Nucleic acids encoding constitutively active cyclic GMP-AMP synthases and immunogenic delivery vehicles thereof

Abstract

The present disclosure relates to compositions for expressing a constitutively active cyclic GMP-AMP synthase in a cell of a mammalian subject and their use for enhancing the immunogenicity of an mRNA vaccine. The mRNA may be encapsulated in a lipid nanoparticle (LNP) or may be complexed with a lipid (RNA-Lipoplex). The present disclosure also relates to compositions further comprising one or both of a lysophosphatidylcholine (LPC) compound and a pathogen-recognizing receptor agonist.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 341,984, filed May 13, 2022, which is incorporated herein by reference in its entirety. ASCII Text File Sequence Listing Submission

[0002] The contents of the electronic Sequence Listing (165532000240SEQLIST.xml; size: 22,341 bytes; and creation date: May 11, 2023) are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to compositions for expressing constitutively active cyclic GMP-AMP synthase in cells of mammalian subjects and their use for enhancing the immunogenicity of mRNA vaccines. The mRNA can be encapsulated in lipid nanoparticles (LNP) or can be 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. Background Art

[0004] Advances in mRNA chemistry and delivery systems have enabled the rapid production of several effective mRNA COVID-19 vaccines (Hou et al., Nature Review Materials, 6: 1078-1094, 2021). However, recent reports have found that T cell reactivity to the SARS-CoV-2 spike protein is significantly lower in vaccinated, uninfected individuals than in individuals previously infected with SARS-CoV-2 (Naranbhai et al., Cell, 185: 1-11, 2022). This is concerning given 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).

[0005] Therefore, there is a need in the art for formulations for enhancing the cellular immune response elicited by mRNA vaccines. In particular, adjuvants suitable for increasing the adaptive immune response to nucleic acid-encoded antigens are desirable. Summary of the invention

[0006] The present disclosure relates to compositions for expressing constitutively active cyclic GMP-AMP synthase in cells of mammalian subjects and their use for enhancing the immunogenicity of mRNA vaccines. The mRNA can be encapsulated in lipid nanoparticles (LNP) or can be 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 is an alignment of the amino acid sequences of primate cGASΔN proteins, including: human (Homo sapiens, SEQ ID NO: 1); Rhesus monkey (Macaca mulatta, SEQ ID NO: 2); olive baboon (Papio anubis, SEQ ID NO: 3); northern white-cheeked gibbon (Nomascus leucogenys, SEQ ID NO: 4); common gibbon (Hylobates lar, SEQ ID NO: 5); Sumatran orangutan (Sumatran orangutan, Pongo abelii, SEQ ID NO: 6); chimpanzee (Pan troglodytes, SEQ ID NO: 7); western gorilla (Gorilla, SEQ ID NO: 8); NO:8); and cGASΔN consensus sequence (SEQ ID NO:9).

[0008] Figure 2 Cartoon depicting activation of the stimulator of interferon genes (STING) innate immune signaling pathway by cyclic GAMP-AMP synthase (cGAS) that has been engineered to be constitutively active by removing its amino-terminal phosphoinositide binding domain. cGASΔN localizes to mitochondria where it binds DNA, leading to the production of cGAMP.

[0009] Figure 3A-3C Characterization of lipid nanoparticles (LNPs) loaded with 22:0LPC and / or cGASΔN mRNA is depicted. Figure 3A It was shown that the diameters of LNPs loaded with 22:0LPC and / or cGASΔN mRNA were all less than 150 nm. Figure 3BIt was shown that the mRNA loading efficiency (actual loading compared to theoretical loading) was similar between LNPs loaded with cGASΔN mRNA only and LNPs loaded with both cGASΔN mRNA and 22:0 LPC. Figure 3C It is shown that the 22:0LPC loading efficiency (actual loading compared to theoretical loading) is similar between LNPs loaded with 22:0LPC only and LNPs loaded with both cGASΔN mRNA and 22:0LPC. Data from LNPs loaded with both cGASΔN mRNA and 22:0LPC ([cGASΔN+22:0LPC] LNPs) are shown with diagonal stripes.

[0010] Figure 4A-4B It is shown that treatment of murine dendritic cells (DCs) with R848 in combination with LNPs loaded with 22:0LPC results in superactivation, defined as the ability of DCs to secrete IL-1β while maintaining viability. Murine bone marrow-derived dendritic cells (BMDCs) were cultured for 48 h with controls (PBS or R848) or LNPs with or without R848. Figure 4A It is shown that BMDCs stimulated with R848 in combination with LNPs loaded with 22:0LPC ([22:0LPC]LNP) or LNPs loaded with both cGASΔN mRNA and 22:0LPC ([cGASΔN+22:0LPC]LNP) induce IL-1β secretion. In contrast, treatment of BMDCs with LNPs loaded with only mRNA encoding cGASΔN ([cGASΔN]LNP) or with [cGASΔN+22:0LPC]LNP was insufficiently stimulatory for inducing IL-1β secretion. Figure 4B The viability of BMDC is shown, as determined by measuring LDH release when collecting cell culture supernatant. Relative to PBS control, most treatment conditions lead to relatively similar cell viability levels, but except for [cGASΔN+22:0LPC]LNP treatment, which leads to reduced cell viability. The changes in cytokine secretion caused by various treatment conditions are compared with only R848 treatment. p<0.0001****. Statistics are completed using one-way ANOVA with Tukey post hoc test for multiple comparisons. Data from DCs processed with LNPs ([cGASΔN+22:0LPC]LNPs) loaded with cGASΔN mRNA and 22:0LPC are shown with oblique stripes.

[0011] Figure 5A-Figure 5C Shown are the effects of various treatments on cytokine secretion by murine DCs. Murine BMDCs were cultured for 48 h with control (PBS or R848) or LNPs with or without R848. Figure 5A and Figure 5BStimulation of BDMC with R848 in the presence or absence of other stimuli is shown to induce the secretion of IL-6 and TNFα. Figure 5C and Figure 5D It is shown that R848 is used to stimulate BMDC to induce the secretion of RANTES and IP-10 in the presence or absence of other stimulants. Interestingly, in the absence of R848, [cGASΔN] LNP stimulates the secretion of RANTES and IP-10, and in the absence of R848, [cGASΔN+22:0LPC] LNP does not stimulate the secretion of RANTES and IP-10. The changes in cytokine secretion caused by various treatment conditions are compared with only R848 treatment. P < 0.05 *, p < 0.0001 ****. Statistics are completed using one-way analysis of variance and Tukey's post hoc test for multiple comparisons. The data from DCs processed with LNPs ([cGASΔN+22:0LPC] LNPs) loaded with cGASΔN mRNA and 22:0LPC are shown with oblique stripes.

[0012] Figure 6A-6C It was shown that human monocyte-derived dendritic cells (moDCs) can mediate cGAS / STING signaling and hyperactivation simultaneously.Human moDCs were cultured with control (PBS or R848) or LNPs with or without R848 for 48 h. Fig. 6A It was shown that moDCs maintained high levels of viability under all treatment conditions, as determined by measuring LDH release. Figure 6B It was shown that R848 and 22:0 LPC are required for inducing IL-1β secretion by moDCs and that addition of cGASΔN mRNA to LNPs is not inhibitory. Figure 6C It is shown that even when combined with super-activating stimulants R848 and 22:0LPC, treating moDC with LNPs loaded with cGASΔN mRNA also stimulates IP-10 secretion. The changes in cytokine secretion caused by various treatment conditions are compared with treatment with only R848. p<0.05*, p<0.0001****. Statistics are completed using one-way ANOVA with Tukey post hoc test for multiple comparisons. Data from DCs treated with LNPs loaded with cGASΔN mRNA and 22:0LPC ([cGASΔN+22:0LPC]LNP) are shown with oblique stripes.

[0013] Figure 7A-7CIt is shown that when hyperactivation is combined with cGAS / STING signaling, cell surface marker expression of human moDCs is maintained or increased.Human moDCs were cultured with controls (PBS or R848) or LNPs with or without R848 for 48 h and then stained for the expression of cell surface proteins involved in DC function. Fig. 7A Shown is the expression of CCR7, a chemokine receptor required for cell migration to lymph nodes, by moDCs resulting from various treatment conditions. Figure 7B Shown is the expression of CD40, a receptor that engages CD40L expressed by T cells during T cell activation and leads to signaling that further enhances T cell activation, by moDCs resulting from various treatment conditions. Figure 7C The expression of CD83 of moDC caused by various treatment conditions is shown, and CD83 is a marker of DC maturation involved in T cell activation. The changes in cell surface marker expression caused by various treatment conditions are compared with R848 stimulation alone. p<0.05*, p<0.01**, <0.0001****. Statistics are completed using one-way ANOVA with Tukey post hoc test for multiple comparisons. Data from DC treated with LNPs ([cGASΔN+22:0LPC]LNP) loaded with cGASΔN mRNA and 22:0LPC are shown with oblique stripes.

[0014] Figure 8A-8D The results show that when hyperactivation is combined with cGAS / STING signaling, antigen presentation and expression of co-stimulatory molecules by human moDCs are maintained or increased. Human moDCs were cultured with controls (PBS or R848) or LNPs with or without R848 for 48 h and then stained for the expression of cell surface proteins involved in DC function. Treatment of moDCs with various combinations of stimuli resulted in increased expression of CD80 ( Fig. 8A ) and CD86( Figure 8B ) co-stimulatory molecules were upregulated. Importantly, when cGAS / STING signaling was combined with hyperactivation, moDCs maintained high levels of CD80 and CD86 expression. Treatment of moDCs with various combinations of stimuli resulted in HLA-ABC( Figure 8C ) and HLA-DR( Fig.8D) is upregulated. HLA-ABC and HLA-DR present antigens to CD8+T cells and CD4+T cells, respectively. When cGAS / STING signaling is combined with super activation (R848+[cGASΔN+22:0LPC]LNP), HLA-ABC and HLA-DR expression are observed to increase further. The changes in cell surface marker expression caused by various treatment conditions are compared with R848 stimulation alone. p<0.05*, p<0.0001****. Statistics are completed using one-way ANOVA with Tukey post hoc test for multiple comparisons. Data from DCs treated with LNPs ([cGASΔN+22:0LPC]LNP) loaded with cGASΔN mRNA and 22:0LPC are shown with diagonal stripes.

[0015] Fig. 9A Shown are the structures of cationic lipids and ionizable lipids suitable for use in lipid-based mRNA delivery vehicles of the present disclosure. Fig. 9B The structures of other types of lipids suitable for lipid-based mRNA delivery vehicles of the present disclosure are shown. See also Hou et al., Nature Review Materials, 6: 1078-1094, 2021, which is incorporated herein by reference. DETAILED DESCRIPTION

[0016] The present disclosure relates to compositions for expressing constitutively active cyclic GMP-AMP synthase in cells of mammalian subjects and their use for enhancing the immunogenicity of mRNA vaccines. The mRNA can be encapsulated in lipid nanoparticles (LNP) or can be 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.

[0017] Inducing an inflammatory response may be desirable, such as for immunotherapy or vaccination. COVID-19 mRNA vaccines (comprising LNPs loaded with mRNA encoding SARS-CoV-2 antigens) have been shown to be effective in reducing the frequency and severity of infection. As described herein, inducing the cGAS-STING innate immune pathway assists a composition comprising LNPs loaded with mRNA encoding a protein antigen, which in an exemplary embodiment is ovalbumin (OVA). Of all possible innate immune signaling pathways, STING signaling is particularly interesting because, unlike some other signaling pathways, cGAS-STING activation does not induce translational repression.

[0018] In order to induce cGAS-STING signaling, a cGAS mutant lacking a portion of the N-terminus of cGAS was designed so that it was constitutively active (cGASΔN). The mRNA encoding the cGAS mutant was packaged in LNPs for cellular uptake and protein expression. The LNP-packaged mRNA encoding cGASΔN has the ability to assist in immune responses via the cGAS-STING pathway. Importantly, mRNA encoding constitutively active cGAS (such as cGASΔN) is included to increase the efficacy of LNPs loaded with mRNA encoding antigens by increasing inflammatory signals. General Techniques and Definitions

[0019] The practice of the present disclosure will employ, unless otherwise indicated, 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" excipient includes one or more excipients.

[0021] As used herein, the phrase "comprising" is open ended, indicating that such embodiments may include additional elements. In contrast, the phrase "consisting of is closed ended, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase "consisting essentially of is partially closed, indicating that these embodiments may further include elements that do not materially change the basic characteristics of these embodiments.

[0022] As used herein, the term "about" with respect to a value encompasses from 90% to 110% of that value (eg, a molecular weight of about 900 Daltons refers to molecular weights from 810 Daltons to 990 Daltons).

[0023] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to achieve a beneficial or desired result, including a clinical result, and thus, an "effective amount" depends on the context in which it is used. For example, in the case of administering an immunogenic composition comprising one or more mRNAs encoding an antigen and constitutively active cGAS, an effective amount contains sufficient mRNA to stimulate an immune response (e.g., antigen-reactive antibodies and / or cellular immune responses) to the antigen.

[0024] The terms "individual" and "subject" refer to mammals. "Mammals" include, but are not limited to, humans, non-human primates (e.g., monkeys), livestock, sports animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats). In some embodiments, the subject is a human patient, such as a human patient suffering from cancer and / or an infectious disease.

[0025] The term "dose" as used herein with reference to an immunogenic composition refers to the measured portion of the immunogenic composition that a subject takes (is administered or receives) at any one time.

[0026] As used herein, the terms "isolated" and "purified" refer to material that is removed from at least one component with which it is naturally associated (e.g., removed from its original environment). As an example, when used with respect to 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 with respect to a recombinant protein, an isolated protein refers to a protein that has been removed from the culture medium of a host cell in which the protein was produced.

[0027] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to preparations that are in a form that permits the biological activity of the active ingredient to be effective, and that contain no additional components that are unacceptably toxic to a subject to which the formulation or composition is administered. Such formulations or compositions are intended to be sterile.

[0028] As used herein, "excipient" includes pharmaceutically acceptable excipients, carriers, vehicles or stabilizers that are not toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Typically, a physiologically acceptable excipient is a pH buffered aqueous solution.

[0029] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or T cell antigen receptor. Antigens may include peptides, polypeptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids and phospholipids; parts thereof and combinations thereof. In the context of the present disclosure, the term "antigen" typically refers to a polypeptide encoded by a nucleic acid sequence of mRNA or DNA. Polypeptide antigens are preferably at least eight amino acid residues in length and may contain one or more post-translational modifications.

[0030] The term "agonist" is used in its broadest sense and includes any molecule that activates signal transduction through a receptor. In some embodiments, the agonist binds to a receptor. For example, a TLR8 agonist binds to TLR8 and activates the TLR8 signal transduction pathway.

[0031] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbon 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 x and y carbon atoms (including the end value).

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

[0033] "Alkenyl" refers to a monovalent hydrocarbon 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 between x and y carbon atoms (inclusive).

[0034] "Stimulation" of a response or parameter includes when compared to conditions that are otherwise identical except for the parameter of interest or alternatively compared to another condition, initiating and / or enhancing the response or parameter (e.g., TLR signaling increases in the presence of a TLR agonist compared to the absence of a TLR agonist). For example, "stimulation" of an immune response means an increase in a response. Depending on the parameter measured, the increase can be from 2 times to 2,000 times or from 5 times to 500 times or more, or from 2, 5, 10, 50 or 100 times to 500, 1,000, 2,000, 5,000 or 10,000 times.

[0035] In contrast, "inhibition" of a response or parameter includes reducing and / or suppressing the response or parameter when compared to conditions that are otherwise identical except for the parameter of interest, or alternatively compared to another condition (e.g., a decrease in abnormal cell proliferation following administration of a composition of the disclosure compared to administration of a placebo composition or no treatment). For example, "inhibition" of an immune response means a decrease in the response. Depending on the parameter measured, the decrease can be from 2-fold to 2,000-fold or from 5-fold to 500-fold or more, or from 2, 5, 10, 50, or 100-fold to 500, 1,000, 2,000, 5,000, 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 conditions that are otherwise identical except for the parameter of interest, or alternatively compared to another condition. For example, "higher levels of DC hyperactivation" means that the level of DC hyperactivation as a result of the treatment condition is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than the level of DC hyperactivation as a result of the control condition. Similarly, "lower levels of DC hyperactivation" means that the level of DC hyperactivation as a result of the treatment condition is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower than the level of DC hyperactivation as a result of the control condition.

[0037] As used herein, the term "immunization" refers to the process of increasing a mammalian subject's immune response to an antigen and thereby improving 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] "Adjuvant" refers to a substance that, when added to a composition comprising an antigen or a nucleic acid encoding an antigen, enhances or potentiates the immune response of a mammalian recipient to the antigen upon exposure.

[0040] The term "treating" or "treatment" of a disease refers to the implementation of a regimen that may include administering one or more therapeutic agents to an individual (human or other individual) in an effort to obtain a beneficial or desired result, including a clinical result, in the individual. Beneficial or desired clinical results include, but are not limited to, alleviating or improving one or more signs or symptoms of the disease, reducing the extent of the disease, stabilizing (i.e., not worsening) the disease state, preventing disease spread, delaying or slowing disease progression, improving or alleviating the disease state, and alleviating (whether partial or complete). "Treatment" may also mean extending survival compared to the expected survival of an individual who has not received treatment. In addition, "treating" and "treatment" may occur by administering a dose of one or more therapeutic agents, or may occur when administering a series of doses of one or more therapeutic agents. "Treatment" or "treatment" does not require complete alleviation of signs or symptoms, and does not require cure, and particularly includes regimens that have only a palliative effect on an individual. By "alleviating" a disease or disorder, it is meant that the extent of the disease or disorder and / or undesirable clinical manifestations are lessened and / or the time course of progression of the disease or disorder is slowed, as compared to the expected untreated outcome.

[0041] The term "constitutively active" as used herein with respect to cGAS 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 enzymatic activity. In some preferred embodiments, a "constitutively active cGAS" is a "truncated cGAS," such as cGASΔN, which comprises a C-terminal DNA-binding enzymatic domain in the absence of an N-terminal disordered domain. That is, cGASΔN is a constitutively active cGAS that lacks the regulation of enzymatic activity conferred by the N-terminal disordered domain of full-length cGAS. I. Constitutively active cyclic GMP-AMP synthase

[0042] Cyclic GMP-AMP synthase, also known as cGAMP synthase or cGAS, is an enzyme receptor for cytoplasmic DNA.cGAS recognizes double-stranded DNA, which leads to dimerization, the formation of liquid-like droplets and the production of the second messenger 2'3' cyclic GMP-AMP (cGAMP) regardless of its sequence, and cGAMP binds to and activates STING, leading to the expression of interferon and other inflammatory mediators. The length of human cGAS is 522 amino acids, including an N-terminal phosphoinositide binding domain (residues 1-59) and a C-terminal DNA binding enzymatic domain (residues 160-522) (Barnett et al., Cell, 176: 1432-1446, 2019). Importantly, it was found that the expression of cGASΔN in human leukemia monocytic cell lines resulted in higher expression levels of interferon and interferon-stimulated genes (Barnett, ibid., 2019).

[0043] The amino acid sequence of human cGAS (GenBank No. NP_612450.2) is: (SEQ ID NO: 10).

[0044] The amino acid sequence of the N-terminal domain of cGAS is: MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSA PDTQERPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWDVPSPSGLPVSAPILVRRDAA (SEQ ID NO: 11).

[0045] The amino acid sequence of the C-terminal domain of cGAS (cGASΔN) is: PGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLNTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQN WLSAKVRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF(SEQ IDNO:1).

[0046] The nucleotide sequence encoding human cGASΔN that was codon-optimized for expression in mouse cells is shown in SEQ ID NO: 17. A DNA template having this nucleotide sequence was used to prepare mRNA encoding human cGASΔN, which was loaded 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 catalytic adjuvants for improving adaptive immune responses elicited by mRNA vaccines. In some preferred embodiments, the constitutively active cGAS is a truncated cGAS (cGASΔN) lacking an N-terminal phosphoinositide binding domain. In some preferred embodiments, the constitutively active cGAS is a truncated cGAS (cGASΔN) comprising a C-terminal DNA binding enzymatic domain.

[0048] Homologs of cGAS are expressed in species of the animal kingdom, and the cGAS amino acid sequence is conserved among 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 various non-human primate cGAS proteins is shown in Figure 1 middle. Table I. Identity percentage matrix created by Clustal 12.1^ ^The numbers in this table are not SEQ ID NOs.

[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 cGAS (SEQ ID NO: 1) comprising a 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 operably combined with a start codon (ATG).

[0050] "Percentage (%) of sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the candidate sequence with the reference polypeptide sequence and introducing spaces (if necessary) to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage of amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. For example, the % sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or comprising a certain % sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 multiplied by a fraction X / Y, where X is the number of amino acid residues scored as identical matches by sequence in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that where 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. II. mRNA encoding antigen

[0051] The compositions and methods of the present disclosure may comprise mRNA encoding an antigen, or may otherwise be suitable for use with a formulation comprising mRNA encoding an antigen. In some embodiments, the antigen is a protein antigen. The terms "polypeptide" and "protein" are used interchangeably herein to refer to an antigen comprising a peptide chain of at least 8 amino acids in length. In some embodiments, the length of the antigen is from 8 to 1800 amino acids, 9 to 1000 amino acids, or 10 to 100 amino acids. The polypeptide may be post-translationally modified, such as modified by phosphorylation, hydroxylation, sulfonation, palmitoylation, and / or glycosylation.

[0052] In some embodiments, antigen is a tumor antigen comprising an amino acid sequence of at least one full-length protein or a fragment thereof. In some embodiments, tumor antigens comprise amino acid sequences or fragments thereof from oncoproteins. In some embodiments, mammalian antigens are neoantigens or are encoded by genes comprising mutations relative to genes present in normal cells from mammalian subjects. Neoantigens are considered to be particularly useful in enabling T cells to distinguish between cancer cells and non-cancerous cells (see, for example, Schumacher and Schreiber, Science, 348: 69-74, 2015). In other embodiments, tumor antigens include viral antigens, such as antigens of oncoviruses.

[0053] In some embodiments, the tumor antigen is a fusion protein comprising two or more polypeptides, wherein each polypeptide comprises an amino acid sequence from a different tumor antigen or a non-continuous amino acid sequence from the same tumor antigen. In some of these embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, wherein each polypeptide comprises a non-continuous amino acid sequence 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 protozoan antigen, a fungal antigen, or a combination thereof. In some embodiments, the microbial antigen includes a surface protein or other antigenic subunit of a microorganism.

[0055] In some preferred embodiments, the mRNA comprises 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 comprises one or both of a 5' cap structure and a poly A tail.

[0056] In some embodiments, the mRNA further encodes a ribosomal skipping sequence, such as a 2A-like (2AL) sequence as shown in 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 in which the mRNA comprises two or more coding regions, the coding regions may be separated by a 2AL sequence. For example, the 2AL sequence may be located between the coding region for constitutively active cyclic GMP-AMP synthase (cGAS) and the coding region for an antigen (e.g., cGASΔN-2AL-antigen or antigen-2AL-cGASΔN). Similarly, in some embodiments in which the mRNA comprises coding regions for two or more antigens, the coding regions may be separated by a 2AL sequence (e.g., antigen 1-2AL-antigen 2). Additional 2AL sequences used in the mRNA of the present disclosure are known in the art (see, e.g., Luke et al., J. Gen. Virol, 89: 1036-1042, 2008, Figure 2 The 2AL sequence of ATCC is incorporated herein by reference). III. Lipid-Based Delivery Vehicles

[0057] 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, LNP comprises at least one lipid selected from the following: ionizable lipids, cationic lipids, phospholipids, pegylated lipids, structural lipids and mixtures thereof. In some embodiments, the at least one lipid comprises ionizable lipids. In some embodiments, the at least one lipid comprises cationic lipids. In some embodiments, the at least one lipid comprises phospholipids. In some embodiments, the at least one lipid comprises pegylated lipids. In some embodiments, the at least one lipid comprises structural lipids. In some embodiments, the at least one lipid comprises ionizable lipids, phospholipids, pegylated lipids and structural lipids.

[0059] In some embodiments, the lipid component of RNA-Lipoplex comprises one or more lipids. In some preferred embodiments, the one or more lipids comprise a first lipid and a second lipid, wherein 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 lipid-based mRNA delivery vehicles of the present disclosure are described in Fig. 9A and Fig. 9B The figure is from Hou et al., Nature Review Materials, 6: 1078-1094, 2021 Figure 2 Modified. IV. Lysophosphatidylcholine compounds

[0061] The compositions and methods of the present disclosure may include a phospholipid, preferably lysophosphatidylcholine. "Lysophosphatidylcholine" (LPC) or "lysophosphatidylcholine molecule" refers to a glycerol molecule that carries a phosphorylcholine group on the hydroxyl group of glycerol and an acyl group on one of the other two hydroxyl groups of glycerol. The remaining hydroxyl groups are not substituted.

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

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

[0064] The alkyl or alkenyl chain together with the carbonyl carbon forms an acyl chain that is one carbon atom longer than the alkyl or alkenyl chain. For example, a (C23 alkyl)-C(=O)-group forms a C24 acyl chain. Thus, when the group "(alkyl or alkylene)" is a C12-C23 alkyl (such as a C12-C19 alkyl or a C20-C23 alkyl), the (C12-C23 alkyl)-C(=O)-group forms a C13-C24 acyl chain (such as a C13-C20 acyl chain or a C21-C24 acyl chain). When the group "(alkyl or alkylene)" is a C12-C23 alkenyl (such as a C12-C19 alkenyl or a C20-C23 alkenyl), the (C12-C23 alkenyl)-C(=O)-group forms a C13-C24 acyl chain (such as a C13-C20 acyl chain or a C21-C24 acyl chain). Acyl chains may be referred to as saturated acyl or unsaturated acyl to distinguish between alkyl-containing acyl and alkenyl-containing acyl. 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, wherein 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. V. Pathogen Recognition Receptor Agonists

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

[0067] The term "TLR7 / 8 agonist" as used herein 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 agonist of the present disclosure is suitable for superactivating human dendritic cells in the presence of LPC.

[0068] In some aspects, TLR7 / 8 agonists are small molecules. In some embodiments, TLR7 / 8 agonists are small molecules or their salts with a molecular weight of 900 Daltons or less. That is, small molecule TLR7 / 8 agonists are not macromolecules like recombinant proteins or synthetic oligonucleotides, and the macromolecules can be controlled by the US FDA Biological Products Evaluation and Research Center (USFDA's Center for Biologics Evaluation and Research). On the contrary, small molecule TLR7 / 8 agonists can be controlled by the FDA Drug Evaluation and Research Center (FDA's Center for Drug Evaluation and Research). In some embodiments, the molecular weight of the small molecule is from about 90 to about 900 Daltons. In some embodiments, TLR7 / 8 agonists include imidazoquinoline compounds. In some preferred embodiments, TLR7 / 8 agonists include resiquimod (R848). B. Other PRR agonists

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

[0070] In other aspects, the PRR agonist comprises a NOD-like receptor (NLR) agonist. In further aspects, the PRR agonist comprises a RIG-I-like receptor (RLR) agonist. In additional aspects, the PRR agonist comprises a C-type lectin receptor (CLR) agonist. VI. Pharmaceutical Preparations

[0071] Some compositions of the present disclosure are pharmaceutical formulations comprising pharmaceutically acceptable excipients. The pharmaceutical formulations of the present disclosure may be in the form of solutions or suspensions. Alternatively, the pharmaceutical formulations may be dehydrated solids (e.g., freeze-dried or spray-dried solids). The pharmaceutical formulations of the present disclosure are preferably sterile and are preferably substantially free of endotoxins. The term "pharmaceutical formulation" may be used interchangeably with the terms "pharmaceutical product" and "medicament" herein. In some embodiments, the pharmaceutical formulations include various components in specific ratios based on the intended purpose of the formulation.

[0072] Pharmaceutically acceptable excipients of the present disclosure include, for example, solvents, buffers, tonicity adjusters, fillers, and preservatives (see, for example, Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the pharmaceutical formulation may include an excipient that serves as one or more of a solvent, buffer, tonicity adjuster, and filler (e.g., sodium chloride in saline can serve as both an aqueous vehicle and a tonicity adjuster).

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

[0074] The pharmaceutical formulation may include a buffer. The buffer controls pH to inhibit degradation of the active agent during processing, storage and optionally reconstitution. Suitable buffers include, for example, salts, including acetate, citrate, phosphate or sulfate. Other suitable buffers include, for example, amino acids, such as 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 in the range of 6 to 9. In some embodiments, the pH is greater than (lower limit) 6, 7 or 8. In some embodiments, the pH is less than (upper limit) 9, 8 or 7. That is, the pH is in the range of from about 6 to 9, wherein the lower limit is less than the upper limit.

[0075] The pharmaceutical composition may contain a tonicity adjuster. Suitable tonicity adjusters include, for example, dextrose, glycerol, sodium chloride, glycerol, and mannitol.

[0076] The pharmaceutical formulation may include fillers. Fillers are particularly useful when the pharmaceutical composition is to be lyophilized before administration. In some embodiments, fillers are protective agents that help stabilize the active agent and prevent degradation of the active agent during freezing or spray drying and / or during storage. Suitable fillers are sugars (monosaccharides, disaccharides and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose and raffinose.

[0077] The pharmaceutical formulation may contain a preservative. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in a preferred embodiment, the pharmaceutical formulation is prepared under sterile conditions and stored in a disposable container, and therefore does not need to contain a preservative.

[0078] The pharmaceutical formulations of the present disclosure are suitable for parenteral administration. That is, the pharmaceutical formulations of the present disclosure are not intended for enteral administration (eg, not administered orally, gastrically, or rectally). VII. How to use

[0079] In some aspects, the present disclosure relates to methods of use of any of the compositions or formulations described herein. The methods of use are suitable for a variety of uses involving stimulating immune responses. In some embodiments, the methods of use include methods for treating cancer. In some embodiments, the methods of use include methods for inhibiting abnormal cell proliferation. In some embodiments, the methods of use include methods for treating or preventing infectious diseases. The method includes administering an effective amount of the formulation or composition described herein to an individual in need to achieve a specific outcome. The individual is a mammalian subject, such as 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 relate to clinical use, while in other embodiments, the methods of use relate to preclinical and / or veterinary use. For preclinical use, the mammalian subject can be a non-human primate (e.g., monkey or ape) or a rodent (e.g., mouse or rat). For veterinary use, the mammalian subject can be a farm animal (e.g., cattle), a sports animal (e.g., horse) or a pet (e.g., a companion animal, such as a dog or cat). A. Stimulate immune response

[0080] In short, the present disclosure provides methods for stimulating an immune response of an individual, the method comprising administering a composition or formulation described herein to an individual in an amount sufficient to stimulate an immune response of the individual. "Stimulating" an immune response (which can be used interchangeably with "eliciting" an immune response) means increasing an immune response, which can be produced by inducing a de novo immune response (e.g., as a result of an initial 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 chemoattractant-related gene expression; and stimulating dendritic cell (DC) maturation. Methods for measuring the stimulation of an immune response are known in the art.

[0081] For example, the present disclosure provides a method for inducing an antigen-specific immune response in an individual by administering a composition or formulation as described herein to an individual in an amount sufficient to induce an antigen-specific immune response in the individual. In a preferred embodiment, the composition or formulation comprises an antigen. In some embodiments, the composition or formulation comprising the antigen is administered to an individual's tissue. The immune response may 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 increasing the titer of an antigen-specific antibody to a level higher than a threshold level (such as a baseline titer or serum protection level before administration). "Inducing" an antigen-specific CTL response means increasing the frequency of an antigen-specific CTL found in peripheral blood to a level higher than a baseline frequency before administration. "Inducing" an antigen-specific Th cell response means increasing the frequency of an antigen-specific Th cell found in peripheral blood to a level higher than a baseline frequency before administration.

[0082] The analysis of immune response (both qualitative and quantitative) can be performed by any method known in the art, including but not limited to measuring antigen-specific antibody production (including measuring specific antibody subclasses), activation of specific lymphocytes (such as B cells and helper T cells) populations, production of cytokines (such as IFN-α, IFN-γ, IL-6, IL-12) and / or release of histamine. The method for measuring antigen-specific antibody response includes enzyme-linked immunosorbent assay (ELISA). The activation of specific lymphocyte populations can be measured by proliferation assay and fluorescence activated cell sorting (FACS). The production of cytokines can also be measured by ELISA. In some embodiments, the method of stimulating immune response includes stimulating interleukin-1β (IL-1β) secretion, interferon-γ (IFN-γ) secretion and / or tumor necrosis factor-α (TNF-α) secretion of 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 contacted with a composition of the disclosure remain viable 40-56 hours (or about 48 hours) after contact.

[0083] In some embodiments, the method is suitable for stimulating an anti-tumor immune response. In other embodiments, the method is suitable for stimulating an anti-microbial immune response. In some embodiments, the anti-microbial response is an anti-bacterial immune response. In some embodiments, the anti-microbial response is an anti-fungal immune response. In some embodiments, the anti-microbial response is an anti-viral immune response. In some embodiments, the anti-microbial response is an anti-protozoan immune response. B. Treat or prevent disease

[0084] The present disclosure further provides methods for treating or preventing a disease in an individual, 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 relates to treating cancer in an individual or otherwise treating a mammalian subject with cancer. In some embodiments, the cancer is a blood cancer, such as a lymphoma, leukemia, or myeloma. In other embodiments, the cancer is a non-blood cancer, such as a sarcoma, carcinoma, or melanoma. In some embodiments, the cancer is malignant.

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

[0087] In some embodiments, the method relates to 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 a further embodiment, the infectious disease is caused by a fungal infection. In a still further embodiment, the infectious disease is caused by a protozoan infection. Of particular importance are infectious diseases caused by animal-derived pathogens that infect humans and other animals (such as mammals or birds). In some embodiments, animal-derived pathogens are transmitted to humans via intermediate species (carriers). List of implementation plans 1. A composition comprising an mRNA encapsulated in a lipid nanoparticle (LNP), wherein the mRNA comprises a coding region for a 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 an ionizable lipid, a pegylated lipid, a structural lipid, a second phospholipid, and mixtures thereof, wherein the first phospholipid comprises a lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain. 2. A composition comprising a first mRNA and a second mRNA encapsulated in a lipid nanoparticle (LNP), wherein the first mRNA comprises a coding region for a constitutively active cyclic GMP-AMP synthase (cGAS), and the second mRNA comprises a coding region for an antigen; and the LNP comprises a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a pegylated lipid, a structural lipid, a second phospholipid, and a mixture thereof, wherein the first phospholipid comprises a lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain. 3. A composition comprising mRNA encapsulated in a lipid nanoparticle (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 for a constitutively active cyclic GMP-AMP synthase (cGAS), and the second coding region is a coding region for an antigen; or the first coding region is a coding region for an antigen, and the second coding region is a coding region for a 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 an ionizable lipid, a pegylated lipid, a structural lipid, a second phospholipid, and mixtures thereof, wherein the first phospholipid comprises lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain. 4. A composition according to any one of embodiments 1-3, wherein the at least one lipid comprises an ionizable lipid, a second phospholipid, a PEGylated lipid, and a structural lipid. 5. The composition of any one of embodiments 1-4, wherein the ionizable lipid comprises: i) 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecanyloxy)hexyl]amino]octanoate (SM-102) or an analogue or derivative thereof; and / or ii) 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexane-1-ammonium (ALC-0315) or an analogue or derivative thereof; and / or iii) (6Z,9Z,28Z,31Z)-heptatriacontane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA) or an analogue or derivative thereof. 6. A composition according to any one of embodiments 1-5, wherein the PEGylated lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and combinations thereof. 7. A composition according to any one of embodiments 1-5, wherein the PEGylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoylglycerol [DMG]. 8. A composition according to any one of embodiments 1-7, wherein the structured lipid is selected from the group consisting of: cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and combinations thereof. 9. A composition according to any one of embodiments 1-7, wherein the structured lipid comprises cholesterol. 10. The composition of any one of embodiments 1-9, wherein the second phospholipid comprises: i) a hydrophilic head portion selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin; and ii) one or more fatty acid tail moieties selected from the group consisting of lauric acid, myristic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. 11. The composition of any one of embodiments 1-9, wherein the second phospholipid is selected from: 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-cholesterol succinate monoacyl-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-racemic-(1-glycerol) sodium salt (DOPG), sphingomyelin and Its combination. 12. The composition of embodiment 11, wherein the second phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). 13. The composition of any one of embodiments 1-12, wherein the composition further comprises at least one excipient. 14. The composition of embodiment 13, wherein the excipient comprises sucrose. 15. A composition comprising: i) mRNA complexed with one or more lipids (RNA-Lipoplex); and ii) lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain, wherein the mRNA comprises a coding region for a constitutively active cyclic GMP-AMP synthase (cGAS), and the one or more lipids comprise a first lipid and a second lipid. 16. A composition comprising: i) a first mRNA and a second mRNA complexed with one or more lipids (RNA-Lipoplex); and ii) lysophosphatidylcholine (LPC) with a single C13-C24 acyl chain; wherein the first mRNA comprises a coding region for a constitutively active cyclic GMP-AMP synthase (cGAS), and the second mRNA comprises a coding region for an antigen, and the one or more lipids comprise a first lipid and a second lipid. 17. A composition comprising: i) mRNA complexed with one or more lipids (RNA-Lipoplex); and ii) lysophosphatidylcholine (LPC) with a single C13-C24 acyl chain, 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 for a constitutively active cyclic GMP-AMP synthase (cGAS), and the second coding region is a coding region for an antigen; or the first coding region is a coding region for an antigen, and the second coding region is a coding region for a constitutively active cyclic GMP-AMP synthase (cGAS), and the one or more lipids comprise a first lipid and a second lipid. 18. A composition according to any one of embodiments 15-17, wherein the first lipid is a cationic lipid and the second lipid is a neutral or anionic lipid. 19. The composition of embodiment 18, wherein the cationic lipid comprises one or both of the following: i) 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) or its analogs or derivatives; and ii) 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) or an analogue or derivative thereof. 20. The composition of embodiment 18 or embodiment 19, wherein the neutral or anionic lipid comprises: i) 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or an analogue or derivative thereof; and / or ii) cholesterol or its analogs or derivatives; and / or iii) 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or an analogue or derivative thereof. 21. A composition according to any one of embodiments 1-20, wherein the acyl chain of the LPC is a C21-C24 acyl chain. 22. A composition according to any one of embodiments 1-20, wherein the acyl chain of the LPC is a C22 acyl chain. 23. A composition according to any one of embodiments 1-22, wherein the acyl chain of the LPC is fully saturated. 24. A composition according to embodiment 23, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)]. 25. The composition of any one of embodiments 1-24, further comprising a TLR7 / 8 agonist. 26. A composition according to embodiment 25, wherein the TLR7 / 8 agonist is a small molecule with a molecular weight of 900 Daltons or less. 27. A composition according to embodiment 26, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound. 28. The composition of embodiment 27, wherein the TLR7 / 8 agonist comprises resiquimod (R848). 29. A composition according to any one of embodiments 25-28, wherein the LPC comprises LPC(22:0) and the TLR7 / 8 agonist comprises resiquimod (R848). 30. The composition of any one of embodiments 1-29, 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). 31. A composition according to any one of embodiments 1-30, wherein the mRNA comprises a 5' cap structure. 32. A composition according to any one of embodiments 1-31, wherein the mRNA comprises a poly A tail. 33. A composition according to any one of embodiments 1-32, wherein the mRNA is a nucleoside-modified mRNA. 34. A nucleic acid comprising: i) a coding region for an antigen, and ii) a coding region for a constitutively active cyclic GMP-AMP synthase (cGAS), optionally wherein the nucleic acid is mRNA, optionally wherein the nucleic acid is DNA. 35. An expression vector comprising the nucleic acid according to embodiment 34. 36. A composition according to any one of embodiments 1-35, wherein the constitutively active cGAS has a greater propensity for self-DNA reactivity than its wild-type counterpart. 37. The composition of any one of embodiments 1-36, wherein the cGAS is a truncated cGAS lacking the amino-terminal phosphoinositide binding domain (cGASΔN). 38. The composition of embodiment 37, 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. 39. The composition of embodiment 38, wherein the cGASΔN comprises: (i) an 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. 40. The composition of embodiment 38, wherein the cGASΔN is encoded by the nucleotide sequence of SEQ ID NO:17. 41. The composition of any one of embodiments 37-40, wherein the coding region of cGASΔN is operably combined with a start codon (ATG). 42. The composition of any one of embodiments 1-41, wherein the antigen is a tumor antigen. 43. A composition according to embodiment 42, wherein the tumor antigen is a tumor-associated antigen. 44. The composition of embodiment 42, wherein the tumor antigen is a neoantigen. 45. The composition of any one of embodiments 1-41, wherein the antigen comprises a microbial antigen. 46. ​​The composition of embodiment 44, wherein the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoan antigen, or a fungal antigen. 47. The composition of any one of embodiments 1-41, wherein the antigen comprises a surface protein of a pathogen or a fragment thereof. 48. A composition according to embodiment 47, wherein the pathogen is capable of causing disease in a human subject. 49. The composition of embodiment 47 or embodiment 48, wherein the pathogen is a virus. 50. The composition of embodiment 49, wherein the virus is SARS-CoV-2. 51. A composition according to embodiment 50, wherein the antigen is the spike (S) glycoprotein of SARS-CoV-2, optionally wherein the spike glycoprotein is a pre-fusion stabilized variant. 52. A composition according to any one of embodiments 1-51, wherein the composition does not contain lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA). 53. The composition of any one of embodiments 1-52, wherein the composition does not comprise oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) or species of oxPAPC. 54. The composition of embodiment 53, wherein the composition does not contain 2-[[(2R)-2-[(E)-7-carboxy-5-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropyloxy]-hydroxyphosphoryl]oxyethyl-trimethylazonium (HOdiA-PC), [(2R)-2-[(E)-7-carboxy-5-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropyl]-hydroxyphosphoryl]oxyethyl-trimethylazonium (HOdiA-PC), [(2R)-2-[(E)-7-carboxy-5-oxohept-6-enoyl]oxy-3-hexadecanoyloxypropyl]-2-(trimethylazonium)ethylphosphoryl ester (KOdiA-PC), 1-palmitoyl-2-(5-hydroxy-8-oxo-octenoyl)-sn-glycero-3-phosphorylcholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxooct-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylammonium (KOOA-PC), [(2R)-3-hexadecanoyloxy-2-(5-oxopentanoyloxy)-sn-glycero-3-phosphorylcholine (HOOA-PC), )propyl] 2-(trimethylazonium)ethyl phosphate (POVPC), [(2R)-2-(4-carboxybutyryloxy)-3-hexadecanoyloxypropyl] 2-(trimethylazonium)ethyl phosphate (PGPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-en-1-ylidene]methyl]oxiran-2-yl]butyryloxy]propyl] 2-(trimethylazonia)ethyl phosphate (PECPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiran-2-yl]butanoyloxy]propyl]2-(trimethylazonia)ethyl phosphate (PEIPC) and / or 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PAzePC). 55. A pharmaceutical formulation comprising the composition according to any one of embodiments 1-54 and a pharmaceutically acceptable excipient. 56. A method for producing superactivated dendritic cells, the method comprising contacting dendritic cells with an effective amount of the composition according to any one of embodiments 1-54, any one of embodiments 25-54, or the formulation according to embodiment 55 to produce superactivated dendritic cells, wherein the superactivated dendritic cells secrete IL-1β without undergoing cell death within about 48 hours of exposure. 57. The method of embodiment 56, wherein the dendritic cells: (i) contacting with the composition in vivo; or (ii) contacting with said composition ex vivo. 58. The method of embodiment 56 or embodiment 57, wherein the hyperactivated dendritic cells: (i) secrete higher levels of one or more of IL-6, TNFα, RANTES and IP-10 than unstimulated dendritic cells or dendritic cells contacted with empty LNPs; and / or (ii) expresses higher levels of 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. 59. A pharmaceutical formulation comprising at least 10^3, 10^4, 10^5 or 10^6 hyperactivated dendritic cells produced by the method according to any one of embodiments 56-58 and a pharmaceutically acceptable excipient. 60. A method of stimulating an immune response to an antigen, the method comprising administering to an individual in need thereof an effective amount of the pharmaceutical formulation according to embodiment 55 or embodiment 59 to stimulate an immune response to the antigen. 61. A method of treating cancer, the method comprising administering to an individual in need thereof an effective amount of the pharmaceutical formulation according to Embodiment 55 or Embodiment 59 to treat the cancer. 62. A method of inhibiting abnormal cell proliferation, the method comprising administering to an individual in need thereof an effective amount of the pharmaceutical formulation according to embodiment 55 or embodiment 59 to inhibit abnormal cell proliferation. 63. A method for treating or preventing an infectious disease, the method comprising administering to an individual in need thereof an effective amount of the pharmaceutical formulation according to embodiment 55 to treat or prevent the infectious disease. 64. The method of embodiment 63, wherein the infectious disease is a viral disease. 65. The method of embodiment 63, wherein the infectious disease is a bacterial disease. 66. The method or pharmaceutical formulation of any one of embodiments 56-62, wherein the dendritic cells are mammalian cells. 67. The method or pharmaceutical formulation of embodiment 66, wherein the mammalian cell is a human cell. 68. The method of any one of embodiments 60-66, wherein the individual is a mammal. 69. The method of embodiment 68, wherein the mammal is a human. 70. The method of embodiment 68, wherein the mammal is a dog or a cat. 71. The composition, formulation, or method of any one of embodiments 1-70, wherein the second phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and the at least one lipid comprises 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecanyloxy)hexyl]amino]caprylate (SM-102) or an analog or derivative thereof and cholesterol. 72. The composition, formulation or method of embodiment 71, wherein the at least one lipid further comprises a PEGylated lipid, optionally wherein the PEGylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoylglycerol [DMG]. 73. The composition, formulation or method of any one of embodiments 1-72, wherein 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. 74. The composition, formulation or method of embodiment 73, wherein the LNP has an effective diameter of less than about 200 nanometers. 75. The composition, formulation or method of embodiment 73, wherein the LNP has an effective diameter of less than about 150 nanometers. Example

[0088] Abbreviations: BMDC (bone marrow-derived dendritic cell); CDS (cytoplasmic 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 dimyristoylglycerol [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-oxooct-6-enoyl)-sn-glycero-3-phosphocholine); IFNγ (interferon-γ); IL-1b / IL1-β / IL-1β (interleukin-1β); KOdiA-PC (1-(palmitoyl)-2-(5-keto-6-octen-diacyl)phosphatidylcholine); KOOA-PC (1-palmitoyl-(5-keto-8-oxo-6-octenoyl)-sn-glycero-3-phosphocholine); LNP (lipid nanoparticle); LPC / Lyso PC (lysophosphatidylcholine); Lyso PC(22:0) (1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine); LPS (lipopolysaccharide); MC3 ((6Z,9Z,28Z,31Z)-triacontriacontane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate, also known as DLin-MC3-DMA); mcg or μg (microgram); moDC (monocyte-derived dendritic cell); MPLA (monophosphoryl lipid A); NLR (NOD-like receptor); OVA (ovalbumin); oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero sn-glycero-3-phosphocholine); PAMPs (pathogen-associated molecular patterns); PBMCs (peripheral blood mononuclear cells); PGPCs (1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine); POVPCs (1-palmitoyl-2-(5'-oxo-pentanoyl)-sn-glycero-3-phosphocholine); PRRs (pathogen recognition receptors); RLRs (RIG-I-like receptors); R848 (resiquimod); SC (subcutaneous); STING (stimulator of interferon genes); TNFα (tumor necrosis factor-α); and TLRs (Toll-like receptors).

[0089] Although for the purpose of clarity and understanding, the disclosure has been described in detail by way of illustration and example, it will be clear to those skilled in the art that certain changes and modifications may be implemented. Therefore, the following examples should not be construed as limiting the scope of the disclosure, which is defined by the appended claims. Example 1: Combination of Lysophosphatidylcholine (LPC) with a Single Acyl Chain and TLR7 / 8 Agonist Superactivates Mammalian Peripheral Blood Mononuclear Cells

[0090] This example describes the hyperactivation of canine and human peripheral blood mononuclear cells (PBMCs) using a combination of lipid DAMPs and small molecule PAMPs. Materials and methods

[0091] PBMCs were isolated from whole blood. PBMCs were isolated from whole blood using density gradient centrifugation with Ficoll-Paque PLUS (Cytivia). Whole blood was diluted with PBS at 1:1, layered on the top of Ficoll-Paque PLUS, and centrifuged at room temperature for 30 minutes at 1000 x g. 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 plated 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 plated at 1 x 10 5 (canine cells) or 1x 10 6 (Human cells) were plated in 96-well flat-bottom tissue culture plates. Lyophilized Vaccigrade R848 (Invivogen) was reconstituted and diluted according to the manufacturer's recommendations and added to the cells at a final concentration of 1 μg / mL. Next, 22:0LYSO PC was added to the cells at a final concentration of 82.5 μM. Additional innate agonists were diluted in R10 medium according to the manufacturer's recommendations and added to the 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; aluminum adjuvant hydroxide (Invivogen) was added at a final concentration of 30 μg / mL. The cells were incubated at 37°C, 5% CO2 for two days. The cell cultures were then used for endpoint analysis.

[0093] Endpoint analysis. After culturing PBMC with PAMPs and DAMPs for two days, supernatants and cell samples were collected for analysis. Cells in the culture were pelleted by centrifugation at 400 x g for 5 minutes. Half of the culture volume in the collection wells was used for ELISA or Lumit TM Cytokine quantification was performed by bioluminescence 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 by human PBMCs was assessed using one of the following kits: ELISA MAXDeluxe Set Human IL-1β Kit (Biolegend), Invitrogen Human IL-1β Kit, or Lumit TM Human IL-1β immunoassay (Promega). IFNγ secretion from human PBMCs was assessed using ELISA MAX Deluxe Set Human IFNγ (Biolegend), and TNFα secretion from human PBMCs was assessed using the Human TNFα Uncoated ELISA Kit (Invitrogen). ELISAs were 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 diluted two-fold to 7.8 pg / mL; and iii) sample incubation was done overnight at 4°C on an orbital shaker. Lumit TM Assay. IL-1β secretion by canine PBMCs was assessed 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) overnight incubation of samples was done at 4°C on an orbital shaker. For all ELISAs, absorbance was measured at 450 nm with calibration at 570 nm using a Spectramax M5e plate reader (Molecular Devices). For Lumit TM For determination, luminescence at all wavelengths was measured 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 on GraphPad Prism 9 (GraphPad Software). The interpolated results for the samples were then adjusted for any dilution of the supernatant.

[0095] Quantification of cell viability.Cell viability was assessed by quantifying the presence of ATP as an indicator of metabolically active cells using CellTiter-Glo luminescent cell viability assay (Promega).Metabolic activity was assessed following the manufacturer's instructions.CellTiter-Glo reagent was mixed with cell pellets and fresh culture medium and then transferred to a white opaque 96-well plate.Luminescence at all wavelengths was measured on a Spectramax M5e plate reader (Molecular Devices) using an integration time of 500ms.The viability percentage relative to the control condition of the PBMCs treated with R848 was calculated.

[0096] Statistical analysis.For each condition, cells from each donor were plated in triplicate for testing.For cytokine quantification, triplicate replicates were used for interpolation, and data were plotted as total concentration (pg / mL) or the change multiple of each donor relative to the control condition of only R848.For vigor quantification, triplicates were repeated to average each donor, and average value was used as a donor measurement value.Multiple donors were tested, and each data point on the bar graph represents the value of the donor.In order to test the difference of test conditions, the test results were compared with the control condition of only R848. P values ​​were calculated using mixed effects one-way ANOVA, and multiple comparisons were corrected using Dunnett's test. Results - Hyperactivated canine PBMCs treated with 22:0LYSO PC and R848

[0097] The combination of 22:0LYSO PC (DAMP) and TLR7 / 8 agonist R848 (PAMP) was found to have potent superstimulatory activity in human moDC. To assess whether this superstimulatory activity translates to other clinically relevant species, the ability of 22:0LYSO PC+R848 to superactivate PBMCs isolated from canine whole blood was evaluated. For each data set, PBMCs from multiple donors were used instead of moDCs due to the lack of canine-specific reagents that can be used to induce true canine moDCs. Briefly, PBMCs were isolated from whole blood using density gradient centrifugation and then cultured for two days with the superactivating stimuli of interest.

[0098] After two days of culture, superactivation was assessed by quantifying IL-1β in the cell culture supernatant and measuring cell viability. When treated with 22:0LYSO PC and R848 together, canine PBMCs secreted comparable or higher levels of IL-1β compared to each other stimulus tested, both in terms of concentration per mL and in terms of the fold change per donor relative to R848 alone. Consistent with previous studies showing that monocytes accounting for 5%-10% of PBMCs can release IL-1β in response to activation with R848, IL-1β secretion levels of canine PBMCs increased in the case of R848 alone compared to untreated cells. As expected, the pyroptosis combination of LPS+aluminum adjuvant triggered high levels of IL-1β. It is worth noting that, although PGPC+R848 triggered similar levels of IL-1β compared to R848 alone, neither GM-CSF nor 2'3'cGAMP induced significant IL-1β secretion of canine PBMCs compared to untreated cells.

[0099] Although IL-1β could be detected in the cell culture supernatant one day after superactivation of canine PBMCs, cell viability was evaluated two days after superactivation to ensure sustained viability following IL-1β secretion. 22:0LYSO+R848 did not significantly reduce relative cell viability. Interestingly, while the combination of PGPC and R848 proved slightly toxic to canine PBMCs, it was not observed to be toxic to human moDCs or human PBMCs. Together, these data demonstrate that 22:0LYSO+R848 elicits high levels of IL-1β secretion from canine PBMCs, indicative of superactivation. Results - Treatment of Superactivated Human PBMCs with 22:0LYSO PC and R848

[0100] Superactivation experiments were also performed with PBMCs isolated from whole blood obtained from human donors. Briefly, PBMCs were isolated from whole blood from multiple human donors by density gradient centrifugation and cultured for two days with the superactivating stimuli of interest.

[0101] Like human moDC and canine PBMC, human PBMC secrete IL-1β at higher or comparable levels than all other stimuli tested. Similar to canine PBMC, human PBMC secrete IL-1β in response to R848 alone due to monocyte activation, and this is increased by the addition of 22:0LYSO PC. As expected, the pyroptosis combination of LPS+aluminum adjuvant elicits high levels of IL-1β. Consistent with the observations in canine PBMC, PGPC+R848 did not induce significantly higher levels of IL-1β than R848 alone. GM-CSF did not induce human PBMC to secrete IL-1β at levels significantly higher than the background level produced by untreated cells.

[0102] The viability of human PBMCs was also assessed two days after super activation to ensure the lasting viability of human PBMCs after IL-1β secretion. No significant reduction in human PBMC viability was observed after treatment with any stimulant. Together, these data demonstrate that both human and canine PBMCs are super activated by 22:0LYSO PC+R848. Interestingly, canine PBMCs are super activated to a greater extent by 22:0LYSO PC+R848 than by PGPC+R848.

[0103] Because activated human PBMC can secrete other cytokines besides IL-1β, the secretion of proinflammatory cytokines IFNγ and TNFα in cell culture supernatants was measured two days after superactivation. Compared with all other stimuli tested, the combination of 22:0LYSO PC+R848 induced the highest change fold for each donor in both IFNγ secretion and TNFα secretion relative to only R848. It is worth noting that although LPS+aluminum adjuvant induces high levels of IL-1β secretion in human PBMC, this stimulant combination does not induce a multiple increase in IFNγ or TNFα secretion. In addition, relative to only R848, neither GM-CSF nor 2'3'cGAMP induces a substantial multiple change in IFNγ secretion. These data show that the combination of 22:0LYSO PC+R848 is superior in inducing human PBMC to secrete proinflammatory cytokines IFNγ and TNFα. Example 2: Both cGASΔN mRNA and 22:0LPC can be loaded into lipid nanoparticles (LNPs) Materials and Methods

[0104] Materials. Lipids for LNP were purchased from Cayman Chemicals (SM102) or Avanti (22:0LPC, DSPC, DMG-PEG2000). Cholesterol was purchased from Sigma. cGASΔN mRNA was custom made and synthesized from linearized template DNA (Trilink) via in vitro transcription. The human cGASΔN sequence was codon optimized for expression in mouse cells, and the synthesized nucleotide sequence is shown in SEQ ID NO: 17. Using Trilink's proprietary Clean Cap mRNA technology, the mRNA sequence was capped with N1-methyl pseudouridine base modification and a 120-residue poly A tail. The sequence contained a Bbsl restriction enzyme site. The sequence information was used to construct a plasmid from which the mRNA was synthesized synthetically. The mRNA was phosphatase treated after synthesis.

[0105] LNP synthesis. Lipid nanoparticles (LNPs) were prepared using a custom LNP lipid mixture (Table 2-1). cGASΔN mRNA was prepared at 0.02 mg / mL in sodium citrate buffer at pH 4. A custom LNP lipid mixture was prepared at 12.5 mM. The ratio of mRNA / 22:0LPC was selected based on experiments that previously determined the in vitro activity of each component. LNPs were synthesized using a NanoAssemblr Ignite instrument (Precision Nanosystems). Lipids in ethanol were combined with mRNA solutions in a 1:3 volume ratio, using a flow rate of 12 mL / min. LNPs were washed in 10 volumes of phosphate buffered saline (PBS) at pH 7.4 to remove residual ethanol, and then concentrated using an Amicon 10K MWCO centrifugal filter. LNPs were filtered through a 0.2 μm filter before use. Table 2-1. LNP formulations 22:0LPC LNP cGASΔN LNP 22:0LPC+cGASΔN LNP Lipid mixture SM102 40％ 40％ 40％ DSPC 0％ 35％ 0％ cholesterol 28.5％ 28.5％ 28.5％ DMG-PEG2000 1.5％ 1.5％ 1.5％ 22:0LPC 35％ 0％ 35％ mRNA N / A cGASΔN cGASΔN

[0106] LNP characterization. Following the manufacturer's protocol, mRNA loading in LNPs was quantified using the RiboGreen assay (ThermoFisher). Samples were diluted to fall within the range of the standard curve. LNPs were lysed using Triton X-100 to assess mRNA encapsulation in LNPs. Total mRNA and encapsulated mRNA were quantified. The size of LNPs was assessed using dynamic light scattering (DLS) on a NanoBrook Omni (Brookhaven). LNPs were diluted 1:10 in PBS prior to running on DLS. Three 90 second measurements were recorded for each sample. result

[0107] The LNPs can be loaded with cGASΔN mRNA and 22:0 LPC at similar levels as LNPs loaded with cGASΔN mRNA or 22:0 LPC. All LNPs (i.e., 22:0 LPC LNPs, cGASΔN LNPs, and cGASΔN+22:0 LPC LNPs) showed similar sizes, with all LNP sizes <150 nm ( Figure 3A ) with a relatively uniform distribution (PDI < 0.3). cGASΔN LNPs and cGASΔN+22:0LPC-loaded LNPs showed similar mRNA loading levels, but the overall loading efficiency (actual / theoretical loading) was slightly reduced when 22:0LPC was also added to the LNPs ( Figure 3B ). For 22:0LPC, 22:0LPC LNP and cGASΔN+22:0LPC LNP showed similar loading levels and efficiencies (actual / theoretical) ( Figure 3C ). Example 3: Mouse dendritic cells achieve a unique activation state by treatment with mRNA encoding cGASΔN and hyperactivating stimuli Materials and Methods

[0108] Generation of mouse bone marrow derived dendritic cells (BMDC). The leg femur and tibia were removed from the mouse, chopped with scissors and flushed into a sterile tube. 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 culture medium (I10) consisting of complete IMDM containing 10% FBS, penicillin and streptomycin, as well as L-glutamine and sodium pyruvate supplements. The cells were then plated in a P12 plate with 8E6 bone marrow cells per well. Recombinant mouse FLT3L (Miltenyi) was added to the culture at 200 ng / mL. Subsequent determinations were performed using differentiated cells on the 9th day. The efficiency of differentiation was monitored by flow cytometry using BD SymphonyA3, and CD11c + MHC-II + Cells were routinely above 80% of viable cells. For each experiment, 5 to 15 mice were used to generate DCs from the bone marrow.

[0109] Superactivation of murine BMDC and cGASΔN delivery. BMDC were harvested on day 9 after differentiation, washed with PBS and cultured at 2×10 5 The cells were plated at a concentration of 10 cells / well in complete IMDM medium (I10). Cells were cultured in the presence or absence of LNPs loaded with 100 μμM 22:0LPC LNPs and loaded with or without approximately 1 μμg / mL of cGASΔN mRNA (Table 3-1). Under certain conditions, R848 was added at a final concentration of 1 μg / mL. 48 hours after stimulation, supernatants were collected for cytokine secretion assessment. IL-1β cytokine secretion of BMDCs was measured using a sandwich ELISA (Invitrogen) following the manufacturer's instructions. Additional cytokines were measured using the LEGENDplex mouse antiviral response plate (Biolegend) according to the manufacturer's instructions. Cell viability was assessed by measuring LDH released into fresh supernatant using the CyQUANT LDH cytotoxicity assay (Invitrogen) according to the manufacturer's instructions. Table 3-1. In vitro administration of mouse BMDC 22:0LPC LNP cGASΔN LNP 22:0LPC+cGASΔN LNP 22:0 LPC dose 100μM N / A 100μM mRNA dosage N / A 0.91 μg / mL 0.91 μg / mL result

[0110] In previous work, DC hyperactivation using R848 and 22:0 LPC was found to activate the transcription factor NF-kB, leading to the production of inflammatory cytokines such as IL-6 and TNFα. In addition, the NLRP3 inflammasome was activated, leading to the production of IL-1β. Unlike these cellular responses, cGAS activation and the resulting STING signaling activates the transcription factor IRF3, which co-regulates some genes with NF-kB but also regulates a unique set of genes independently of NF-kB. cGASΔN is a modified cGAS that is constitutively active. Using LNPs formulated with cGASΔN mRNA with and without 22:0 LPC, it was hypothesized that cGAS signaling and hyperactivation could induce a complementary set of inflammatory signals.

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

[0112] Flt3L BMDCs treated with R848 and [cGASΔN+22:0 LPC] LNPs can be superactivated. The potential of DCs to be superactivated in response to treatment with R848+[cGASΔN+22:0 LPC] LNPs was assessed; superactivation was expressed as the ability of DCs to secrete IL-1β while remaining viable. DCs treated with PBS (unstimulated) or with R848 alone produced little or no IL-1β. As expected, combining R848 and 22:0 LPC treatment resulted in IL-1β production after 48 hours ( Figure 4A Treatment of cells with LNPs containing cGASΔN mRNA did not induce IL-1β. Combination of cGASΔN treatment with 22:0 LPC also did not result in IL-1β release ( Figure 4A cGAS / STING signaling may not be sufficient to generate the pro-form of IL-1β protein required for 22:0LPC-mediated mature IL-1β release. However, combining all three stimuli (R848, cGASΔN, and 22:0LPC) resulted in IL-1β release ( Figure 4A ). This result demonstrates that cGASΔN treatment does not inhibit hyperactivation. Next, cell viability was assessed using an LDH release assay to confirm that the cells were hyperactivated and not pyroptotic. In the treatment conditions tested, DCs had a similar viability profile to unstimulated cells, confirming that DCs were hyperactivated ( Figure 4BTogether, these data suggest that DCs can be simultaneously treated with R848, 22:0LPC, and cGASΔN mRNA and are hyperactivated.

[0113] [cGASΔN and 22:0LPC] LNP+R848 treatment activates NF-kB signaling and the cGAS-STING pathway in Flt3L BMDCs. To further confirm the activity of the individual stimuli, IL-6 and TNFα production were quantified. These two genes are primarily regulated by NF-kB and are therefore regulated by R848 stimulation. When R848 was added as a treatment, IL-6 and TNFα were produced ( Figure 5A-Figure 5B ). Addition of LNPs containing 22:0 LPC to R848 treatment increased the secretion of both IL-6 and TNFα. cGASΔN alone did not stimulate IL-6 and TNFα secretion ( Figure 5A-Figure 5B cGAS / STING signaling primarily activates IRF3 rather than NF-kB. In addition, secretion of RANTES and IP-10, 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 expression of RANTES and IP-10, confirming that our mRNA transcripts encode active cGAS that was successfully delivered to cells and expressed. Figure 5C-5D ). Unexpectedly, when cGASΔN was delivered together with 22:0LPC, RANTES and IP-10 expression was suppressed compared to single cGASΔN treatment. When cells were stimulated with R848 alone or in combination with 22:0LPC in hyperactivation, cells produced RANTES and IP-10, and no 22:0LPC-mediated suppression was observed. Although unexpected suppression was observed when cGASΔN was combined with 22:0LPC, combining all three resulted in expression of RANTES and IP-10 ( Figure 5C-5D ).

[0114] In summary, treatment of murine DCs with various combinations of R848, 22:0 LPC, and cGASΔN demonstrated several things. First, cGASΔN mRNA was successfully formulated in LNPs for delivery into DCs and translated into protein. The cGASΔN protein was active, as indicated 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 the key hyperactivating cytokine IL-1β. 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 suggest that cGASΔN treatment can be combined with hyperactivating stimuli to direct DCs to produce an inflammatory environment for two processes simultaneously. This combination results in a unique DC activation state that can have potential advantages in immune responses. Example 4: Treatment of human dendritic cells with cGASΔN and hyperactivating stimuli leads to activation of the NLRP3 inflammasome and cGAS-STING pathway Materials and Methods

[0115] Human monocyte-derived dendritic cells (moDC) are produced. Human monocytes are isolated from Leukopaks purchased from Miltenyi Inc. (San Jose, California) using the StraightFromLeukopak CD14 microbead kit according to the manufacturer's instructions. The monocytes are then aliquoted and frozen in fetal bovine serum containing 10% dimethyl sulfoxide. For studies using monocyte-derived dendritic cells (moDC) cultures, monocytes are thawed and cultured in RPMI culture medium (R10 culture medium) containing 10% FBS, 50 units / mL penicillin, 50mg / mL streptomycin, 2mM L-glutamine, 1mM sodium pyruvate, 50mMβ-mercaptoethanol, 10mM HEPES and Gibco MEM non-essential amino acids. In order to differentiate monocytes into moDC, recombinant human GM-CSF (50ng / mL) and IL-4 (25ng / mL) are added to R10 culture medium. Cells were cultured with GM-CSF and IL-4 for 6 days, with additional cell feeding on day 3 with R10 medium containing GM-CSF and IL-4. On day 6 of differentiation, moDCs were collected for use, and a portion was stained to confirm successful differentiation. Routinely, >80% of live cells were CD11c+CD209+ by flow cytometry using BD Symphony A3.

[0116] Superactivation and cGASΔN delivery of moDC. Six days after differentiation from monocytes, moDC were collected and counted. Cells were plated in 96-well plates at 100,000 cells / well. moDC was treated with 1ug / mL R848 and LNPs containing 22:0LPC and / or cGASΔNmRNA. 48 hours after stimulation, cell culture supernatants were collected. Cytokine expression was measured using LEGENDplex human antiviral response plates (Biolegend) according to the manufacturer's instructions. Cell viability was assessed by measuring LDH released into fresh supernatant using CyQUANT LDH cytotoxicity assay (Invitrogen) according to the manufacturer's instructions. Cells were collected and stained to measure the expression of DC molecules (such as CD40, CD80, CD83, CD86, CCR7, HLA-ABC and HLA-DR) involved in T cell activation, which were evaluated by flow cytometry using BD Symphony A3. result

[0117] It was hypothesized that combining cGASΔN-mediated STING signaling with hyperactivation could help create immunotherapies and vaccines that could provide better protection than currently available options. Therefore, the combination of cGASΔN therapy with hyperactivation of human DCs was tested. As a source of DCs, human monocytes were differentiated into DCs by culturing for six days in R10 medium containing recombinant GM-CSF and IL-4. The cells were confirmed to be DCs by CD11c and CD209 staining on day 6, and the cells were used for treatment with the following three stimuli of interest: R848, 22:0LPC, and cGASΔN.

[0118] moDCs treated with R848 and [cGASΔN+22:0LPC] LNPs were hyperactivated and allowed cGAS-STING pathway activation. We first began by confirming the main conclusions obtained using murine DCs. The combination of these three treatments (R848, 22:0LPC, and cGASΔN) had a similar effect on human DC viability compared to DCs treated with PBS (unstimulated). Fig. 6A When IL-1β release was measured, it was observed that very little IL-1β was produced by treatment with PBS (no stimulation) or R848 ( Figure 6B As expected, the combination of R848 and 22:0LPC delivered via LNP induced IL-1β secretion. Similar to mice, cGASΔN treatment alone or in combination with 22:0LPC did not induce IL-1β secretion ( Figure 6B When cGASΔN was combined with the hyperactivating combination of R848 and 22:0LPC, IL-1β secretion was maintained or increased ( Figure 6B). IP-10 secretion was quantified to ensure that cGASΔN mRNA formulated in LNPs could induce signaling. Only cGASΔN treatment induced IP-10 expression. In contrast to the mouse data, addition of 22:0 LPC to cGASΔN LNPs did not inhibit IP-10 expression ( Figure 6C ). In addition, in contrast to the murine data, R848 treatment (alone or in combination with 22:0LPC) was insufficient to induce IP-10 expression ( Figure 6C When all three stimuli were combined, cGASΔN, R848, and 22:0LPC were able to induce IP-10 expression ( Figure 6C ). For the donors shown, a reduction in IP-10 was observed when all three stimuli were combined compared to cGASΔN treatment alone. However, this reduction was not consistently observed in the 4 donors tested. Regardless of this reduction, the combination of cGASΔN, R848, and 22:0LPC resulted in significantly higher IP-10 expression than that detected under unstimulated conditions ( Figure 6C Together, these results suggest that, like in murine DC, human DC are able to mediate cGAS / STING activation and are also hyperactivated.

[0119] moDCs treated with R848 and [cGASΔN+22:0LPC] LNPs upregulate surface proteins required for T cell activation. In addition to producing a specific cytokine environment, DCs also need to express cell surface proteins that are important in T cell activation. One such protein is the chemokine receptor CCR7 required for DC trafficking from peripheral tissues to lymph nodes, where they can prime T cells. DCs were stained for CCR7 expression after treatment with various combinations of R848, 22:0LPC, and cGASΔN. As previously observed, treatment with R848 alone increased CCR7 expression, and superactivation treatment with a combination of R848 and 22:0LPC resulted in a further increase in CCR7 ( Fig. 7A ). cGASΔN treatment also increased CCR7 expression compared to unstimulated cells, and addition of 22:0LPC had no significant effect on CCR7 expression ( Fig. 7A Interestingly, combining cGASΔN treatment with R848 and 22:0LPC resulted in the greatest changes in CCR7 expression ( Fig. 7A Importantly, combining cGASΔN with hyperactivation did not inhibit CCR7 expression, and increased CCR7 expression may enable DCs to migrate to lymph nodes more efficiently.

[0120] We continued to examine other key cell surface proteins on dendritic cells that are important for their function. CD40 is a receptor that engages CD40L on CD4 T cells and ultimately enhances T cell responses. CD83 is also involved in T cell activation and is often used as an indicator of DC activation. CD40 expression was upregulated when DCs were treated with R848 with or without 22:0LPC compared to unstimulated cells, and further enhancement was observed when cells were treated with cGASΔN with or without 22:0LPC (Figure 2—figure supplement 1A). Figure 7B Combining cGAS signaling with hyperactivation also resulted in an increase in CD40 expression compared to unstimulated cells ( Figure 7B Compared to CD40 staining, CD83 expression was more variable, but combining cGASΔN treatment with hyperactivation treatment still did not abolish CD83 expression ( Figure 7C ).

[0121] CD80 and CD86 are co-stimulatory molecules that engage T cells during antigen presentation and serve as confirmation signals that the presented non-self antigen is indeed dangerous. When DCs were activated with R848 or cGASΔN or when DCs were superactivated with R848 and 22:0LPC, CD80 and CD86 expression increased ( Figure 8A-B When cGASΔN and hyperactivation treatments were combined, DCs continued to upregulate CD80 and CD86 compared to unstimulated cells ( Figure 8A-B ). Thus, while executing two cellular processes, DCs are still able to supply co-stimulatory signals to T cells.

[0122] Finally, DCs were stained for HLA-ABC and HLA-DR, which are class I and class II MHC molecules that present antigens to CD8 and CD4 T cells, respectively. R848 treatment with or without the addition of 22:0LPC increased the expression of HLA-ABC compared to unstimulated cells ( Figure 8C Treatment of DCs with cGASΔN with or without 22:0LPC also had a similar effect on HLA-ABC expression ( Figure 8C Surprisingly, when cGASΔN treatment and hyperactivation were combined, a significant increase in HLA-ABC was observed compared to the other stimulatory combinations. When HLA-DR staining was analyzed, it was observed that R848 treatment increased HLA-DR expression compared to unstimulated cells ( Fig.8D When superactivated with a combination of R848 and 22:0LPC, further increases were observed ( Fig.8D cGASΔN treatment was more effective than R848 treatment alone or as part of hyperactivation ( Fig.8D). Addition of 22:0LPC to cGASΔN treatment further increased HLA-DR expression compared to cGASΔN treatment alone ( Fig.8D Finally, combining cGASΔN treatment with hyperactivation resulted in the highest HLA-DR expression of all experimental conditions ( Fig.8D ). Combining all three stimuli results in the highest expression of MHC class I and class II molecules, which may enable more efficient presentation of antigens to T cells.

[0123] Studies using human DCs have shown that human DCs generally have similar responses to cGASΔN, R848, and 22:0LPC compared to mouse DCs. An unexpected difference is when cells are treated with cGASΔN with or without 22:0LPC. When 22:0LPC was added, mouse DCs showed IP-10 inhibition, while human DCs did not have the same results. More importantly, the combination of cGASΔN, R848, and 22:0LPC did not prevent human DCs from being superactivated and producing cytokines (such as IL-1β and IP-10). Further analysis of human DCs for cell surface expression of key proteins involved in T cell activation showed that the combination of three stimuli did not inhibit expression. These proteins are still inducible, and in some cases such as HLA-ABC and HLA-DR, expression is further increased compared to cGASΔN treatment or superactivation alone. Taken together, these data collected from human DC treatments suggest that cGASΔN treatment can be combined with hyperactivation to induce a cellular state in which both processes complement each other and could potentially have additive effects on cellular functions for the induction of adaptive immune responses. Example 5: Combining cGASΔN mRNA and hyperactivation as a vaccination strategy to improve immune response

[0124] By utilizing mRNA encoding cGASΔN, DCs can be activated to produce chemical signals that promote T cell effector responses. Hyperactivation of DCs produces a complementary set of chemical signals. In particular, hyperactivation induces proinflammatory cytokines and adds IL-1β, a key cytokine for memory T cell formation, to their cytokine repertoire. By combining stimuli to engage cGAS / STING signaling and hyperactivation, a broader set of inflammatory signals can be generated to maximize the immune response triggered by DCs.

[0125] To test this model, vaccination strategies using various combinations of cGASΔN, R848 and 22:0LPC as adjuvants were compared in vivo. The goal of vaccination is to guide an immune response to a complex source of a target antigen or target antigen (e.g., model antigens, tumor-associated antigens, new antigens, microbial-derived antigens, etc.). The immune response is easily detected using recognized immunological methods and common reagents (e.g., tetramer staining, ELISpot assay, ELISA, flow cytometry, etc.). An immunogenic composition is prepared, for example, by loading an mRNA transcript encoding the target antigen into a lipid nanoparticle (LNP). These LNPs are suitable for administration to mammalian subjects to achieve in vivo expression of the target antigen for inducing 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 to model antigens. It is believed that cGASΔN, R848 and 22:0LPC can improve DC function by enhancing de novo T cell activation and memory T cell reactivation. These improvements can have positive downstream effects on effector and memory T cell responses. When B cells undergo germinal center reactions that require T cell engagement, improved T cell activation can also have further positive downstream effects on B cells. Table 5-1. Immunization strategies method

[0127] In vivo immunization was performed with [cGASΔN+22:0LPC]LNPs with mRNA antigen added to LNPs. C57BL / 6J mice were immunized subcutaneously with the LNPs detailed in Table 5-1. In combination with LNPs containing adjuvant cGASΔN mRNA or non-auxiliary GFP mRNA, LNPs loaded with OVA mRNA were used to deliver transcripts for in vivo production of OVA antigens. The OVA mRNA dose was fixed at 5μg / mouse, while the adjuvant cGASΔN mRNA was administered at approximately 1.5μg / mouse, and 22:0LPC was administered at approximately 60μg / mouse (Table 5-1). Mice were given a primary immunization on day 0 and a booster immunization of the same dose on day 7. To evaluate short-term effector responses, blood and secondary lymphoid organs were collected on day 14. To evaluate long-term memory responses, blood and secondary lymphoid organs were collected on day 40. Blood was collected for measuring antibody and T cell responses. Serum was collected from the blood using serum separator tubes, while blood for cell analysis was collected using K2EDTA tubes. After blood collection, mice were euthanized, and draining lymph nodes and spleens were collected and processed into single cell suspensions. The expected responses are shown in Table 5-1.

[0128] OVA-specific T cell tetramer evaluation. The OVA-specific T cells in the blood and draining lymph nodes of mice receiving OVA LNP immunity were evaluated 14 and 40 days after the primary immunization. The CD8+T cells with specificity for SIINFEKL (SEQ ID NO: 17) (MHC-I restricted T cell epitope of ovalbumin (OVA)) were quantified in blood and dLN using SIINFEKL-tetramer staining. In brief, red blood cells were lysed using RBC lysis buffer, and two lysis were completed to remove all RBCs in the blood. The cells were washed and then stained for vitality (Live / Dead), SIINFEKL-tetramer binding (MBL) and CD3, CD4 and CD8 expression. The cells were fixed with 4% paraformaldehyde, and counting beads were added before running to allow the total cell count to be determined. Data were collected using BD FACS Symphony and analyzed using Flowjo (BD).

[0129] Effector and memory T cell measurements. The frequencies of T effector cells and T memory cells were assessed by flow cytometry in the blood and draining lymph nodes of mice immunized with OVALNPs. Briefly, cell suspensions were stained with CD3, CD4, CD8, CD62L, and CD44 antibodies to measure T effector cells (CD44 低 CD62L - ) and T memory cells (CD44 高 CD62L + ) and the data were analyzed using Flowjo (BD).

[0130] OVA-specific antibody evaluation. The OVA-specific antibodies in the serum of mice immunized with OVALNP were evaluated 14 and 40 days after the primary immunization. OVA-specific total IgG, IgG1 and IgG2b were evaluated by ELISA. In brief, the ELISA plate was coated with 10 μg / mL of Endofit ovalbumin (Invivogen) overnight, then washed and blocked with 2% bovine serum albumin. The plate was washed again, and then the serum was added to the plate at a dilution of 1:500, followed by a total of 7 serum dilutions at a dilution of 1:5. The sample was washed and then incubated with a detection antibody specific for IgG, IgG1 or IgG2b conjugated to HRP (Southern Biotech) to detect total, Th2 dominant and Th1 dominant OVA-specific antibodies, respectively. The plate was washed, then incubated with TMB, and the stop solution was added immediately after the color development was completed.

[0131] OVA specific T cell response. Determine OVA specific T cell response from secondary lymphoid organ activity. After immunization, draining lymph nodes and spleen were collected from mice at early (14th day) and late (40th day) time points. The harvested lymph nodes and spleen were dissociated into single cell suspensions, which were used for ELISPOT determination. ELISPOT was used to detect IFNγ and IL-5 secretion of T cells, which indicated Th1 and Th2 responses, respectively. Cells from draining lymph nodes and spleen were plated in RPMI medium (R10 medium) containing 10% FBS, 50 units / mL penicillin, 50mg / mL streptomycin, 2mM L-glutamine, 1mM sodium pyruvate, 50mMβ-mercaptoethanol, 10mM HEPES and Gibco MEM non-essential amino acids. Cells were plated at 200,000 cells / well in 96-well ELISPOT plates and restimulated with 10mcg / mL OVA peptivator or 1mcg / mL OVA peptide antigen. As controls, additional plated cells were either left unstimulated or stimulated with an irrelevant antigen (not used for vaccination). Completion of the assay produced spots that could be visually quantified where T cells secreted cytokines as a means of quantifying the number of T cells that responded to restimulation. result

[0132] The above assays were used to evaluate short-term (2 weeks after vaccination) and long-term (>4 weeks after vaccination) adaptive T cell immunity, analyzing effector responses and memory responses. Table 5-1 lists the experimental groups to be included in the study. As a negative control, mice in Group 1 received a sham injection without antigen and adjuvant to serve as a baseline, in which little antigen-specific immune response was expected to be induced. Group 2 mice received mRNA encoding a model antigen (OVA) formulated in LNPs, which represents a standard LNP vaccination regimen. The remaining groups all received LNPs loaded with mRNA encoding OVA in combination with adjuvants. Depending on the type of stimulus administered, one or more of the following is expected: i) Activation of cells (inducing NFkB signaling); ii) Super-activation of DCs (inducing NLRP3 pathway activation); and iii) Induction of IRF3 signaling in DCs. For example, Group 4 mice received R848+22:0LPC, which is expected to super-activate DCs, thereby activating the NLRP3 pathway in addition to the NFkB pathway, leading to conventional pro-inflammatory cytokine secretion (IL-6 and TNFα) and IL-1β production. Group 5 mice received LNPs encoding cGASΔN, which is expected to induce a type I IFN response via IRF3. Group 6 and 7 mice received two of the three stimuli, while Group 8 mice received all three stimuli (R848, 22:0LPC, and cGASΔN).

[0133] It is believed that cGASΔN expression in vivo can stimulate DC signals that are particularly beneficial to immediate effector immune responses. It is also believed that hyperactivation conditions can benefit effector responses, even in the absence of type I IFN signaling. In addition, the IL-1β signaling induced by hyperactivation leads to improved memory formation. Therefore, by combining cGAS signaling and hyperactivation, it is believed that very strong effector responses and lasting memory responses can be triggered. By immunizing with sham treatment (group 1), it may be observed that there is almost no effector and memory response, and it is considered that immunizing with antigen mRNA alone or in combination with R848 (groups 2 and 3) will lead to smaller effector responses and minimal memory formation. Group 4 processes hyperactivated DC, and it is expected to be an improvement relative to group 2 processing. In contrast, group 5 and group 6 processes only participate in cGAS signaling in DC, and are therefore believed to cause strong effector responses, but memory responses are not as persistent as group 4 processing. Group 7 is a hyperactivation treatment condition. In view of the fact that group 7 and group 8 participate in both cGAS and hyperactivation pathways, it is believed that they will produce the strongest effector and memory responses. Because hyperactivating lipids are involved in cellular processes beyond NLRP3 activation, it is thought that Group 8 treatments including 22:0LPC would result in superior effector and memory responses.

[0134] The three auxiliary stimulants can be formulated in various ways. For example, cGASΔN mRNA can be encapsulated in LNP alone or in combination with 22:0LPC. R848 can be used as a separate component, in LNP or in LNP containing one or two other stimulants. In addition, the temporal order of applying the stimulant can be changed. For example, the following may be beneficial: in the primary immunization injection, the LNP loaded with antigen mRNA is combined with the LNP loaded with cGASΔN mRNA and R848 to provide a strong effector signal via type I IFN, followed by a booster injection of the LNP loaded with antigen mRNA and the LNP loaded with 22:0LPC and R848 to provide the memory signal required for the generation of lasting immunity.

[0135] Studying the response of T cells and B cells to vaccination generated in vivo data that are believed to demonstrate that combining hyperactivation with cGAS signaling will result in improved antigen-specific immune responses. Evaluation of how the administration of various DC stimulants affects B cell responses was accomplished by measuring total antigen-specific IgG antibodies in the sera of immunized mice, as well as antibody isotypes such as IgG1 (associated with TH2 responses) and IgG2b (associated with TH1 responses). Based on previous studies using cGASΔN LNPs, mice immunized with LNPs loaded with mRNA encoding antigen and mRNA encoding cGASΔN were expected to produce a TH1-dominant response. sequence SEQ ID NO:1 >Human PGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLNTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO:2 >Rhesus monkey PGARKLRAVLEKLRLSHQDISKAAKVVNGVVGHLLRRLKCEPEFGGVEQLHTGSYYEHVKISAPNEFDVMFKLQVPRIQLEEYSDTGAYYFVKFKRNPEGNPLSLFLEDEILSASKMLSKFRKIIKEEINNIKDVQPDADVIMKRKRPGSPAVTLLINKEISVDITLALELKTSWPASTQEGLAIKNWLSAKVRQQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILSNHGKSKTCCENKEMKCCRKDCLKLMKYLLEQLKEKFKDKKHLDKFSSYHVKTAFFHLCTENPQDSQWDPKDLGLCFDNCVTYFLECLRTEQLKNYFIPGFNLFSSNLIDKTSKEFLSKQIEYERNNEFPVFGEF SEQ ID NO:3 >East African baboon PGARKLRAVLEKLRLSHQDISKAAKVVNGVVGHLLHRLKCESEFEGVEQLHTGSYYEHVKISAPNEFDVMFKLQVPRIQLEEYSDTGAYYFVKFKRNPKGNPLSQFLEDEILSASKMLSQFRKIIKEEINNIKDTDVIMKRKRRGSPAVTLLINKEISVDITLALELKTSWPASTQEGLAIKNWLSAKVRQQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILSNHGKSKTCCENKEMKCCRKDCLKLMKYLLEQLKEKFKDKKHLDKFSSYHVKTAFFHLCTENPQDSQWDPKDLGLCFDNCVTHFLQCLRTEKLANYFIPGFNLFSSNLIDKSSKEFLSKQIEYERNNEFPVFGEF SEQ ID NO:4 > Northern white - cheeked gibbon PGARKLRAVLEKLKLSRQEISEAAEVVNGVVDHLLRRLQKCDSEFRGVGLLRTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTGAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINNIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESNSNSWPASTQEGLPIKDWLSGKVRRQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKTKTCCENKNVKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTENPQDSQWDPKNLGFCFDKCVIYFLKCLRTEQLGNYFIPGFNLFSSNLIDKRSKEFLSKQIEYERNNEYPVFG SEQ ID NO:5 > White - handed gibbon PGARKLPAVLEKLKLSRQ EICEAAEVVNGVVDHLLRRLQKCDSEFRGVGLLRTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTGAYYFVKFKRNPKENPLCQFLEGEILSASKMLSKFRKIIKEEINNIKDTDVILKRKRGGSPAVTLLISEKISVDITLALESKSSSWPASTQEGLPIKDWLSGKVRTQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGETKTCCENKKVKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTENPKDSQWDPKDLGLCFDKCVIYFLQCLSTEQLGNYFIPGFNLFSSNLIDKRSKEFLSKQIEYERNNEYPVFGKC SEQ ID NO:6 > Sumatran orangutan PGASKLRAVLEKLKLSRLEISKAAEVVNRVVDHLLRRLQKYNSEFRGVGLLRTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTGAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINNIKDTDVIMKRKRGGSPAVTLLISEEISVDITLALESKSSWPASTQEGLPIKNWLSAKVRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCEDKEVKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDPKDLGLCFDNCVTYFLQCLRTEQLGNYFIPGFNLFSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO:7 > Chimpanzee PGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLNTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINNIKDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLPIKNWLSAKVRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCEIKEAKCCRKDCLKLMKYLLEQLKEKFKDKKHLDKFSSYHVKTAFFHVCTENPQDSQWDRKDLGLCFDNCVTYFLQCLRIEKLENYFIPEFNLFSSDLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO:8 > Gorilla PGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLNTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINNIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRRQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEAKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKQLGLCFDNCVTYFLQCLRTERLENYFIPEFNLFSRNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO:9 > CGASΔN_ consensus sequence PGAXKLXAVLEKLXLSXXXIXXAAXXVXXVVXHLLXRLXXXXXXFXGVXXLXTGSYYEHVKISAPNEFDVMFKLXVPRIQLEEYSXTXAYYFVKFKRNPXXNPLXXFLEXEILSASKMLSXFRKIIKEEINXIKXXXXXXDVIXKRKRXGSPAVTLLIXXXISVDITLALEXXXXSWPASTQEGLXIXXWLSXKVRXQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILXNHGXXKTCCEXKXXKCCRKDCLKLMKYLLEQLKEXFKDKKHLDKFSSYHVKTAFFHXCTXNPXDSQWDXKXLGXCFDXCVTXFLXCLXXEXLXNYFIPXFNLFSXXLIDKXSKEFLXKQIEYERNNEXPVFXXX SEQ ID NO:10 >Full - length human CGAS MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQERPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWDVPSPGLPVSAPILVRRDAAPGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLNTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO:11 >Human CGAS N - terminus MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQERPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWDVPSPGLPVSAPILVRRDAA SEQ ID NO:12 >T2A ribosomal skipping peptide of β-tetrasomal virus of the genus Acanthocephalus [GSG]EGRGSLLTCGDVEENPGP SEQ ID NO:13 >Porcine Teschovirus-1P2A ribosomal jumping peptide [GSG]ATNFSLLKQAGDVEENPGP SEQ ID NO:14 >Horse nasitis virus E2A ribosomal jumping peptide [GSG]QCTNYALLKLAGDVESNPGP SEQ ID NO:15 >Foot-and-mouth disease virus F2A ribosomal jumping peptide [GSG]VKQTLNFDLLKLAGDVESNPGP SEQ ID NO:16 >Ovalbumin peptide SIINFEKL SEQ ID NO:17 >Synthesis of CGASΔN

Claims

1. A composition comprising an mRNA encapsulated in a lipid nanoparticle (LNP), wherein the mRNA comprises a coding region for a 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 an ionizable lipid, a pegylated lipid, a structural lipid, a second phospholipid, and mixtures thereof, wherein the first phospholipid comprises a lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain.

2. A composition comprising a first mRNA and a second mRNA encapsulated in a lipid nanoparticle (LNP), wherein the first mRNA comprises a coding region for a constitutively active cyclic GMP-AMP synthase (cGAS), and the second mRNA comprises a coding region for an antigen; and the LNP comprises a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a pegylated lipid, a structural lipid, a second phospholipid, and a mixture thereof, wherein the first phospholipid comprises a lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain.

3. A composition comprising mRNA encapsulated in a lipid nanoparticle (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 for a constitutively active cyclic GMP-AMP synthase (cGAS), and the second coding region is a coding region for an antigen; or the first coding region is a coding region for an antigen, and the second coding region is a coding region for a 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 an ionizable lipid, a pegylated lipid, a structural lipid, a second phospholipid, and mixtures thereof, wherein the first phospholipid comprises lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain.

4. The composition of any one of claims 1-3, wherein the at least one lipid comprises an ionizable lipid, a second phospholipid, a PEGylated lipid, and a structural lipid.

5. The composition of any one of claims 1-4, wherein the ionizable lipid comprises: i) 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecanyloxy)hexyl]amino]octanoate (SM-102) or an analogue or derivative thereof; and / or ii) 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexane-1-ammonium (ALC-0315) or an analogue or derivative thereof; and / or iii) (6Z,9Z,28Z,31Z)-heptatriacontane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA) or an analogue or derivative thereof.

6. The composition according to any one of claims 1-5, wherein the PEGylated lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and combinations thereof.

7. The composition of any one of claims 1-5, wherein the PEGylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoylglycerol [DMG].

8. The composition according to any one of claims 1-7, wherein the structured lipid is selected from the group consisting of cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and combinations thereof.

9. The composition of any one of claims 1-7, wherein the structured lipid comprises cholesterol.

10. The composition of any one of claims 1-9, wherein the second phospholipid comprises: i) a hydrophilic head portion selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin; and ii) one or more fatty acid tail moieties selected from the group consisting of lauric acid, myristic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

11. The composition of any one of claims 1-9, wherein the second phospholipid is selected from: 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-cholesterol succinate monoacyl-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-racemic-(1-glycerol) sodium salt (DOPG), Sphingomyelin and Its combination.

12. The composition of claim 11, wherein the second phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

13. The composition according to any one of claims 1-12, wherein the composition further comprises at least one excipient.

14. The composition of claim 13, wherein the excipient comprises sucrose.

15. A composition comprising: i) mRNA complexed with one or more lipids (RNA-Lipoplex); and ii) lysophosphatidylcholine (LPC) with a single C13-C24 acyl chain, wherein the mRNA comprises a coding region for a constitutively active cyclic GMP-AMP synthase (cGAS), and the one or more lipids comprise a first lipid and a second lipid.

16. A composition comprising: i) a first mRNA and a second mRNA complexed with one or more lipids (RNA-Lipoplex); and ii) lysophosphatidylcholine (LPC) with a single C13-C24 acyl chain; wherein the first mRNA comprises a coding region for a constitutively active cyclic GMP-AMP synthase (cGAS), and the second mRNA comprises a coding region for an antigen, and the one or more lipids comprise a first lipid and a second lipid.

17. A composition comprising: i) mRNA complexed with one or more lipids (RNA-Lipoplex); and ii) lysophosphatidylcholine (LPC) with a single C13-C24 acyl chain, 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 for a constitutively active cyclic GMP-AMP synthase (cGAS), and the second coding region is a coding region for an antigen; or the first coding region is a coding region for an antigen, and the second coding region is a coding region for a constitutively active cyclic GMP-AMP synthase (cGAS), and the one or more lipids comprise a first lipid and a second lipid.

18. The composition of any one of claims 15-17, wherein the first lipid is a cationic lipid and the second lipid is a neutral or anionic lipid.

19. The composition of claim 18, wherein the cationic lipid comprises one or both of: i) 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) or its analogs or derivatives; and ii) 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) or an analogue or derivative thereof.

20. A composition according to claim 18 or claim 19, wherein the neutral or anionic lipid comprises: i) 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or an analogue or derivative thereof; and / or ii) cholesterol or its analogs or derivatives; and / or iii) 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or an analogue or derivative thereof.

21. The composition of any one of claims 1-20, wherein the acyl chain of the LPC is a C21-C24 acyl chain.

22. The composition of any one of claims 1-20, wherein the acyl chain of the LPC is a C22 acyl chain.

23. The composition of any one of claims 1-22, wherein the acyl chains of the LPC are fully saturated.

24. The composition of claim 23, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

25. The composition of any one of claims 1-24, further comprising a TLR7 / 8 agonist.

26. The composition of claim 25, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less.

27. The composition of claim 26, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

28. The composition of claim 27, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

29. The composition of any one of claims 25-28, wherein the LPC comprises LPC(22:0), and the TLR7 / 8 agonist comprises resiquimod (R848).

30. The composition of any one of claims 1-29, 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).

31. The composition of any one of claims 1-30, wherein the mRNA comprises a 5' cap structure.

32. The composition of any one of claims 1-31, wherein the mRNA comprises a poly A tail.

33. The composition of any one of claims 1-32, wherein the mRNA is a nucleoside-modified mRNA.

34. A nucleic acid comprising: i) a coding region for an antigen, and ii) a coding region for a constitutively active cyclic GMP-AMP synthase (cGAS), optionally wherein the nucleic acid is mRNA, optionally wherein the nucleic acid is DNA.

35. An expression vector comprising the nucleic acid according to claim 34.

36. The composition of any one of claims 1-35, wherein the constitutively active cGAS has a greater propensity for self-DNA reactivity than its wild-type counterpart.

37. The composition of any one of claims 1-36, wherein the cGAS is a truncated cGAS lacking the amino-terminal phosphoinositide binding domain (cGASΔN).

38. The composition of claim 37, 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.

39. The composition of claim 38, wherein the cGASΔN comprises: (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.

40. The composition of claim 38, wherein the cGASΔN is encoded by the nucleotide sequence of SEQ ID NO:

17.

41. The composition of any one of claims 37-40, wherein the coding region of cGASΔN is operably combined with a start codon (ATG).

42. The composition of any one of claims 1-41, wherein the antigen is a tumor antigen.

43. The composition of claim 42, wherein the tumor antigen is a tumor-associated antigen.

44. The composition of claim 42, wherein the tumor antigen is a neoantigen.

45. The composition of any one of claims 1-41, wherein the antigen comprises a microbial antigen.

46. ​​The composition of claim 44, wherein the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoan antigen, or a fungal antigen.

47. The composition of any one of claims 1-41, wherein the antigen comprises a surface protein of a pathogen or a fragment thereof.

48. The composition of claim 47, wherein the pathogen is capable of causing disease in a human subject.

49. A composition according to claim 47 or claim 48, wherein the pathogen is a virus.

50. The composition of claim 49, wherein the virus is SARS-CoV-2.

51. The composition of claim 50, wherein the antigen is the spike (S) glycoprotein of SARS-CoV-2, optionally wherein the spike glycoprotein is a pre-fusion stabilized variant.

52. The composition of any one of claims 1-51, wherein the composition does not comprise lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA).

53. The composition of any one of claims 1-52, wherein the composition does not comprise oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) or species of oxPAPC.

54. The composition of claim 53, wherein the composition does not comprise 2-[[(2R)-2-[(E)-7-carboxy-5-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropyloxy]-hydroxyphosphoryl]oxyethyl-trimethylazonium (HOdiA-PC), [(2R)-2-[(E)-7-carboxy-5-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropyl]-hydroxyphosphoryl]oxyethyl-trimethylazonium (HOdiA-PC), [(2R)-2-[(E)-7-carboxy-5-oxohept-6-enoyl]oxy-3-hexadecanoyloxypropyl]-2-(trimethylazonium)ethylphosphoryl]oxyethyl-trimethylazonium ester (KOdiA-PC), 1-palmitoyl-2-(5-hydroxy-8-oxo-octenoyl)-sn-glycero-3-phosphorylcholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxooct-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylammonium (KOOA-PC), [(2R)-3-hexadecanoyloxy-2-(5-oxopentanoyloxy)-sn-glycero-3-phosphorylcholine (HOOA-PC), )propyl] 2-(trimethylazonium)ethyl phosphate (POVPC), [(2R)-2-(4-carboxybutyryloxy)-3-hexadecanoyloxypropyl] 2-(trimethylazonium)ethyl phosphate (PGPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-en-1-ylidene]methyl]oxiran-2-yl]butyryloxy]propyl] 2-(trimethylazonia)ethyl phosphate (PECPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiran-2-yl]butanoyloxy]propyl]2-(trimethylazonia)ethyl phosphate (PEIPC) and / or 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PAzePC).

55. A pharmaceutical formulation comprising the composition of any one of claims 1-54 and a pharmaceutically acceptable excipient.

56. A method for producing superactivated dendritic cells, the method comprising contacting dendritic cells with an effective amount of a composition according to any one of claims 1-54, any one of claims 25-54, or a formulation according to claim 55 to produce superactivated dendritic cells, wherein the superactivated dendritic cells secrete IL-1β without undergoing cell death within about 48 hours of exposure.

57. The method of claim 56, wherein the dendritic cells are: (i) contacting with the composition in vivo; or (ii) contacting with said composition ex vivo.

58. The method of claim 56 or claim 57, wherein the hyperactivated dendritic cells: (i) secrete higher levels of one or more of IL-6, TNFα, RANTES and IP-10 than unstimulated dendritic cells or dendritic cells contacted with empty LNPs; and / or (ii) expresses higher levels of 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.

59. A pharmaceutical formulation comprising at least 10^3, 10^4, 10^5 or 10^6 hyperactivated dendritic cells produced by the method according to any one of claims 56-58 and a pharmaceutically acceptable excipient.

60. A method of stimulating an immune response to an antigen, the method comprising administering to an individual in need thereof an effective amount of a pharmaceutical formulation according to claim 55 or claim 59 to stimulate an immune response to the antigen.

61. A method of treating cancer, the method comprising administering to an individual in need thereof an effective amount of the pharmaceutical formulation of claim 55 or claim 59 to treat the cancer.

62. A method of inhibiting abnormal cell proliferation, the method comprising administering to an individual in need thereof an effective amount of the pharmaceutical formulation according to claim 55 or claim 59 to inhibit abnormal cell proliferation.

63. A method for treating or preventing an infectious disease, the method comprising administering to an individual in need thereof an effective amount of the pharmaceutical formulation according to claim 55 to treat or prevent the infectious disease.

64. The method of claim 63, wherein the infectious disease is a viral disease.

65. The method of claim 63, wherein the infectious disease is a bacterial disease.

66. The method or pharmaceutical formulation of any one of claims 56-62, wherein the dendritic cells are mammalian cells.

67. The method or pharmaceutical formulation of claim 66, wherein the mammalian cell is a human cell.

68. The method of any one of claims 60-66, wherein the individual is a mammal.

69. The method of claim 68, wherein the mammal is a human.

70. The method of claim 68, wherein the mammal is a dog or a cat.

71. The composition, formulation or method of any one of claims 1-70, wherein the second phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and the at least one lipid comprises 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate (SM-102) or an analog or derivative thereof and cholesterol.

72. The composition, formulation or method of claim 71, wherein the at least one lipid further comprises a PEGylated lipid, optionally wherein the PEGylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoylglycerol [DMG].

73. The composition, formulation or method of any one of claims 1-72, wherein 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.

74. The composition, formulation or method of claim 73, wherein the LNP has an effective diameter of less than about 200 nanometers.

75. The composition, formulation or method of claim 73, wherein the LNP has an effective diameter of less than about 150 nanometers.

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