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 56 to 58, and a pharmaceutically acceptable additive. Embodiment 60. A method for stimulating an immune response against an antigen, the method comprising administering an effective amount of the pharmaceutical preparation according to Embodiment 55 or Embodiment 59 to an individual in need of stimulating an immune response against the antigen to stimulate the immune response against the antigen. Embodiment 61. 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 55 or Embodiment 59 to treat the cancer. Embodiment 62. 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 55 or Embodiment 59 to inhibit abnormal cell growth. Embodiment 63. 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 55 to treat or prevent the infectious disease. Embodiment 64. The method according to Embodiment 63, wherein the infectious disease is a viral disease. Embodiment 65. The method according to Embodiment 63, wherein the infectious disease is a bacterial disease. Embodiment 66. The method or pharmaceutical preparation according to any one of Embodiments 56 to 62, wherein the dendritic cell is a mammalian cell. Embodiment 67. The method or pharmaceutical preparation according to Embodiment 66, wherein the mammalian cell is a human cell. Embodiment 68. The individual is a mammal. The method according to any one of Embodiments 60 to 66. Embodiment 69. The method according to Embodiment 68, wherein the mammal is a human. Embodiment 70. The method according to Embodiment 68, wherein the mammal is a dog or a cat. Embodiment 71. The second 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. The composition, preparation, or method according to any one of Embodiments 1 to 70. Embodiment 72. 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 71. Embodiment 73. 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 72. Embodiment 74. The LNP has an effective diameter of less than about 200 nanometers, the composition, formulation, or method according to embodiment 73. Embodiment 75. The LNP has an effective diameter of less than about 150 nanometers, the composition, formulation, or method according to embodiment 73.

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 is described in some detail by way of illustration and examples, 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, and the scope of the present invention disclosure is clearly indicated by the appended claims.

[0090] Example 1: The combination of lysophosphatidylcholine (LPC) having a single acyl chain and a TLR7 / 8 agonist hyperactivates mammalian peripheral blood mononuclear cells This example describes the hyperactivation of canine and human peripheral blood mononuclear cells (PBMCs) with lipid DAMPs in combination with low molecular weight PAMPs.

[0091] Materials and Methods Isolation of PBMCs from whole blood. PBMCs were isolated from whole blood using density gradient centrifugation with Ficoll-Paque PLUS (Cytivia). Whole blood was diluted 1:1 with PBS, overlaid on top of Ficoll-Paque PLUS, and centrifuged at 1000×g for 30 minutes at room temperature. PBMCs were collected, washed twice in PBS, and incubated with Ack lysis buffer (Lonza) to remove any remaining red blood cells.

[0092] Cell culture and stimulation. Immediately after isolation, PBMCs were seeded in RPMI medium (R10 medium) containing 10% FBS, 50 units / mL penicillin, 50 mg / mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 mM β-mercaptoethanol. Cells were seeded at 1×10 5 (for canine cells) or 1×10 6(Human cells) were seeded. Lyophilized Vaccigrade R848 (Invivogen) was reconstituted, diluted according to the manufacturer's recommendations, and added to the cells at a final concentration of 1 μg / mL. Immediately thereafter, 22:0 LYSO PC was added to the cells at a final concentration of 82.5 μM. Additional native agonists were diluted in R10 medium according to the manufacturer's recommendations and added to the above cells as follows: human GM-CSF (Peprotech) was added at a final concentration of 10 ng / mL; 2’3’ cGAMP (Invivogen) was added at a final concentration of 15 μg / mL; LPS, serotype O55:B5 (Enzo Life Sciences) was added at a final concentration of 1 μg / mL; alum hydroxide (Invivogen) was added at a final concentration of 30 μg / mL. The cells were incubated at 37 °C, 5% CO 2 for 2 days. The cell culture was then used for endpoint analysis.

[0093] Endpoint analysis. After culturing PBMC with PAMPs and DAMPs for 2 days, the supernatant and cell samples were collected for analysis. The cells in the culture were pelleted by centrifugation at 400 × g for 5 minutes. Half of the volume of the medium in the well was collected for cytokine quantification by enzyme-linked immunosorbent assay (ELISA) or Lumit TM Bioluminescent assay, while the remaining medium and cells were used to quantify cell viability by assessing metabolic activity.

[0094] Quantification of cytokine secretion. IL-1β secretion from human PBMC was evaluated using one of the following kits: ELISA MAX Deluxe Set Human IL-1β kit (Biolegend), Invitrogen Human IL-1β kit, or Lumit TMHuman IL-1β Immunoassay (Promega). IFNγ secretion from human PBMCs was evaluated using the ELISA MAX Deluxe Set Human IFNγ (Biolegend), and TNFα secretion from human PBMCs was evaluated using the Human TNFα Uncoated ELISA kit (Invitrogen). ELISA was performed according to the manufacturer's instructions with the following modifications: i) the total sample + buffer volume for incubation was reduced from 100 μL to 50 μL; ii) the top standard was prepared at 500 pg / mL and serially diluted 2-fold down to 7.8 pg / mL; and iii) sample incubation was completed overnight at 4°C on an orbital shaker. Lumit TM The assay was performed according to the manufacturer's instructions. IL-1β secretion from canine PBMCs was performed using the Canine IL-1β / IL-1F2 DuoSet ELISA (R&D) according to the manufacturer's instructions with the following modifications: i) the total sample + buffer volume for incubation was reduced from 100 μL to 50 μL; ii) sample incubation was completed overnight at 4°C on an orbital shaker. For all ELISAs, absorbance was measured at 450 nm corrected at 570 nm using a Spectramax M5e plate reader (Molecular Devices). Lumit TM For the assay, luminescence was measured at all wavelengths using a Spectramax M5e plate reader (Molecular Devices) with an integration time of 500 ms. To determine the cytokine concentration in the supernatant, sample concentrations were interpolated using a standard curve via 4PL analysis in GraphPad Prism 9 (GraphPad Software). The interpolated results for the samples were then adjusted for any dilutions made to the supernatant.

[0095] Quantification of cell viability. Cell viability was evaluated by providing the presence of ATP as an indicator of metabolically active cells using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Metabolic activity was evaluated according to the manufacturer's instructions below. The CellTiter-Glo reagent was mixed with the cell pellet and then fresh medium was transferred to a white opaque 96-well plate. Luminescence was measured at all wavelengths with a Spectramax M5e plate reader (Molecular Devices) using an integration time of 500 ms. Percent viability was calculated relative to the control condition of PBMCs treated with R848.

[0096] Statistical analysis. For each condition, cells from each donor were seeded in triplicate for testing. For cytokine quantification, triplicate values were used for interpolation and the data were plotted as total concentration (pg / mL) or fold change per donor relative to the control condition of R848 alone. For viability quantification, the triplicates for each donor were averaged and the mean was used as one donor measurement. Multiple donors were tested. Each data point on the bar graph represents the value of a donor. To test for differences in the test conditions, the test results were compared to the control condition of R848 alone. P-values were calculated using a mixed-effects one-way ANOVA and corrected for multiple comparisons using Dunnett's test.

[0097] Results - Treatment with 22:0 LYSO PC and R848 hyperactivates canine PBMCs The combination of 22:0 LYSO PC(DAMP) and the TLR7 / 8 agonist, R848(PAMP), was found to have potent hyperstimulatory activity in human moDC. To evaluate whether this hyperstimulatory activity translates to other clinically relevant species, the ability of 22:0 LYSO PC + R848 to hyperactivate PBMC isolated from canine whole blood was evaluated. For each dataset, PBMC from multiple donors were used in place of moDC due to the lack of canine-specific reagents available to induce bona fide canine moDC. Briefly, PBMC were isolated from whole blood using density gradient centrifugation and then cultured for 2 days with the stimulant of interest for hyperactivation.

[0098] After 2 days of culture, hyperactivation was evaluated by quantification of IL-1β in the cell culture supernatant and measurement of cell viability. When treated together with 22:0 LYSO PC and R848, canine PBMC secreted levels of IL-1β that were comparable or higher in both concentration per mL and fold change per donor compared to all other stimulants tested. Consistent with previous studies showing that monocytes (which constitute 5 - 10% of PBMC) can release IL-1β in response to activation with R848, canine PBMC increased the level of IL-1β secretion with R848 alone compared to untreated cells. The pyroptotic combination of LPS + Alum induced high levels of IL-1β as expected. Notably, PGPC + R848 induced levels of IL-1β similar to R848 alone, but neither GM-CSF nor 2’3’cGAMP induced significant IL-1β secretion from canine PBMC compared to untreated cells.

[0099] IL-1β can be detected one day after the overactivation of canine PBMCs in cell culture supernatants, but cell viability was evaluated two days after overactivation to ensure that viability was sustained after IL-1β secretion. 22:0 LYSO + R848 did not significantly reduce relative cell viability. Interestingly, PGPC in combination with R848 was found to be somewhat toxic to canine PBMCs, but was not observed to be toxic to human moDCs or human PBMCs. Collectively, these data indicate that 22:0 LYSO + R848 induces high levels of IL-1β secretion (which indicates overactivation) from canine PBMCs.

[0100] Results - Treatment with 22:0 LYSO PC and R848 overactivates human PBMCs Overactivation experiments were also performed on PBMCs isolated from whole blood obtained from human donors. Briefly, PBMCs were isolated from whole blood by density gradient centrifugation from multiple human donors and cultured for two days with the stimulant of interest for overactivation.

[0101] Human PBMCs secreted IL-1β at higher levels compared to all other stimulants tested, similar to human moDCs and canine PBMCs. Similar to canine PBMCs, human PBMCs secreted IL-1β in response to R848 due to monocyte activation, which was increased by the addition of 22:0 LYSO PC. The pyroptotic combination of LPS + Alum induced high levels of IL-1β as expected. Consistent with the observations in canine PBMCs, PGPC + R848 did not induce substantially higher levels of IL-1β than R848 alone. GM-CSF did not induce levels of IL-1β secretion from human PBMCs that significantly exceeded the background levels produced by untreated cells.

[0102] The viability of human PBMCs was also evaluated 2 days after hyperactivation to ensure the viability of human PBMCs after IL-1β secretion. No significant decrease in human PBMC viability was observed after treatment with any of the above stimulants. Summarizing these data, PBMCs from both humans and dogs are shown to be hyperactivated by 22:0 LYSO PC + R848. Interestingly, canine PBMCs are hyperactivated to a greater extent by 22:0 LYSO PC + R848 than by PGPC + R848.

[0103] Since activated human PBMCs can secrete other cytokines in addition to IL-1β, the secretion of the pro-inflammatory cytokines IFNγ and TNFα in the cell culture supernatant was measured 2 days after hyperactivation. The combination of 22:0 LYSO PC + R848 induced the highest fold change per donor in both IFNγ secretion and TNFα secretion compared to R848 alone and all other stimulants tested, while LPS + Alum induced high levels of IL-1β secretion from human PBMCs and this combination of stimulants did not induce a fold increase in IFNγ or TNFα secretion. Furthermore, neither GM-CSF nor 2’3’cGAMP induced a substantial fold change in IFNγ secretion beyond that of R848 alone. These data indicate that the combination of 22:0 LYSO PC + R848 is superior in inducing the secretion of the pro-inflammatory cytokines IFNγ and TNFα from human PBMCs.

[0104] Example 2: Both cGASΔN mRNA and 22:0 LPC can be encapsulated into lipid nanoparticles (LNPs) 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. 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 mouse cells. The synthetic nucleotide sequence is shown as SEQ ID NO: 17. The mRNA sequence was capped with N1-methylpseudouridine base modification and a 120-residue polyA tail using Trilink's proprietary Clean Cap mRNA technology. The sequence contains a Bbsl restriction enzyme site. The sequence information was used to construct a plasmid from which the mRNA was synthesized. The mRNA was treated with phosphatase after synthesis.

[0105] LNP Synthesis. Lipid nanoparticles (LNP) were prepared using the ordered LNP lipid mix (Table 2-1). cGASΔN mRNA was prepared at 0.02 mg / mL in sodium citrate buffer (pH 4). The ordered LNP lipid mix was prepared at 12.5 mM. The mRNA / 22:0 LPC ratio was selected based on previous experiments to determine the in vitro activity of each component. LNP 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. 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. LNP were filtered through a 0.2 μm filter before use.

Table 2-1

[0106] 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 prior to performing DLS. Three 90-second measurements were recorded for each sample.

[0107] Results LNP can be loaded with cGASΔN mRNA and 22:0 LPC at levels similar to LNP loaded with either cGASΔN mRNA or 22:0 LPC alone. All LNP (22:0 LPC LNP, cGASΔN LNP, and cGASΔN + 22:0 LPC LNP) showed similar sizes, with sizes <150 nm for all LNP with a relatively uniform distribution (PDI < 0.3) (Figure 3A). cGASΔN LNP and cGASΔN + 22:0 LPC-loaded LNP showed similar mRNA loading levels, but there was a slight reduction in overall loading efficiency (actual loading / theoretical loading) when 22:0 LPC was also added to the LNP (Figure 3B). 22:0 LPC LNP and cGASΔN + 22:0 LPC LNP showed similar loading levels and efficiency (actual / theoretical) with respect to 22:0 LPC (Figure 3C).

[0108] Example 3: Mouse dendritic cells achieve a distinct activation state upon treatment with mRNA encoding cGASΔN and a stimulant for overactivation. Materials and Methods Generation of mouse bone marrow-derived dendritic cells (BMDCs). The femurs and tibias of the legs were removed from the mice, cut with scissors, and rinsed 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. The cells were counted and resuspended in a 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 8E6 bone marrow cells per well of a P12 plate. Recombinant mouse FLT3L (Miltenyi) was added to the culture at 200 ng / mL. The differentiated cells were used for subsequent assays on day 9. When the efficiency of differentiation was monitored by flow cytometry using a BD Symphony A3, CD11c + MHC-II + cells always exceeded 80% of the viable cells. For each experiment, 5 - 15 mice were used to generate DCs from the bone marrow.

[0109] Hyperactivation of mouse BMDCs and delivery of cGASΔN. BMDCs were harvested on day 9 after differentiation, washed with PBS, and reseeded in complete IMDM medium (I10) at a concentration of 2×10 5 cells / well. The cells were cultured in the presence or absence of LNP loaded with 100 μM 22:0 LPC LNP and with or without approximately 1 μg / mL of cGASΔN mRNA (Table 3-1). Under certain conditions, R848 was added at a final concentration of 1 μg / mL. After 48 hours of stimulation, the supernatant was collected for cytokine secretion evaluation. IL-1β cytokine secretion by BMDCs was measured using a sandwich ELISA (Invitrogen) according to the manufacturer's instructions. Additional cytokines were measured using a LEGENDplex Mouse Anti-viral Response Panel (Biolegend) according to the manufacturer's instructions. Cell viability was evaluated by measuring LDH release into the fresh supernatant using a CyQUANT LDH Cytotoxicity Assay (Invitrogen) according to the manufacturer's instructions.

Table 3-1

[0110] Results In previous studies, the inventors found that DC overactivation using R848 and 22:0 LPC activates the transcription factor NF-kB, leading to the production of inflammatory cytokines (e.g., IL-6 and TNFα). Furthermore, the NLRP3 inflammasome is activated, leading to IL-1β production. Separately from these cellular responses, cGAS activation and the resulting STING signaling activate the transcription factor IRF3, which co-regulates some genes together with NF-kB but also regulates a distinct set of genes independent of NF-kB. cGASΔN is a constitutively active modified cGAS. Using LNPs formulated with cGASΔN mRNA with and without 22:0 LPC, the inventors hypothesized that cGAS signaling and overactivation could induce a complementary set of inflammatory signals.

[0111] To test the inventors' hypothesis, mouse bone marrow cells were differentiated into DCs using FLT3L. The BMDCs were then stimulated with R848, 22:0 LPC, and cGASΔN mRNA in various combinations. As described in Example 2, cGASΔN mRNA and 22:0 LPC were formulated with LNP either separately or in combination.

[0112] Flt3L BMDCs treated with R848 and [cGASΔN + 22:0 LPC] LNP can be hyperactivated. DCs were evaluated for their potential to be hyperactivated in response to treatment with R848 + [cGASΔN + 22:0 LPC] LNP; hyperactivation was typed by the ability of the DCs to secrete IL-1β while remaining viable. DCs treated with PBS (unstimulated) or R848 alone produced minimal or no IL-1β. As predicted, combining R848 and 22:0 LPC treatment resulted in IL-1β production after 48 hours (Figure 4A). Treatment of cells with cGASΔN mRNA-containing LNP did not induce IL-1β. cGASΔN treatment combined with 22:0 LPC also did not result in IL-1β release (Figure 4A). cGAS / STING signaling is probably insufficient to generate the pro-form of the IL-1β protein required for 22:0 LPC-mediated release of mature IL-1β. However, all three stimulants, R848, cGASΔN, and 22:0 LPC, resulted in the release of IL-1β (Figure 4A). This result indicated that cGASΔN treatment did not inhibit hyperactivation. Next, cell viability was evaluated using the LDH release assay to confirm that the cells were hyperactivated but not undergoing pyroptosis. Across the treatment conditions tested, the DCs had a survival profile similar to that of unstimulated cells, confirming that the DCs were hyperactivated (Figure 4B). Collectively, these data suggest that DCs can be co-treated with R848, 22:0 LPC, and cGASΔN mRNA and can be hyperactivated.

[0113] [cGASΔN and 22:0 LPC] LNP + R848 treatment activates the NF-kB signaling and cGAS-STING pathway in Flt3L BMDCs. To further confirm the activity of individual stimulants, IL-6 and TNFα production was quantified. These two genes are mainly regulated by NF-kB and thus by R848 stimulation. When R848 was added as a treatment, IL-6 and TNFα were produced (Figures 5A - 5B). Adding LNP containing 22:0 LPC to R848 treatment increased the secretion of both IL-6 and TNFα. Alone, cGASΔN did not stimulate IL-6 and TNFα secretion (Figures 5A - 5B). cGAS / STING signaling mainly activates IRF3 and does not activate NF-kB. Furthermore, the secretion of RANTES and IP-10 (which are known to be regulated by cGAS / STING signaling) was measured. Cells treated with PBS (unstimulated) did not express RANTES or IP-10. In contrast, cGASΔN stimulation induced the expression of RANTES and IP-10, confirming that the inventors' mRNA transcript encoding active cGAS was successfully delivered to and expressed in the cells (Figures 5C - 5D). Unexpectedly, when cGASΔN was delivered together with 22:0 LPC, the expression of RANTES and IP-10 was inhibited compared to single cGASΔN treatment. When cells were stimulated with R848 alone or in combination with 22:0 LPC in hyperactivation, the cells produced RANTES and IP-10 and no 22:0 LPC-mediated inhibition was observed. Although a surprising inhibition was observed when GASΔN was combined with 22:0 LPC, all three combinations resulted in the expression of RANTES and IP-10 (Figures 5C - 5D).

[0114] Collectively, treatment of mouse DCs with various combinations of R848, 22:0 LPC, and cGASΔN showed several things. First, cGASΔN mRNA was successfully formulated into LNPs for delivery to DCs and translated into protein. The cGASΔN protein was active, as shown by the ability of the treatment to induce RANTES and IP-10 production. Second, in combination with hyperactivating stimuli, cGASΔN treatment did not reduce the secretion of IL-1β, a key hyperactivation cytokine. Finally, when cells were stimulated with all three stimuli, inflammatory genes regulated by one stimulus were not impaired by the effects of the other two stimuli. These data indicate that cGASΔN treatment can be combined with hyperactivating stimuli to instruct DCs to simultaneously generate the inflammatory environment of both processes. This combination results in a unique DC activation state that may have potential benefits in the immune response.

[0115] Example 4: Treatment of human dendritic cells with cGASΔN and hyperactivating stimuli results in activation of the NLRP3 inflammasome and the cGAS-STING pathway Materials and methods Generation of human monocyte-derived dendritic cells (moDC). Human monocytes were isolated from Leukopaks purchased from Miltenyi Inc. (San Jose, CA) using the StraightFrom Leukopak CD14 MicroBead Kit according to the manufacturer's instructions. The monocytes were then aliquoted and frozen in fetal bovine serum containing 10% dimethyl sulfoxide. For studies in monocyte-derived dendritic cell (moDC) cultures, the 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. To differentiate the monocytes into moDC, recombinant human GM-CSF (50 ng / mL) and IL-4 (25 ng / mL) were added to the R10 medium. The cells were cultured with GM-CSF and IL-4 for 6 days, and on day 3, the cells were replenished with additional nutrients in R10 medium containing GM-CSF and IL-4. On day 6 of differentiation, the moDC were collected for use and a portion was stained to confirm the success of differentiation. Conventionally, >80% of the living cells are CD11c+CD209+ by flow cytometry using a BD Symphony A3.

[0116] Overactivation of moDCs and delivery of cGASΔN. Six days after differentiation from monocytes, moDCs were collected and counted. Cells were seeded at 100,000 cells / well in 96-well plates. moDCs were treated with LNP containing 1 μg / mL R848 and 22:0 LPC and / or cGASΔN mRNA. Forty-eight hours after stimulation, cell culture supernatants were collected. Cytokine expression was measured using the LEGENDplex Human Anti-viral Response Panel (Biolegend) according to the manufacturer's instructions. Cell viability was evaluated by measuring LDH release into fresh supernatants using the CyQUANT LDH Cytotoxicity Assay (Invitrogen) according to the manufacturer's instructions. Cells were collected and stained to measure the expression of DC molecules involved in T cell activation (e.g., CD40, CD80, CD83, CD86, CCR7, HLA-ABC, and HLA-DR expression) and evaluated by flow cytometry using the BD Symphony A3.

[0117] Results We hypothesized that the combination of cGASΔN-mediated STING signaling and overactivation could help construct immunotherapies and vaccines that may provide better protection than currently available options. Therefore, we tested the combination of cGASΔN treatment and overactivation of human DCs. As a source of DCs, human monocytes were differentiated into DCs by culturing in R10 medium containing recombinant GM-CSF and IL-4 for 6 days. Cells were confirmed to be DCs by CD11c and CD209 staining on day 6 and were used for treatment with the three stimulants of interest to us: R848, 22:0 LPC, and cGASΔN.

[0118] MoDCs treated with R848 and [cGASΔN + 22:0 LPC] LNP are hyperactivated and enable cGAS-STING pathway activation. The inventors first began by confirming the major conclusions obtained using mouse DCs. The combination of these three treatments (R848, 22:0 LPC, and cGASΔN) had a similar effect on human DC viability compared to DCs treated with PBS (unstimulated) (Figure 6A). When measuring IL-1β release, the inventors observed that treatment with PBS (no stimulation) or R848 resulted in minimal IL-1β (Figure 6B). As predicted, the combination of R848 and 22:0 LPC delivered via LNP induced IL-1β secretion. Similar to mice, cGASΔN treatment did not induce IL-1β secretion, either alone or in combination with 22:0 LPC (Figure 6B). When cGASΔN was combined with the hyperactivation combination of R848 and 22:0 LPC, IL-1β secretion was maintained or increased (Figure 6B). IP-10 secretion was quantified to confirm that cGASΔN mRNA formulated in LNP could induce signaling. cGASΔN treatment alone induced IP-10 expression. In contrast to the mouse data, addition of 22:0 LPC to cGASΔN LNP did not inhibit IP-10 expression (Figure 6C). Also, in contrast to the mouse data, R848 treatment (either alone or in combination with 22:0 LPC) was insufficient to induce IP-10 expression (Figure 6C). When all three stimulants were combined, cGASΔN, R848, and 22:0 LPC were able to induce IP-10 expression (Figure 6C). For the donors shown, a reduction in IP-10 was observed when all three stimulants were combined compared to cGASΔN treatment alone. However, this reduction was not consistently observed in the four donors tested. Despite this reduction, the combination of cGASΔN, R848, and 22:0 LPC resulted in IP-10 expression, which was significantly higher than that detected under unstimulated conditions (Figure 6C).Collectively, these results suggested that human DCs, like mouse DCs, can mediate cGAS / STING activation while not being overactivated.

[0119] MoDCs treated with R848 and [cGASΔN + 22:0 LPC] LNP upregulate surface proteins required for T cell activation. In addition to the generation of a specific cytokine environment, DCs need to express cell surface proteins that are important in T cell activation. One such protein is the chemokine receptor CCR7, which is required for DC migration to lymph nodes, where they can stimulate T cells from peripheral tissues. DCs were stained for CCR7 expression after treatment with various combinations of R848, 22:0 LPC, and cGASΔN. As previously observed, treatment with R848 alone increased CCR7 expression, and hyperactivation treatment with R848 in combination with 22:0 LPC resulted in a further increase in CCR7 (Figure 7A). Treatment with cGASΔN also increased CCR7 expression compared to unstimulated cells, and the addition of 22:0 LPC had no significant effect on CCR7 expression (Figure 7A). Interestingly, the combination of cGASΔN treatment with R848 and 22:0 LPC resulted in the largest shift in CCR7 expression (Figure 7A). Importantly, the combination of cGASΔN and hyperactivation did not inhibit CCR7 expression, and the increased CCR7 expression could enable more efficient migration of DCs to lymph nodes.

[0120] The inventors then continued to examine other important cell surface proteins on dendritic cells that are important for their function. CD40 is a receptor that binds CD40L on CD4 T cells and ultimately enhances the T cell response. CD83 is also involved in T cell activation and is commonly used as an indicator of DC activation. Compared to unstimulated cells, CD40 expression was upregulated when DCs were treated with R848 with or without 22:0 LPC, and further enhancement was observed when the cells were treated with cGASΔN with or without 22:0 LPC (Figure 7B). The combination of cGAS signaling and hyperactivation also resulted in an increase in CD40 expression compared to unstimulated cells (Figure 7B). CD83 expression was more variable compared to CD40 staining, but repeatedly, the combination of cGASΔN treatment and hyperactivation treatment did not abolish CD83 expression (Figure 7C).

[0121] CD80 and CD86 are costimulatory molecules that bind T cells during antigen presentation and serve as confirmation signals that the presented non-self antigen is actually dangerous. When DCs are activated with R848 or cGASΔN, or when DCs are hyperactivated with R848 and 22:0 LPC, CD80 and CD86 expression increases (Figures 8A - 8B). When the combination of cGASΔN and hyperactivation treatment is combined, DCs continue to upregulate CD80 and CD86 compared to unstimulated cells (Figures 8A - 8B). Thus, when both cellular processes are involved, DCs can still supply costimulatory signals to T cells.

[0122] Finally, DCs were stained for HLA-ABC and HLA-DR, which are class I and class II MHC molecules presenting antigen to CD8 and CD4 T cells, respectively. Compared to unstimulated cells, R848 treatment with or without the addition of 22:0 LPC increased the expression of HLA-ABC (Figure 8C). Treatment of DCs with cGASΔN with or without 22:0 LPC also had a similar effect on HLA-ABC expression (Figure 8C). Surprisingly, combining cGASΔN treatment and hyperactivation resulted in a significant increase in HLA-ABC compared to other combinations of stimuli. When analyzing HLA-DR staining, R848 treatment was observed to increase HLA-DR expression compared to unstimulated cells (Figure 8D). A further increase was then observed when hyperactivated with the combination of R848 and 22:0 LPC (Figure 8D). cGASΔN treatment was effective either more than R848 treatment alone or as part of hyperactivation (Figure 8D). Addition of 22:0 LPC to cGASΔN treatment further enhanced HLA-DR expression compared to cGASΔN treatment alone (Figure 8D). Finally, the combination of cGASΔN treatment and hyperactivation resulted in the highest HLA-DR expression among all experimental conditions (Figure 8D). The combination of all three stimuli resulted in the highest expression of MHC class I and class II molecules. This may enable more effective antigen presentation to T cells.

[0123] Studies using human DCs suggest that they generally respond similarly to cGASΔN, R848, and 22:0 LPC as compared to mouse DCs. One unexpected difference was when cells were treated with cGASΔN with or without 22:0 LPC. Mouse DCs showed inhibition of IP-10 when 22:0 LPC was added, while human DCs did not have the same result. More importantly, the combination of cGASΔN, R848, and 22:0 LPC did not prevent human DCs from overactivation and cytokine (e.g., IL-1β and IP-10) production. Further analysis of human DCs regarding the cell surface expression of important proteins involved in T cell activation showed that the combination of the three stimulants did not inhibit the expression. These proteins were still inducible and in some cases (e.g., HLA-ABC and HLA-DR), the expression was further increased as compared to cGASΔN treatment alone or overactivation. Overall, these data collected from the treatment of human DCs suggest that cGASΔN treatment can be combined with overactivation to induce a cellular state in which the two processes complement each other and potentially have an additional effect on cellular functions for the induction of an adaptive immune response.

[0124] Example 5: Combination of cGASΔN mRNA and overactivation as a vaccination strategy to improve immune response By utilizing mRNA encoding cGASΔN, DCs can be activated to produce chemical signals that promote T cell effector responses. Overactivation of DCs produces a complementary set of chemical signals. In particular, overactivation induces pro-inflammatory cytokines and adds IL-1β, a cytokine extremely important for memory T cell formation, to their cytokine repertoire. By combining stimulants to involve cGAS / STING signaling and overactivation, a broader set of inflammatory signals can be generated to maximize the immune response initiated by DCs.

[0125] To test this model, vaccination strategies utilizing various combinations of cGASΔN, R848, and 22:0 LPC as adjuvants are compared in vivo. The purpose of vaccination is to direct an immune response against the target antigen or a complex source of the target antigen (e.g., model antigen, tumor-associated antigen, neoantigen, microbe-derived antigen, etc.). The immune response is readily detected using well-established immunological methods and common reagents (e.g., tetramer staining, ELISpot assay, ELISA, flow cytometry, etc.). The immunogenic composition is prepared, for example, by loading an mRNA transcript encoding the target antigen into lipid nanoparticles (LNPs). These LNPs are suitable for administration to mammalian subjects to achieve expression of the target antigen in vivo for initiation of an adaptive immune response.

[0126] The purpose of this experiment is to test whether cGASΔN (delivered as mRNA in LNP) can be used as a vaccine adjuvant to improve the immune response against a model antigen. cGASΔN, R848, and 22:0 LPC are intended to improve DC function by enhancing de novo T cell activation and memory T cell reactivation. These improvements may have positive downstream effects on effector and memory T cell responses. Improved T cell activation may also have further positive downstream effects on B cells when the B cells undergo germinal center reactions that require T cell engagement.

Table 5-1

[0127] Methods [cGASΔN + 22:0 LPC] LNP + In vivo immunization with mRNA antigen in LNP. C57BL / 6J mice are subcutaneously immunized with the LNP as detailed in Table 5-1. Using OVA mRNA-loaded LNP, the transcript is delivered in vivo for the production of OVA antigen in combination with LNP containing either the adjuvant cGASΔN mRNA or non-adjuvanting GFP mRNA. The above OVA mRNA dose is fixed at 5 μg / mouse, while the adjuvant cGASΔN mRNA is administered at approximately 1.5 μg / mouse and 22:0 LPC is administered at approximately 60 μg / mouse (Table 5-1). Mice are primed on day 0 and boosted with the same dose on day 7. For the evaluation of short-term effector responses, on day 14, blood and secondary lymphoid organs are collected. For the evaluation of long-term memory responses, on day 40, blood and secondary lymphoid organs are collected. Blood is collected for the evaluation of antibody and T cell responses. Serum is collected from blood using serum separation tubes while blood for cell analysis is collected using K2EDTA tubes. After blood collection, the mice are euthanized and their draining lymph nodes and spleens are collected and processed into single cell suspensions. The predicted responses are shown in Table 5-1.

[0128] OVA-specific T cell tetramer assessment. OVA-specific T cells in the blood and draining lymph nodes of mice receiving OVA LNP immunization are evaluated 14 and 40 days after the first immunization. CD8+ T cells specific for SIINFEKL (SEQ ID NO: 17) (the MHC-I restricted T cell epitope of ovalbumin (OVA)) are quantified in the blood and dLN using SIINFEKL-tetramer staining. Briefly, red blood cells are lysed using RBC lysis buffer and the lysis is completed twice to remove all RBCs in the blood. The cells are washed and then stained for viability (live / dead), SIINFEKL-tetramer binding (MBL), and CD3, CD4, and CD8 expression. The cells are fixed with 4% paraformaldehyde and counting beads are added prior to run to enable determination of the total cell number. Data are collected using a BD FACS Symphony and analyzed using Flowjo (BD).

[0129] Effector and memory T cell measurement. The frequencies of T effector and T memory cells are evaluated by flow cytometry in the blood and draining lymph nodes of mice receiving OVA LNP immunization. Briefly, cell suspensions are stained with CD3, CD4, CD8, CD62L and CD44 antibodies to measure the frequencies of T effector cells (CD44 Low CD62L neg ) and T memory cells (CD44 high CD62L + ) using a BD FACS Symphony instrument and the data are analyzed using Flowjo (BD).

[0130] OVA-specific antibody evaluation. OVA-specific antibodies in the sera of mice immunized with OVA LNPs are evaluated 14 days and 40 days after the first immunization. Total OVA-specific IgG, IgG1, and IgG2b are evaluated by ELISA. Briefly, ELISA plates are coated overnight with 10 μg / mL Endofit Ovalbumin (Invivogen), then washed and blocked with 2% bovine serum albumin. The plates are washed again, then serum is added to the plates at a 1:500 dilution, followed by a total of 7 serum dilutions at a 1:5 dilution. The samples are washed and then incubated with detection antibodies specific for IgG, IgG1, or IgG2b (which are conjugated to HRP (Southern Biotech)) to detect all, Th2-skewed, and Th1-skewed OVA-specific antibodies, respectively. The plates are washed and then incubated with TMB, and once the color development is complete, a stop solution is added.

[0131] OVA-specific T cell response. The OVA-specific T cell response is determined from secondary lymphoid organ activity. After immunization, draining lymph nodes and spleens are collected from mice at early (day 14) and late (day 40) time points. The collected lymph nodes and spleens are dissociated into single cell suspensions, which are used in the ELISPOT assay. The ELISPOT is used to detect IFNγ and IL-5 secretion by T cells. These indicate Th1 and Th2 responses, respectively. Cells from the draining lymph nodes and spleens are seeded 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. The cells are seeded at 200,000 cells / well in a 96-well ELISPOT plate and restimulated with 10 mcg / mL OVA peptivator or 1 mcg / mL OVA peptide antigen. As a control, additional seeded cells are left unstimulated or stimulated with an irrelevant antigen (not used in vaccination). Completion of the assay results in spots that can be visually provided when cytokines are secreted by T cells in response to restimulation, as a method for quantifying the number of T cells responding to restimulation.

[0132] Results Using the above assay, effector and memory responses are analyzed to evaluate adaptive T cell immunity at short (2 weeks post-vaccination) and long ( > 4 weeks post-vaccination) time points. Table 5-1 lists the experimental groups to be included in the study. As a negative control, mice in Group 1 receive a sham injection containing neither antigen nor adjuvant to serve as a baseline where little or no antigen-specific immune response is expected. Mice in Group 2 receive mRNA encoding a model antigen (OVA) formulated in LNP (this represents a standard LNP vaccination protocol). All remaining groups receive LNP loaded with mRNA encoding OVA in combination with an adjuvant. Depending on the type of stimulant administered, one or more of the following are predicted: i) activating cells (inducing NFkB signaling); ii) over-activating DCs (inducing NLRP3 pathway activation); and iii) inducing IRF3 signaling in DCs. For example, mice in Group 4 receive R848 + 22:0 LPC (this is predicted to hyper-activate DCs that activate the NLRP3 pathway in addition to the NFkB pathway, resulting in conventional pro-inflammatory cytokine secretion (IL-6 and TNFα) and IL-1β production). Mice in Group 5 receive LNP encoding cGASΔN (this is predicted to induce a type I IFN response via IRF3). Mice in Groups 6 and 7 receive two of the three stimulants, while mice in Group 8 receive all three stimulants (R848, 22:0 LPC, and cGASΔN).

[0133] In vivo cGASΔN expression is intended to stimulate DC signals that are particularly beneficial for intermediate effector immune responses. The hyperactivation conditions are also intended to contribute to effector responses despite the absence of type I IFN signaling. Additionally, hyperactivation-induced IL-1β signaling results in improved memory formation. Thus, by combining cGAS signaling and hyperactivation, a very strong effector response and a persistent memory response are intended to be induced. Immunization with sham treatment (group 1) will likely result in little or no observed effector and memory responses, and immunization with antigen mRNA alone, or in combination with R848 (groups 2 and 3), is intended to result in a small effector response and minimal memory formation. The treatment of group 4 is predicted to hyperactivate DCs and be an improvement over the treatment of group 2. In comparison, the treatments of groups 5 and 6 involve only cGAS signaling in DCs and are thus intended to result in a strong effector response but with less persistent memory responses than the treatment of group 4. Group 7 is a hyperactivation treatment condition. Groups 7 and 8 are intended to produce the best effector and memory responses, provided they involve both cGAS and the hyperactivation pathway. The hyperactivating lipid involves cellular processes beyond NLRP3 activation, and thus the treatment of group 8 (which includes 22:0 LPC) is intended to result in excellent memory and memory responses.

[0134] The three adjuvant-acting stimulants can be formulated in various ways. For example, cGASΔN mRNA can be encapsulated alone in LNP or in combination with 22:0 LPC. R848 can be administered as an individual component, within LNP, or within LNP containing one or both of the other stimulants. Furthermore, the time series of administration of the stimulants can vary. For example, it may be beneficial to administer antigen mRNA-loaded LNP in combination with cGASΔN mRNA-loaded LNP and R848 in the first injection to provide a strong effector signal via type I IFN, and subsequently, a boost injection of antigen mRNA-loaded LNP in combination with 22:0 LPC-loaded LNP and R848 provides the memory signal necessary to generate a persistent immunity.

[0135] Studying the T cell response and B cell response to vaccination is intended to generate in vivo data showing that the combination of hyperactivation and cGAS signaling results in an improvement in the antigen-specific immune response. Evaluation of how the administration of various DC stimulants affects the B cell response is achieved by measuring the total antigen-specific IgG antibodies in the sera of immunized mice, as well as antibody isotypes (e.g., IgG1 (associated with TH2 response) and IgG2b (associated with TH1 response)). Based on previous studies using cGASΔN LNP, the inventors predict that mice immunized with LNP filled with mRNA encoding an antigen and mRNA encoding cGASΔN will produce a TH1-biased response.

[0136]

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 a first phospholipid and at least one lipid selected from the group consisting of ionizable lipids, PEGylated lipids, structural lipids, second phospholipids, and mixtures thereof, wherein the first phospholipid comprises lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain.

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 comprises an ionizable lipid, a second phospholipid, 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 second phospholipid described above is 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 second phospholipid described above is 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. i) mRNA complexed with 1 or more lipids (RNA-Lipoplex); and ii) Lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain, A composition comprising, The composition wherein the mRNA comprises a constitutively active cyclic GMP-AMP synthase (cGAS) coding region, and the 1 or more lipids comprise a first lipid and a second lipid.

14. The composition according to claim 13, further comprising a second mRNA (RNA-Lipoplex) complexed with one or more lipids, wherein the second mRNA comprises an antigen coding region.

15. The composition according to claim 13, A composition 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 the antigen, or the first coding region is the coding region of the antigen and the second coding region is the coding region of the constitutively active cyclic GMP-AMP synthase (cGAS).

16. The composition according to claim 13, wherein the first lipid is a cationic lipid and the second lipid is a neutral or anionic lipid.

17. The composition according to claim 1, wherein the acyl chain of the LPC is a C21-C24 acyl chain and / or the acyl chain of the LPC is completely saturated.

18. The composition according to claim 17, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

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

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

21. The composition according to claim 20, 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.

22. 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 21, comprising:

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

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

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

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

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

28. 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 27 to generate hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing cell death within approximately 48 hours of exposure.

29. The aforementioned hyperactivated dendritic cells (i) secreting one or more of IL-6, TNFα, RANTES, and IP-10 at higher levels than unstimulated dendritic cells that have not been in contact with the composition; and / or (ii) Cells expressing at least one cell surface marker selected from the group consisting of CCR7, CD40, CD80, CD83, CD86, MHC class II, MHC class I, and combinations thereof, at higher levels than unstimulated dendritic cells. The method according to claim 28.

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

31. Use of an effective amount of the pharmaceutical formulation according to claim 27 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.

32. Use of an effective amount of the pharmaceutical formulation according to claim 27 for the manufacture of a pharmacopoeia for treating cancer or inhibiting abnormal cell proliferation in an individual who requires treatment of cancer or inhibition of abnormal cell proliferation.

33. Use of an effective amount of the pharmaceutical preparation according to claim 27 for the manufacture of a pharmaceutical for treating or preventing an infectious disease in an individual who requires treatment or prevention of an infectious disease.

34. The pharmaceutical formulation according to claim 27, for stimulating an immune response to an antigen in an individual that requires stimulation of an immune response to an antigen.

35. The pharmaceutical formulation according to claim 27 for treating cancer or inhibiting abnormal cell proliferation in an individual who requires treatment of cancer or inhibition of abnormal cell proliferation.

36. The pharmaceutical preparation according to claim 27 for treating or preventing an infectious disease in an individual who requires treatment or prevention of an infectious disease.

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