MRNA encoding constitutively active cyclic GMP-AMP synthase and lipid delivery vehicle 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 pathogen-recognizing receptor agonists.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 341,987, filed May 13, 2022, which is incorporated herein by reference in its entirety. Reference to an electronic sequence listing

[0002] The contents of the electronic Sequence Listing (165532000540SEQLIST.xml; size: 22,326 bytes; and creation date: May 10, 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 pathogen recognition receptor agonists. 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 pathogen recognition receptor agonists. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 is an alignment of the amino acid sequences of primate cGASΔN proteins: the primates include humans (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 (Sumatranorangutan, Pongo abelii, SEQ ID NO: 6); chimpanzee (Pan troglodytes, SEQ ID NO: 7); western gorilla (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 Depicted using GenVoy ILM TM Characterization of lipid nanoparticles (LNPs) produced from ionizable lipid mixtures (Precision NanoSystems). Figure 3A It was shown that the diameters of LNPs loaded with OVA, GFP or cGASΔN mRNA were all less than 200 nm. Figure 3B It was shown that the sizes of LNPs loaded with OVA, GFP or cGASΔN mRNA were all relatively uniform and all exhibited a polydispersity index of less than 0.3. Figure 3C It was shown that LNPs can be loaded with OVA, GFP or cGASΔN mRNA with similar efficiency levels.

[0010] Figure 4A-4C Murine dendritic cells (DCs) are shown to secrete inflammatory cytokines stimulated by the cGAS-STING pathway in response to treatment with LNPs loaded with mRNA encoding cGASΔN (cGASΔN LNPs). Figure 4A It was shown that bone marrow-derived dendritic cells (BMDCs) stimulated with cGASΔNLNPs produced significantly higher levels of RANTES than cells treated with empty LNPs or LNPs loaded only with mRNA encoding a model antigen. Figure 4B It was shown that treatment of BMDCs with cGASΔN LNPs resulted in significantly higher levels of IP-10 secretion than treatment with empty LNPs or LNPs loaded only with mRNA encoding a model antigen. Figure 4C It is shown that BMDCs treated with cGASΔN LNPs produced significantly higher levels of IFNα than cells treated with empty LNPs or LNPs loaded with mRNA encoding the model antigen alone. The x-axis labels for all three graphs are as follows Figure 4C As shown. p<0.05*, p<0.01**, p<0.001***, p<0.0001****. Statistics were performed using one-way ANOVA with Tukey post hoc test for multiple comparisons. Data are representative of two experiments.

[0011] Figure 5A-5F The results show that treatment with LNPs loaded with mRNA encoding cGASΔN (cGASΔNLNPs) activated murine DCs, resulting in increased expression of multiple cell surface markers. DCs treated with cGASΔN LNPs expressed significantly higher levels of the co-stimulatory molecule CD40 ( Figure 5A )、CD86( Figure 5B ) and CD69( Figure 5C ), as indicated by an increase in mean fluorescence intensity (MFI). It was also found that DCs expressed significantly higher levels of the antigen presenting molecule MHC-II ( MHC-II ) when treated with cGASΔN LNPs compared to treatment with empty LNPs or LNPs loaded with mRNA encoding a model antigen alone. Figure 5D ) and H2kB(MHC-I)( Figure 5EIn addition, it was found that DCs had higher levels of expression of the CCR7 migration molecule when treated with cGASΔN LNPs compared to treatment with empty LNPs or LNPs loaded only with mRNA encoding a model antigen ( Fig. 5F ). The x-axis labels for all six plots are as follows Figure 5E and Fig. 5F As shown. p<0.05*, p<0.01**, p<0.001***, p<0.0001****. Statistics were performed using one-way ANOVA with Tukey post hoc test for multiple comparisons. Data are representative of two experiments.

[0012] Figure 6A-6C It was shown that LNPs prepared from different lipid formulations and loaded with different mRNAs exhibited similar characteristics. Fig. 6A Figure 2 shows the expression of GenVoy ILM when loaded with OVA, GFP or cGASΔN mRNA. TM The diameter of LNPs prepared from ionizable lipid mixtures (Precision NanoSystems) or custom lipid mixtures was less than 150 nm. Figure 6B Shown using GenVoy ILM TM The sizes of LNPs prepared from either the monolayer or customized lipid mixtures were relatively uniform, with a polydispersity index of less than 0.3. Figure 6C Shown using GenVoy ILM TM LNPs prepared with 1% or customized lipid mixtures allowed efficient encapsulation of OVA, GFP, and cGASΔN mRNA. Data are representative of three experiments.

[0013] Figure 7A-7F LNPs containing cGASΔN mRNA are shown to activate the cGAS-STING inflammatory pathway in human monocyte-derived dendritic cells (moDCs). Human moDCs were cultured with LNPs loaded with OVA, GFP, cGASΔN mRNA, or a combination of LNPs for 24 h, after which supernatants were collected and cytokine secretion was assessed. Human moDCs treated with LNPs loaded with cGASΔN mRNA produced significantly more IL-6 ( Fig. 7A ), more TNFα( Figure 7B ) and significantly more IP-10 ( Figure 7C Furthermore, moDCs secreted significantly more IFNλ1 when treated with LNPs loaded with cGASΔN mRNA compared to moDCs treated with LNPs loaded with mRNA encoding the model antigen alone ( Fig.7D ), significantly more IFNβ ( Fig. 7E ) and more IFNα2( Figure 7F ). The x-axis labels for all six plots are as follows Fig. 7E and Figure 7F =As shown. p<0.05*, p<0.01**, p<0.0001****. Statistics were performed using two-way ANOVA with Tukey post hoc test for multiple comparisons. Data are representative of two experiments. Circles show control conditions without LNPs. Squares show conditions including the use of GenVoy ILM TM The diamonds show conditions that included LNPs formed using a custom lipid mixture. The white squares and white diamonds show conditions that included LNPs formed using GenVoy ILM TM Conditions for LNPs containing cGASΔN mRNA formed with customized lipid mixtures.

[0014] Figure 8A-8D The results show that treatment with LNPs containing cGASΔN mRNA activated human moDCs. Human moDCs were cultured with LNPs loaded with OVA, GFP, cGASΔN mRNA, or a combination of LNPs for 24 h, after which cell surface marker expression was measured. Compared with treatment with LNPs loaded with mRNA encoding a model antigen alone, CD83 ( Fig. 8A ) expression was significantly increased. Similarly, when moDCs were treated with LNPs loaded with cGASΔN mRNA (both total population and single cell levels), the T cell co-stimulatory molecule CD40 ( Figure 8B ) and CD80( Figure 8C ) expression was significantly increased. In addition, when moDCs were treated with LNPs containing cGASΔN mRNA, compared with treatment with LNPs loaded with mRNA encoding the model antigen alone, MHC class II (HLA-DR) ( Fig.8D ) expression. p<0.05*, p<0.01**. Statistics were performed using two-way ANOVA with Tukey post hoc test for multiple comparisons. Data are representative of two experiments. Circles show control conditions without LNPs. Squares show conditions including the use of GenVoy ILM TM The diamonds show conditions that included LNPs formed using a custom lipid mixture. The white squares and white diamonds show conditions that included LNPs formed using GenVoy ILM TM Conditions for LNPs containing cGASΔN mRNA formed with customized lipid mixtures.

[0015] Figure 9A-9BIt is shown that LNPs containing cGASΔN mRNA activated inflammatory pathways in a human leukemic monocytic cell line (THP-1 cells). Briefly, THP-1 cells were cultured for 24 h with LNPs or a combination of LNPs loaded with OVA, GFP, cGASΔN mRNA, after which the supernatant was collected for measurement of cytokine secretion, and cells were collected for evaluation of cell surface molecule expression. THP-1 cells treated with LNPs containing cGASΔN mRNA secreted significantly more IP-10 ( Fig.9A ). CD40 expression on THP-1 cells was increased after treatment with LNPs containing cGASΔN mRNA compared to LNPs loaded only with mRNA encoding the model antigen ( Fig. 9B ). p<0.01**, p<0.0001****. Statistics were performed using two-way ANOVA with Tukey post hoc test for multiple comparisons. Data are representative of two experiments. Circles show control conditions without LNPs. Squares show conditions including the use of GenVoy ILM TM The diamonds show conditions that included LNPs formed using a custom lipid mixture. The white squares and white diamonds show conditions that included LNPs formed using GenVoy ILM TM Conditions for LNPs containing cGASΔN mRNA formed with customized lipid mixtures.

[0016] Figure 10A-10B It is shown that treatment of moDCs with R848 in combination with LNPs loaded with mRNA encoding cGASΔN resulted in hyperactivation, defined as the ability of DCs to secrete IL-1β while maintaining viability. moDCs were either left unstimulated (PBS-treated) or treated with LNPs loaded with mRNA encoding cGASΔN or OVA in the presence of medium, R848, or R848 and MCC950. Fig. 10A It was shown that all treatment conditions favored viability, indicating that cells did not undergo pyroptosis in response to any of the treatment conditions. Fig. 10B It is shown that only treatment with LNPs loaded with mRNA encoding cGASΔN stimulated IL-1β secretion from moDCs when delivered in combination with R848. Treatment of moDCs with the selective NLRP3 inflammasome inhibitor MCC950 abolished IL-1β production induced by treatment with LNPs loaded with cGASΔN mRNA in combination with R848. p<0.0001****. Statistics were performed using two-way ANOVA with Tukey post hoc test for multiple comparisons.

[0017] Figures 11A-11DIt is shown that the administration of LNPs loaded with cGASΔN mRNA increases the response of T cells to model antigens encoded by mRNA delivered via LNPs. Mice were immunized twice (primary and booster regimen) with LNPs loaded with 5mcg of mRNA encoding ovalbumin (OVA), which were delivered in combination with LNPs loaded with 1mcg or 5mcg of cGASΔN mRNA or loaded with 1mcg or 5mcg of green fluorescent protein (GFP) mRNA. Blood was collected 7 days after the boost, and the binding of "SIINFEKL-tetramer" to T cells was subsequently evaluated. SIINFEKL is the amino acid sequence of a T cell epitope within OVA (SEQ ID NO: 17). In the use of GenVoy ILM TM In the LNPs prepared by the formulation, treatment of mice with a combination of LNPs loaded with cGASΔN mRNA and LNPs loaded with mRNA encoding OVA significantly increased the frequency of SIINFEKL-specific CD8+ T cells in the blood compared to treatment with LNPs loaded with mRNA encoding OVA and GFP or mRNA encoding GFP and cGASΔN ( Fig.11A ) and absolute number ( Fig. 11C Similarly, in LNPs prepared using a custom lipid mixture, treatment of mice with a combination of LNPs loaded with cGASΔN mRNA and LNPs loaded with mRNA encoding OVA significantly increased the frequency of SIINFEKL-specific CD8+ T cells in the blood compared with treatment with LNPs loaded with mRNA encoding OVA and GFP ( Fig. 11B ) and absolute number ( Fig.11D ) both. p<0.01**, p<0.001***. Two-way ANOVA with Tukey post hoc test for multiple comparisons was performed on GenVoy ILM TM Statistics for Fig. 1. Statistics for custom lipid mixtures were done using unpaired t-test.

[0018] Figures 12A-12F Treatment with LNPs loaded with cGASΔN mRNA was shown to reduce Th2-specific antibodies without affecting total IgG responses. Mice were immunized twice with LNPs loaded with 5mcg of OVA mRNA delivered together with LNPs loaded with 1mcg or 5mcg of cGASΔN mRNA or LNPs loaded with 1mcg or 5mcg of GFP mRNA. Blood was collected 7 days after boost and serum was separated to quantify antibody responses. TM In the LNP prepared by the formulation ( Fig. 12A ) and in LNPs prepared using custom lipid mixtures ( Fig. 12B), mice treated with LNPs loaded with OVA and cGASΔN mRNA showed no significant changes in OVA-specific total IgG compared to mice treated with LNPs loaded with OVA mRNA and GFP mRNA. Fig. 12C As shown in the figure, using the GenVoy ILM TM The presence of cGASΔN mRNA in the formulated LNPs significantly reduced OVA-specific IgG1 (p<0.05 for LNPs containing cGASΔN mRNA vs. LNPs containing GFP mRNA at 1 and 5 mcg for 1:500 and 1:2500 dilutions). Fig.12D As shown in , in mice treated with LNPs containing cGASΔN mRNA prepared using a custom lipid mixture, OVA-specific IgG1 responses were reduced (p<0.05 for LNPs containing cGASΔN mRNA compared to LNPs containing GFP mRNA at 5mcg for 1:500, 1:2500 and 1:12500 dilutions). In contrast, when LNPs were prepared with GenVoy (E) and custom lipid mixture (F) formulations, mice treated with LNPs containing OVA mRNA and cGASΔN LNPs did not show significant changes in measured OVA-specific IgG2a compared to OVA + GFP LNP treatment. The legend at the top applies to all panels. Statistics were completed using two-way ANOVA with Tukey post hoc tests for multiple comparisons.

[0019] Fig.13A Shown are the structures of cationic lipids and ionizable lipids suitable for use in lipid-based mRNA delivery vehicles of the present disclosure. Fig. 13B 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

[0020] 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 pathogen recognition receptor agonists. In some embodiments, the compositions of the present disclosure do not include lysophosphatidylcholine (LPC) with a single C13-C24 acyl chain.

[0021] 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.

[0022] 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

[0023] 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.

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

[0025] 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.

[0026] 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).

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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 the host cells in which the protein was produced.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] "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).

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

[0037] "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).

[0038] "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.

[0039] 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.

[0040] 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.

[0041] 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.

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

[0043] "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.

[0044] The term "treating" or "treatment" disease refers to the implementation of such a scheme, which 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 transmission, delaying or slowing disease progression, improving or alleviating the disease state, and alleviating (whether partial or complete). "Treatment" may also mean extending the survival period compared to the expected survival period of an individual who has not received treatment. In addition, "treating" and "treatment" may occur by administering one or more therapeutic agents of a dose, 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 schemes that only have 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.

[0045] 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

[0046] Cyclic GMP-AMP synthase, also known as cGAMP synthase or cGAS, is an enzyme receptor for cytoplasmic DNA. cGAS recognizes double-stranded DNA, which, regardless of its sequence, leads to dimerization, the formation of liquid-like droplets, and the production of the second messenger 2'3' cyclic GMP-AMP (cGAMP), which binds to and activates STING, leading to the expression of interferon and other inflammatory mediators. Specifically, STING activation leads to the signal transduction cascade that ultimately activates IRF3 and NF-kB transcription factors. IRF3 activation leads to the expression of genes (such as IP-10 and type I interferon) that induce antiviral immune responses. NF-kB activation induces the expression of inflammatory cytokines (such as IL-6) that enhance inflammatory immune responses.

[0047] Human cGAS is 522 amino acids long and includes 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 expression of cGASΔN in a human leukemic monocytic cell line resulted in higher expression levels of interferon and interferon-stimulated genes (Barnett, supra, 2019).

[0048] The amino acid sequence of human cGAS (GenBank No. NP_612450.2) is: MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQERPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWDVPSPGLPVSAPILVRRDAAPGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLNTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF(SEQ ID NO:10).

[0049] The amino acid sequence of the N-terminal domain of cGAS is: MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQERPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWDVPSPGLPVSAPILVRRDAA(SEQ ID NO:11).

[0050] The amino acid sequence of the C-terminal domain of cGAS (cGASΔN) is: PGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLNTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQN WLSAKVRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF(SEQ IDNO:1).

[0051] 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 Examples 1, 2, and 3.

[0052] 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.

[0053] 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.

[0054] 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).

[0055] "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 alignment of the candidate sequence with the reference polypeptide sequence, introducing gaps (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

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] In some embodiments, the mRNA further encodes a ribosomal skipping sequence, such as a 2A-like 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., cGASΔNs-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

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

[0063] In some embodiments, 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.

[0064] 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.

[0065] The structures of lipids suitable for use in lipid-based mRNA delivery vehicles of the present disclosure are described in Fig.13A and Fig. 13B The figure is from Hou et al., Nature Review Materials, 6: 1078-1094, 2021 Figure 2 Modified. IV. 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 DAMPs (such as oxPAPC or PGPC).

[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. V. Pharmaceutical Preparations

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

[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, glycerin, 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). VI. 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 In this section, Embodiment 1 refers to Embodiment 1' and Embodiment 1". 1 '. A composition comprising a first mRNA encapsulated in a first lipid nanoparticle (LNP) and a second mRNA encapsulated in a second LNP, wherein the first mRNA comprises a coding region for a constitutively active cyclic GMP-AMP synthase (cGAS), wherein the second mRNA comprises a coding region for an antigen, and Wherein both the first LNP and the second LNP comprise phospholipids and at least one lipid selected from the group consisting of ionizable lipids, PEGylated lipids, structured lipids, and mixtures thereof. 1". A composition comprising mRNA encapsulated in lipid nanoparticles (LNPs), wherein the mRNA comprises a coding region for a constitutively active cyclic GMP-AMP synthase (cGAS), and the LNPs comprise phospholipids and at least one lipid selected from the group consisting of ionizable lipids, pegylated lipids, structural lipids, and mixtures thereof, optionally wherein the composition further comprises an additional mRNA encapsulated in additional lipid nanoparticles, and the additional mRNA comprises a coding region for an antigen of interest, and the LNPs comprise phospholipids and at least one lipid selected from the group consisting of ionizable lipids, pegylated lipids, structural lipids, and mixtures thereof. 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 phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a pegylated lipid, a structural lipid, and a mixture thereof. 3. A composition comprising mRNA encapsulated in lipid nanoparticles (LNPs), 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 phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a pegylated lipid, a structured lipid and a mixture thereof. 4. A composition according to any one of embodiments 1-3, wherein the at least one lipid comprises an ionizable lipid, a PEGylated lipid, and a structured 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) ALC-0315 or an analog 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 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 according to any one of embodiments 1-9, wherein the 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 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, optionally wherein the excipient comprises sucrose. 14. A composition according to any one of embodiments 1-13, wherein the composition does not contain lysophosphatidylcholine (LPC), optionally wherein the LPC has a single C13-C24 acyl chain. 15. A composition comprising: mRNA complexed with one or more lipids (RNA-Lipoplex); and 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: A first mRNA and a second mRNA complexed with one or more lipids (RNA-Lipoplex); and 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: mRNA complexed with one or more lipids (RNA-Lipoplex); and 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. The composition according to any one of embodiments 1-20, further comprising a TLR7 / 8 agonist. 22. A composition according to embodiment 21, wherein the TLR7 / 8 agonist is a small molecule with a molecular weight of 900 Daltons or less. 23. A composition according to embodiment 22, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound. 24. The composition of embodiment 23, wherein the TLR7 / 8 agonist comprises resiquimod (R848). 25. The composition of any one of embodiments 1-24, wherein the mRNA or the first mRNA and the second mRNA comprise a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR). 26. The composition of any one of embodiments 1-25, wherein the mRNA comprises a 5' cap structure. 27. A composition according to any one of embodiments 1-26, wherein the mRNA comprises a poly A tail. 28. The composition of any one of embodiments 1-27, wherein the mRNA is a nucleoside-modified mRNA. 29. A composition according to any one of embodiments 1-28, wherein the constitutively active cGAS has a greater propensity for self-DNA reactivity than its wild-type counterpart. 30. The composition of any one of embodiments 1-29, wherein the cGAS is a truncated cGAS lacking the amino-terminal phosphoinositide binding domain (cGASΔN). 31. The composition of embodiment 30, 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. 32. The composition of embodiment 31, 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. 33. The composition of embodiment 31, wherein the cGASΔN is encoded by the nucleotide sequence of SEQ ID NO:17. 34. The composition of any one of embodiments 30-33, wherein the coding region of cGASΔN is operably combined with a start codon (ATG). 35. The composition of any one of embodiments 1-34, wherein the antigen is a tumor antigen. 36. A composition according to embodiment 35, wherein the tumor antigen is a tumor-associated antigen. 37. The composition of embodiment 35, wherein the tumor antigen is a neoantigen. 38. The composition of any one of embodiments 1-34, wherein the antigen comprises a microbial antigen. 39. The composition of embodiment 38, wherein the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoan antigen, or a fungal antigen. 40. The composition of any one of embodiments 1-34, wherein the antigen comprises a surface protein of a pathogen or a fragment thereof. 41. The composition of embodiment 40, wherein the pathogen is capable of causing disease in a human subject. 42. The composition of embodiment 40 or embodiment 41, wherein the pathogen is a virus. 43. The composition of embodiment 42, wherein the virus is SARS-CoV-2. 44. A composition according to embodiment 43, wherein the antigen is the spike (S) glycoprotein of SARS-CoV-2, optionally wherein the spike glycoprotein is a pre-fusion stabilized variant. 45. A composition according to any one of embodiments 1-44, wherein the composition does not contain lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA). 46. ​​The composition of any one of embodiments 1-45, wherein the composition does not comprise oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) or species of oxPAPC. 47. The composition of embodiment 46, 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). 48. A pharmaceutical formulation comprising the composition according to any one of embodiments 1-47 and a pharmaceutically acceptable excipient. 49. 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 1-47, any one of embodiments 21-47, or the formulation according to embodiment 48 to produce superactivated dendritic cells, wherein the superactivated dendritic cells secrete IL-1β without undergoing cell death within about 48 hours of exposure. 50. The method of embodiment 49, wherein the dendritic cells are: (i) contacting with the composition in vivo; or (ii) contacting with said composition ex vivo. 51. The method of embodiment 49 or embodiment 50, wherein the hyperactivated dendritic cells: (i) secrete higher levels of one or more of RANTES, IP-10 and IFNα 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 CD40, CD86, CD69, MHC class II, MHC class I, CCR7, and combinations thereof. 52. 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 49-51 and a pharmaceutically acceptable excipient. 53. 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 48 or embodiment 52 to stimulate an immune response to the antigen. 54. 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 48 or Embodiment 52 to treat the cancer. 55. 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 48 or embodiment 52 to inhibit abnormal cell proliferation. 56. 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 48 to treat or prevent the infectious disease. 57. The method of embodiment 56, wherein the infectious disease is a viral disease. 58. The method of embodiment 56, wherein the infectious disease is a bacterial disease. 59. The method or pharmaceutical formulation of any one of embodiments 49-55, wherein the dendritic cells are mammalian cells. 60. The method or pharmaceutical formulation of embodiment 59, wherein the mammalian cell is a human cell. 61. The method of any one of embodiments 53-59, wherein the individual is a mammal. 62. The method of embodiment 61, wherein the mammal is a human. 63. The method of embodiment 61, wherein the mammal is a dog or a cat. 64. The composition, formulation or method of any one of embodiments 1-63, wherein the 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]caprylate (SM-102) or an analog or derivative thereof and cholesterol. 65. The composition, formulation or method of embodiment 64, wherein the at least one lipid further comprises a PEGylated lipid, optionally wherein the PEGylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoylglycerol [DMG]. 66. The composition, formulation or method of any one of embodiments 1-65, 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. 67. The composition, formulation or method of embodiment 66, wherein the LNP has an effective diameter of less than about 200 nanometers. 68. The composition, formulation or method of claim 67, 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: Murine DCs can be activated in vitro by LNPs containing cGASΔN mRNA Materials and Methods

[0090] Material. GenVoy ILM TMThe ionizable lipid mixture was purchased from Precision Nanosystems. CleanCapOVA and GFP mRNA were purchased from Trilink with a 120-residue poly-A tail, 5-methoxyuridine base modification, and codon optimization for expression in mammalian systems. cGASΔN mRNA was custom-made from Trilink and synthesized from linearized template DNA via in vitro transcription. The nucleotide sequence of 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 an N1-methyl pseudouridine base modification and a 120-residue poly-A tail. The sequence contains a Bbsl restriction enzyme site. The mRNA was phosphatase treated after synthesis.

[0091] LNP synthesis using GenVoy ILM TM Lipid nanoparticles (LNPs) were prepared from an ionizable lipid mixture (Precision Nanosytems). OVA mRNA, GFP mRNA, and cGASΔN mRNA (Trilink) were each prepared at 0.17 mg / mL in sodium citrate buffer at pH 4. GenVoy ILM TM . LNPs were synthesized using a NanoAssemblr Ignite instrument (Precision Nanosystems). Lipids and mRNA solutions in ethanol were combined individually 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), 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.

[0092] 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. Triton X-100 was used to cleave the LNPs to assess mRNA encapsulation in the LNPs. Total mRNA and encapsulated mRNA were quantified. The size of the LNPs was assessed using dynamic light scattering (DLS) on a NanoBrook Omni (Brookhaven). Prior to running on the DLS, the LNPs were diluted 1:10 in PBS. Three 90 second measurements were recorded for each sample.

[0093] Mouse bone marrow-derived FLT3L-DCs were generated. The femur and tibia were removed from the mice, chopped with scissors and flushed into sterile tubes. The bone marrow suspension was treated with ACK lysis buffer for 1 minute and then passed through a 40 μm cell strainer. The cells were counted and resuspended in a culture medium (I10) consisting of complete IMDM containing 10% FBS, penicillin and streptomycin, and supplements of L-glutamine and sodium pyruvate. The cells were then plated at 8 x 10 per well. 6 Bone marrow cells were plated in P12 plates. Recombinant mouse FLT3L (Miltenyi) was added to the culture at 200 ng / mL. Differentiated cells were used for subsequent assays on day 8. The efficiency of differentiation was monitored by flow cytometry using BD Symphony A3, 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.

[0094] Mouse FLT3L-DC stimulation. FLT3L-DCs were harvested 8 days after differentiation, counted and treated with lipid nanoparticles (LNPs) based on mRNA content. Cells were treated with 1 μg / mL mRNA in 200 μl total medium / well. Approximately 24 hours after incubation, cells and culture supernatants were used for downstream readouts. 150 mL of cell supernatant was collected from each well to measure cytokine secretion by DCs. LEGENDplex was used according to the manufacturer's protocol. TM Mouse antiviral response plate (Biolegend). Data were collected using BD FACS Symphony and analyzed using the cloud-based software provided by Biolegend. In order to evaluate DC activation after LNP exposure, FLT3L DC was collected 24 hours after LNP addition and stained to measure the expression of the following cell surface markers: CD11c, MHC-II, CD24, SIRP1a, CD40, CD86, CD69 and H2kb (MHC-I). The figure shows the mean and SD of triplicate samples. Data represent two experiments. result

[0095] LNPs loaded with mRNA encoding cGASΔN (cGASΔN LNPs) exhibited similar size and loading profiles to LNPs loaded with mRNA encoding model antigens. LNPs loaded with cGASΔN mRNA showed similar size and mRNA loading profiles to LNPs loaded with model antigen mRNAs (OVA and GFP). All mRNA-loaded LNPs had an average effective diameter of less than 150 nm ( Figure 3A ), with a relatively uniform size profile, exhibiting a polydispersity index of less than 0.3 ( Figure 3B ). All mRNA loaded into GenVoy ILM LNPs, where the measured mRNA loading showed 90%-110% of the theoretical mRNA loading ( Figure 3C ).

[0096] cGASΔN LNP induces IFN-I dependent response in mouse DC. FLT3L DC was stimulated with empty LNP (no mRNA) or LNP containing only GFP mRNA or OVA mRNA or cGASΔN mRNA. Alternatively, DC was stimulated with LNP containing OVA mRNA plus empty LNP or LNP containing OVA mRNA plus LNP containing cGASΔN or LNP containing cGASΔN plus LNP containing GFP mRNA. Based on the concentration of mRNA, LNP was added to cell culture at 1 μg / mL. 24 hours after stimulation, the presence of cytokines in cell supernatants was measured using a multiplex cytokine bead array Legendplex assay. Interestingly, it was found that after cGASΔN LNP treatment, the levels of RANTES, IP-10 and IFNα secreted by BMDC were significantly higher than those when treated with LNP loaded with mRNA antigen only ( Figure 4A , Figure 4B , Figure 4C ). These data indicate that constitutively active cGASΔN is successfully expressed and functional in mouse DCs. NFkB-dependent proinflammatory cytokines, including IL-6 and TNFα, were not secreted, suggesting that constitutively active cGASΔN induces NFkB activation in specific cell types. The induction of these cytokines after LNP treatment was compared with stimulation with LPS and R848 PAMPs. Interestingly, expression of IP-10, RANTES, and IFNα was stronger or expressed at similar levels by cGASΔNLNP treatment compared with higher doses of LPS or R848, suggesting that expression of constitutively active cGASΔN can strongly induce the IRF3 pathway.

[0097] cGASΔN LNPs induce murine DC activation. To assess DC activation, LNP-treated DCs were stained for activation markers on total cDCs and cDC1 and cDC2 subsets of DCs 24 h after treatment. When assessed by median fluorescence intensity (MFI), cGASΔN LNPs were used instead of empty LNPs or GFP LNPs or LNPs containing OVA ( Figure 5A , Figure 5B , Figure 5C) cells, the expression of T cell co-stimulatory surface molecules CD40, CD86, and CD69 increased. This trend also applied to the cDC1 subset. In addition, when cDCs were treated with cGASΔN LNPs, the expression of MHCII and H2kB antigen presenting molecules on cDCs increased compared to standard mRNA antigen LNPs ( Figure 5D , Figure 5E This also applies to the cDC1 subset. In addition, CCR7 surface expression, indicative of the potential of DC to migrate to LNs, was increased on cDCs when treated with cGASΔNLNPs compared to standard mRNA antigen LNPs ( Fig. 5F ). This trend also applies to the cDC1 subset. Overall, these data suggest that cGASΔN LNPs induce cGAS-STING pathway activation within DCs, leading to an activated DC state and type I IFN production. Example 2: Human cells can be activated to an inflammatory state in vitro by LNPs containing cGASΔN mRNA Materials and Methods

[0098] Materials. Lipids for LNPs were purchased from Cayman Chemicals (SM102) or Avanti (22:0 LPC, DSPC, DMG-PEG2000). Cholesterol was purchased from Sigma. GenVoy ILM lipids were purchased from Precision Nanosystems. CleanCap OVA and GFP mRNAs were purchased from Trilink off the shelf with 120 poly A tails and base modification 5-methoxyuridine, optimized for mammalian systems. 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 mice. The sequence was capped with base modification N1-methylpseudouridine and 120 poly A tail using Trilink's proprietary Clean Cap mRNA technology. The sequence contains a Bbsl restriction enzyme site. The sequence information was used to construct plasmids from which the mRNA was synthesized synthetically. The mRNA was treated with phosphatase after synthesis.

[0099] LNP synthesis. Lipid nanoparticles (LNPs) were prepared using GenVoy ILM LNP lipid mixtures (Precision Nanosytems) or custom LNP lipid mixtures (Table 2-1). OVA mRNA, GFP mRNA, and cGASΔN mRNA (Trilink) were prepared at 0.17 mg / mL each in sodium citrate buffer at pH 4. GenVoy ILM and custom LNP lipid mixtures were used at 12.5 mM. 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 Lipids <![CDATA[GenVoy ILM TM ]]> Custom lipid blends Ionizable lipids Proprietary lipids 50% SM102 40% DSPC 10％ 35％ cholesterol 37.5％ 28.5％ Stabilizer lipid Proprietary lipids 2.5% DMG-PEG2000 1.5%

[0100] 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. Triton x-100 was used to lyse the LNPs to assess mRNA encapsulation in the LNPs. Total mRNA and encapsulated mRNA were quantified. The size of the LNPs was assessed using dynamic light scattering (DLS) on a NanoBrook Omni (Brookhaven). Prior to running on the DLS, the LNPs were diluted 1:10 in PBS. Three 90 second measurements were recorded for each sample.

[0101] Human monocyte-derived dendritic cells (moDC) are produced. Human monocytes are separated from Leukopaks purchased from Miltenyi using the StraightFrom Leukopak CD14 microbead kit (Miltenyi). The separation is completed following 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 cell (moDC) cultures, monocytes are thawed and cultured in RPMI medium (R10 medium) containing 10% FBS, 50 units / mL penicillin, 50mg / mL streptomycin, 2mM L-glutamine, 1mM sodium pyruvate, 50mM β-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 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. Six days after differentiation, moDCs were collected and counted. Cells were plated at 1E5 cells / well in R10 medium in 96-well flat-bottom plates.

[0102] THP-1 cell generation. THP1-Null2 (Invivogen) cells were thawed and maintained in RPMI medium containing 10% heat-inactivated FBS, 25 mM HEPES, 100 U / mL penicillin / streptomycin, 100 μg / mL normocin, and supplemented with 100 μg / mL zeocin every other passage. Cells were plated in T75 culture flasks at 0.4-0.6×10 6 Cells were passaged between 10 cells / mL, and cells were passaged twice a week. Cells were maintained until passage 20, after which new vials were thawed. Before treatment with LNPs, THP1-Null2 cells were collected from flasks and plated at 100,000 cells / well in RPMI medium containing 10% heat-inactivated FBS, 25 mM HEPES, and 100 U / mL penicillin / streptomycin in 96-well flat-bottom tissue culture plates.

[0103] Human cell activation. Based on the concentration of mRNA content, THP-1 cells and moDC were treated with lipid nanoparticles (LNP). The cells were treated with 0.2 μg / mL of mRNA delivered in LNP with a total stimulus volume of 200 μL / well. After overnight incubation, cells and culture supernatants were used for downstream readout. 150 microliters of cell supernatant were collected. One day after adding LNP, cells and supernatants from THP-1 cultures were collected to measure IL-6 (Lumit assay of Promega) and IP-10 (ELISA kit from Biolegend) cytokines. Activation markers were stained using THP-1 cells for flow cytometry analysis. In order to measure cytokine secretion from moDC, LegendPlex antiviral response plates (Biolegend) were used according to the manufacturer's protocol. Data were collected using BD FACS Symphony and analyzed using cloud-based software provided by Biolegend. Both moDC and THP-1 cells were collected 1 day after adding LNPs and stained to measure the expression of the following cell surface activation markers: CD11c, CD209, CD40, CD80, CD83, CD86, HLA-ABC and HLA-DR. Two different human donor samples and THP-1 cells were studied, and each biological condition was tested in triplicate. The figures show data from two human samples or THP-1 cells. The data represent two experiments.

[0104] HEK-Blue IL-1β reporter assay. Based on the concentration of mRNA content, moDCs were treated with lipid nanoparticles (LNPs). The cells were treated with 1μg / mL OVA mRNA, 1μg / mL cGASΔN mRNA, or 0.2μg / mL mRNA delivered in LNPs with a total stimulus volume of 200μL / well. The LNPs were delivered with culture medium, R848, or R848+MCC950 (an inhibitor of the NLRP3 inflammasome). In the presence of MCC950, IL-1β should not be secreted in an inflammasome-dependent manner. HEK-Blue IL-1β reporter cells (Invivogen) were used to assess the functional IL-1β secreted by human moDCs in response to treatment with cGASΔN LNPs for 48 hours. HEK-Blue IL-1β reporter cells are derived from HEK293 cells and express NK-kB / AP-1 inducible SEAP (secreted embryonic alkaline phosphatase) reporters that trigger SEAP production immediately after the IL-1β receptor encounters mature IL-1β. SEAP production is monitored by color change using QUANTI-Blue solution. HEK-Blue IL-1β reporter cells are thawed and maintained in DMEM culture medium containing 10% heat-inactivated FBS, 2mM L-glutamine, 100U / mL penicillin, 100μg / mL streptomycin, 100μg / mL normocin, and supplemented with 100μg / mL zeocin every other generation. Cells are passaged between 0.4-0.6X10^6 cells / mL in T75 culture bottles, and cell passage is performed twice a week. Cells are maintained until the 20th generation, and then new vials are thawed. Prior to treatment with LNPs, HEK-Blue IL-1β reporter cells were collected from flasks and plated at 50,000 cells / well in 96-well flat-bottom tissue culture plates in DMEM medium containing 10% heat-inactivated FBS, 2mM L-glutamine, 100U / mL penicillin, and 100μg / mL streptomycin. 50μL of medium from cGASΔN-activated moDCs or 50μL of medium containing known concentrations of recombinant human IL-1β to prepare a standard curve was added to the wells containing HEK-Blue IL-1β reporter cells. The cells were incubated overnight, after which 20μL of supernatant containing SEAP from the incubated samples was mixed with 180μL of QUANTI-Blue solution. The reaction was run at 37C for 1 hour, after which the absorbance at 630nm was measured spectrophotometrically. result

[0105] To determine whether cGASΔN LNPs would allow greater cell activation than standard LNPs, LNPs were synthesized to contain mRNA encoding the Ova antigen or mRNA encoding constitutively active cGASΔN. As an experimental control, LNPs were prepared with mRNA encoding GFP. These LNPs were tested alone or in combination on THP-1 cells (human myeloid cell line) and primary human moDCs. Two lipid formulations were included: GenVoy (Precision Nanosystems) and a custom lipid mixture to determine whether activity was LNP formulation dependent. Based on the concentration of mRNA, LNPs were added to cell cultures at 0.2 μg / mL.

[0106] cGASΔN LNPs exhibited similar size and loading profiles to LNPs loaded with model antigens. LNPs loaded with cGASΔN mRNA showed similar size and mRNA loading profiles to LNPs loaded with model antigen mRNAs (OVA and GFP). All mRNA-loaded LNPs had an average effective diameter of less than 150 nm ( Fig. 6A ), with a relatively uniform size profile, exhibiting a polydispersity index of less than 0.3 ( Figure 6B ). Of all the mRNA loaded into GenVoy ILM LNPs, 70%-90% of the theoretical loading amount of mRNA was encapsulated in the LNPs ( Figure 6C Both GenVoy and the custom lipid blend allowed the production of LNPs with similar sizes, but the optimized GenVoy formulation exhibited increased size uniformity ( Figure 6B ), as indicated by the lower polydispersity index. Both GenVoy and the custom lipid blend allowed similar loading levels for all mRNAs, with the GenVoy formulation encapsulating mRNA slightly more efficiently.

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

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

[0109] cGASΔN LN activates inflammatory pathways in THP-1. After one day of incubation, the presence of cytokines (IL-6 and IP-10) that are dependent on NF-kB or IRF3 activation, respectively, was measured in the supernatant of THP-1 cells. At both time points, THP-1 cells treated with cGAS LNPs did not differentially express IL-6, indicating that NF-kB was not activated. In contrast, IP-10, a gene controlled by IRF3, was expressed within one day of cGAS LNP treatment ( Fig.9A ). cGAS LNPs induced IP-10 expression, while other control LNPs barely induced IP-10. Detectable IP-10 suggests that packaging transcripts of constitutively active cGAS into LNPs may be a way to activate the innate immune system.

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

[0111] Treatment with cGASΔN LNP+R848 results in moDC hyperactivation. moDCs were stimulated with R848 and cGASΔN to determine whether the combination of NF-kB stimulation and cGAS-STING pathway activation would allow for more effective inflammatory activity. Specifically, the ability of moDCs to be superactivated by this combination was tested. Superactivation is expressed as the ability of DCs to secrete IL-1β while remaining viable. moDCs were either left unstimulated (PBS-treated) or treated with cGASΔN LNPs or OVA LNPs in the presence of culture medium, R848, or R848 and MCC950. MCC950 is an NLRP3 inflammasome inhibitor, so cells treated with MCC950 were unable to produce IL-1β in an inflammasome-dependent manner. Importantly, all cells treated with LNPs showed similar viability to moDCs treated with PBS alone, indicating that no cells underwent pyroptosis in response to LNP treatment ( Fig. 10A To determine whether these cells produced any active IL-1β in response to treatment with R848 and cGASΔN, supernatants from moDC cultures were added to HEK-Blue IL-1β reporter cells that produce SEAP in response to IL-1β. As expected, moDCs treated with PBS, R848, or R848+MCC950 did not produce IL-1β ( Fig. 10B To determine whether hyperactivation was dependent on the LNP lipid composition, LNPs containing OVA were incubated with R848, a condition that does not allow IL-1β production. Interestingly, when delivered in combination with R848, LNPs loaded with cGASΔN stimulated IL-1β secretion in moDCs ( Fig. 10B ). This response was NLRP3 inflammasome-dependent, as treatment with MCC950 abolished IL-1β production.

[0112] In summary, several conclusions were drawn from the data collected from treating THP-1 and human moDC with LNPs. First, LNPs were successfully synthesized. In addition, the expression of constitutively active cGAS resulted in upregulation of genes associated with the cGAS-STING pathway in a formulation-independent manner. Inflammatory cytokines were expressed, and upregulation of activation markers was observed on the cell surface. Constitutively active cGAS encoded as mRNA and packaged in LNPs can be successfully used to initiate innate immune signaling in target cells to promote inflammatory responses. Finally, constitutively active cGAS in combination with R848 allowed moDC hyperactivation, and IL-1β secretion occurred in live DCs. Example 3: Mouse cells can be activated in vivo by LNPs containing cGASΔN mRNA Materials and Methods

[0113] Materials. Lipids for LNPs were purchased from Cayman Chemicals (SM102) or Avanti (22:0LPC, DSPC, DMG-PEG2000). Cholesterol was purchased from Sigma. GenVoy ILM lipids were purchased from Precision Nanosystems. CleanCap OVA and GFP mRNAs were purchased from Trilink off the shelf with 120 poly A tails and base modified 5-methoxyuridine, optimized for mammalian systems. 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 mice. The sequence was capped with base modified N1-methyl pseudouridine and 120 poly A tails using Trilink's proprietary Clean Cap mRNA technology. 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 treated with phosphatase after synthesis.

[0114] LNP synthesis. Lipid nanoparticles (LNP) were prepared as in Example 2 using GenVoy ILM LNP lipid mixtures (Precision Nanosytems) or typical LNP lipid mixtures (Table 2-1, Example 2). OVA mRNA, GFP mRNA and cGASΔN mRNA (Trilink) were prepared at 0.17 mg / mL in sodium citrate buffer at pH 4. GenVoy ILM and typical LNP lipid mixtures were used at 12.5 mM. LNP was synthesized using NanoAssemblr Ignite instrument (PrecisionNanosystems). Lipids in ethanol were combined with mRNA solution in a 1:3 volume ratio, using a flow rate of 12 mL / min. LNP was 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. LNP was filtered through a 0.2 μm filter before use.

[0115] LNP characterization. Following the manufacturer's protocol, mRNA loading in LNPs was quantified using RiboGreen assay (ThermoFisher) as in Example 2. Samples were diluted to fall within the range of the standard curve. Triton X-100 was used to cleave LNPs to assess mRNA encapsulation in LNPs. Total mRNA and encapsulated mRNA were quantified. Dynamic light scattering (DLS) was used to assess the size of LNPs on NanoBrook Omni (Brookhaven). LNPs were diluted 1:10 in PBS before running on DLS. Three 90 second measurements were recorded for each sample.

[0116] In vivo immunization was performed with LNPs containing cGASΔN mRNA and OVA antigen mRNA. BL6 mice were subcutaneously immunized with LNPs according to the following table (Table 3-1). Antigens were delivered using LNPs loaded with OVA mRNA and LNPs containing adjuvant cGASΔN mRNA or non-auxiliary GFP mRNA. Mice were administered with the mRNA doses shown in the table. The OVA mRNA dose was fixed at 5 μg / mouse, while the adjuvant mRNA varied from 1 μg / mouse to 10 μg / mouse. Mice (n=5 / group) were given a primary immunization on day 0, and a booster immunization of the same dose was given on day 7. Seven days after the booster, blood was collected for antibody and T cell responses. Serum was collected from the blood using serum separation tubes, and blood for cell analysis was collected using K2EDTA tubes. After collecting the blood, the mice were euthanized, and the draining lymph nodes and spleen were collected and processed into single cell suspensions. Table 3-1. LNPs used for in vivo assessment of T cell and B cell responses to cGASΔN LNPs

[0117] OVA-specific T cell tetramer evaluation. The OVA-specific T cells in the blood of mice receiving OVA LNP immunity were evaluated 7 days after the boost. CD8+T cells with specificity for SIINFEKL (SEQ ID NO: 16) (OVA MHC-I epitope) were quantified in the blood using tetramer analysis. In brief, the red blood cells in the blood were lysed using RBC lysis buffer, and two lysis were completed to completely remove any RBC 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 for total cell counting. Data were collected using BD FACS Symphony and analyzed using Flowjo (BD).

[0118] OVA-specific antibody evaluation. The OVA-specific antibodies in the serum of mice immunized with OVA LNP were evaluated 7 days after the boost. OVA-specific total IgG, IgG1 and IgG2b were evaluated using ELISA. In brief, the ELISA plate was coated overnight with 10 μg / mL of Endofit ovalbumin (Invivogen), 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 1:5 dilution, for a total of 7 serum dilutions. The samples were washed and then incubated with a detection antibody specific for IgG, IgG1 or IgG2b conjugated to HRP (Southern Biotech) to detect total, Th2 dominant or Th1 dominant OVA-specific antibodies, respectively. The plate was washed and then incubated with TMB, and the stop solution was added immediately after the color development was completed. result

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

[0120] cGASΔN LNPs exhibited similar size and loading profiles to LNPs loaded with model antigens. LNPs loaded with cGASΔN mRNA were prepared according to Example 2 and characterized following Figure 6A-6C All LNPs showed similar sizes and mRNA loading profiles to those loaded with model antigen mRNA (OVA and GFP). All mRNA loaded LNPs had an average effective diameter of less than 150 nm, with a relatively uniform size profile, exhibiting a polydispersity index of less than 0.3. Of all mRNAs loaded into GenVoy ILM LNPs, 70%-90% of the theoretical loading of mRNA was encapsulated in the LNPs.

[0121] Treatment with OVA LNP and cGASΔN LNP increased OVA-specific CD8+T cells. After two immunizations (Table 3-1) with OVA LNP (5 μg mRNA / mouse) delivered together with cGASΔN LNP (1 or 5 μg mRNA / mouse) or control GFP LNP (1 or 5 μg mRNA / mouse), the presence of OVA-specific T cells in the blood was analyzed. Blood was collected 7 days after reinforcement, processed to remove red blood cells, and then stained for Live / Dead, CD3, CD8, CD4 and SIINFEKL-tetramer reactivity (OVA-specific CD8+T cell epitopes as shown in SEQ IDNO:16). Determine the frequency of SIINFEKL-specific cells in live CD8+T cells, and determine the total absolute number of SIINFEKL-specific CD8+T cells collected in the blood. When mice were treated with GenVoyLNP-OVA LNPs (5 μg mRNA) and cGASΔN or GFP LNPs (1 or 5 μg mRNA), the frequency of SIINFEKL-specific (OVA-specific) CD8+ T cells was significantly increased when GASΔN mRNA was delivered in addition to OVA compared to when GFP mRNA was delivered in addition to OVA ( Fig.11A ). Mice that did not receive OVA LNPs had no SIINFEKL-specific CD8+ T cells. This trend was not formulation-specific, as mice that received OVA LNPs (5 μg mRNA) and cGASΔN (5 μg mRNA) prepared with a custom lipid mixture had significantly higher frequencies of SIINFEKL-specific T cells in their blood than mice that received OVA LNPs + GFP LNPs (5 μg mRNA) ( Fig. 11B This trend also applies to the use of GenVoy ( Fig. 11C ) or custom lipid mixtures ( Fig.11D Interestingly, lowering the dose of cGASΔN mRNA did not seem to have a large effect on the frequency of OVA-specific T cells, suggesting that a small amount of this cGASΔN mRNA combined with a standard lipid formulation containing antigen mRNA may be sufficient to significantly enhance antigen-specific T cell responses.

[0122] Treatment with cGASΔN LNPs reduced Th2-specific OVA-specific antibodies without affecting Th1-specific IgG responses. OVA-specific antibodies were quantified in the blood after two immunizations with OVA LNPs (5 μg mRNA / mouse) delivered together with cGASΔN LNPs (1 or 5 μg mRNA / mouse) or control GFP LNPs (1 or 5 μg mRNA / mouse) (Table 3-1). Blood was collected 7 days after the boost, serum was separated, and total IgG, IgG1 (associated with Th2 response), and IgG2a (associated with Th1 response) were then analyzed. Traditionally, immunization with LNPs loaded with mRNA antigens produces a strong antibody response, so the antibody response was evaluated to determine whether the addition of cGASΔN LNPs to the treatment would reduce the expected response. Interestingly, mice treated with OVA and cGASΔN LNPs prepared using the GenVoy formulation did not show significant changes in measured OVA-specific total IgG compared to OVA+GFP LNP treatment ( Fig. 12A Mice that did not receive OVALNPs did not produce OVA-specific antibodies. This was not a formulation-specific response: the presence of cGASΔN in LNPs prepared using the custom lipid mix had no significant effect on the measured total OVA-specific IgG ( Fig. 12B ). OVA-specific IgG1 was also evaluated, as IgG1 is typically associated with Th2 immune responses. The presence of cGASΔN in LNPs prepared using the GenVoy formulation significantly reduced OVA-specific IgG1 (p<0.05 for cGASΔN LNPs vs. GFP LNPs at 1 ug and 5 ug for 1:500 and 1:2500 dilutions) ( Fig. 12C This reduced OVA-specific IgG1 response was also measured in mice treated with cGASΔN LNPs prepared using a custom lipid mix (p<0.05 for cGASΔN LNPs vs. GFP LNPs at 5ug for 1:500, 1:2500, and 1:12500 dilutions) ( Fig.12D IgG2a levels were also assessed, as IgG2a is associated with a more inflammatory Th1 response. Importantly, compared to OVA+GFP treated mice, mice treated with GenVoy formulation ( Fig.12E ) or custom lipid mixtures ( Fig.12F )-prepared OVA LNPs and cGASΔN LNP-treated mice did not show significant changes in OVA-specific IgG2a.

[0123] In summary, the data collected from the in vivo immunization experiments show that treating mice with cGASΔN LNPs in combination with antigen LNPs significantly improved antigen-specific adaptive immune responses. Mice receiving cGASΔN LNPs showed a significant increase in the number of antigen-specific T cells without sacrificing antibody responses. In fact, the reduction in antigen-specific IgG1 without affecting total antigen-specific IgG levels shows a bias towards Th1-related immune responses, which is beneficial for inducing protective immune responses (which are required for immunotherapy). Taken together, these data show that adding low doses of cGASΔN LNPs to standard antigen LNP formulations can result in significantly more effective immune responses to LNP vaccines. sequence SEQ ID NO:1 >People PGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLN TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKE GNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO:2 > Macaques PGARKLRAVLEKLRLSHQDISKAAKVVNGVVGHLLRRLKCEPEFGGVEQLH TGSYYEHVKISAPNEFDVMFKLQVPRIQLEEYSDTGAYYFVKFKRNPEGNPLSLFLEDEILSASKMLSKFRKIIKEEINNIKDVQPDADVIMKRKRPGSPAVTLLINKEISVDITLALELKTSWPASTQEGLAIKNWLSAKVRQQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILSNHGKSKTCCENKEMKCCRKDCLKLMKYLLEQLKEKFKDKKHLDKFSSYHVKTAFFHLCTENPQDSQWDPKDLGLCFDNCVTYFLECLRTEQLKNYFIPGFNLFSSNLIDKTSKEFLSKQIEYERNNEFPVFGEF SEQ ID NO:3 > Papio anubis PGARKLRAVLEKLRLSHQDISKAAKVVNGVVGHLLHRLKCESEFEGVEQLH TGSYYEHVKISAPNEFDVMFKLQVPRIQLEEYSDTGAYYFVKFKRNPKGNPLSQFLEDEILSASKMLSQFRKIIKEEINNIKDTDVIMKRKRRGSPAVTLLINKEISVDITLALELKTSWPASTQEGLAIKNWLSAKVRQQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILSNHGKSKTCCENKEMKCCRKDCLKLMKYLLEQLKEKFKDKKHLDKFSSYHVKTAFFHLCTENPQDSQWDPKDLGLCFDNCVTHFLQCLRTEKLANYFIPGFNLFSSNLIDKSSKEFLSKQIEYERNNEFPVFGEF SEQ ID NO:4 > Nomascus leucogenys PGARKLRAVLEKLKLSRQEISEAAEVVNGVVDHLLRRLQKCDSEFRGVGLLRTGSYYEH VKISAPNEFDVMFKLEVPRIQLEEYSNTGAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINNIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESNSNSWPASTQEGLPIKDWLSGKVRRQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKTKTCCENKNVKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTENPQDSQWDPKNLGFCFDKCVIYFLKCLRTEQLGNYFIPGFNLFSSNLIDKRSKEFLSKQIEYERNNEYPVFG SEQ ID NO:5 >Hoolock gibbon PGARKLPAVLEKLKLSRQ EICEAAEVVNGVVDHLLRRLQKCDSEFRGVGLLRTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTGAYYFVKFKRNPKENPLCQFLEGEILSASKMLSKFRKIIKEEINNIKDTDVILKRKRGGSPAVTLLISEKISVDITLALESKSSSWPASTQEGLPIKDWLSGKVRTQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGETKTCCENKKVKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTENPKDSQWDPKDLGLCFDKCVIYFLQCLSTEQLGNYFIPGFNLFSSNLIDKRSKEFLSKQIEYERNNEYPVFGKC SEQ ID NO:6 >Sumatran orangutan PGASKLRAVLEKLKLSRLEISKAAEVVNRVVDHLLRRLQKYNSEFRGVGLLRT GSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTGAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINNIKDTDVIMKRKRGGSPAVTLLISEEISVDITLALESKSSWPASTQEGLPIKNWLSAKVRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCEDKEVKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDPKDLGLCFDNCVTYFLQCLRTEQLGNYFIPGFNLFSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO:7 >Chimpanzee PGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLN TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINNIKDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLPIKNWLSAKVRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCEIKEAKCCRKDCLKLMKYLLEQLKEKFKDKKHLDKFSSYHVKTAFFHVCTENPQDSQWDRKDLGLCFDNCVTYFLQCLRIEKLENYFIPEFNLFSSDLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO:8 >Gorilla PGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLN TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINNIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRRQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEAKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKQLGLCFDNCVTYFLQCLRTERLENYFIPEFNLFSRNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO:9 >CGASΔN_ consensus sequence PGAXKLXAVLEKLXLSXXXIXXAAXXVXXVVXHLLXRLXXXXXXFXGVXXLX TGSYYEHVKISAPNEFDVMFKLXVPRIQLEEYSXTXAYYFVKFKRNPXXNPLXXFLEXEILSASKMLSXFRKIIKEEINXIKXXXXXXDVIXKRKRXGSPAVTLLIXXXISVDITLALEXXXXSWPASTQEGLXIXXWLSXKVRXQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILXNHGXXKTCCEXKXXKCCRKDCLKLMKYLLEQLKEXFKDKKHLDKFSSYHVKTAFFHXCTXNPXDSQWDXKXLGXCFDXCVTXFLXCLXXEXLXNYFIPXFNLFSXXLIDKXSKEFLXKQIEYERNNEXPVFXXX SEQ ID NO:10 >Human CGAS_full length MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQERPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCST KPRPPPGPWDVPSPGLPVSAPILVRRDAAPGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLNTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENP LSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHG KSKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF 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 nasal infection 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 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 phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a pegylated lipid, a structural lipid, and a mixture thereof.

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 phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a pegylated lipid, a structural lipid, and a mixture thereof.

3. A composition comprising mRNA encapsulated in lipid nanoparticles (LNPs), 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 phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a pegylated lipid, a structured lipid and a mixture thereof.

4. The composition of any one of claims 1-3, wherein the at least one lipid comprises an ionizable lipid, a PEGylated lipid, and a structured 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 according to any one of claims 1 to 9, wherein the phospholipids comprise: 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 according to any one of claims 1 to 9, wherein the 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 phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

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

14. The composition of any one of claims 1-13, wherein the composition does not comprise lysophosphatidylcholine (LPC), optionally wherein the LPC has a single C13-C24 acyl chain.

15. A composition comprising: mRNA complexed with one or more lipids (RNA-Lipoplex); and 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: A first mRNA and a second mRNA complexed with one or more lipids (RNA-Lipoplex); and 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: mRNA complexed with one or more lipids (RNA-Lipoplex); and 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 according to any one of claims 1-20, further comprising a TLR7 / 8 agonist.

22. The composition of claim 21, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less.

23. The composition of claim 22, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

24. The composition of claim 23, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

25. The composition of any one of claims 1-24, wherein the mRNA or the first mRNA and the second mRNA comprise a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR).

26. The composition of any one of claims 1-25, wherein the mRNA comprises a 5' cap structure.

27. The composition of any one of claims 1-26, wherein the mRNA comprises a poly A tail.

28. The composition of any one of claims 1-27, wherein the mRNA is a nucleoside-modified mRNA.

29. The composition of any one of claims 1-28, wherein the constitutively active cGAS has a greater propensity for self-DNA reactivity than its wild-type counterpart.

30. The composition of any one of claims 1-29, wherein the cGAS is a truncated cGAS lacking the amino-terminal phosphoinositide binding domain (cGASΔN).

31. The composition of claim 30, 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.

32. The composition of claim 31, 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.

33. The composition of claim 31, wherein the cGASΔN is encoded by the nucleotide sequence of SEQ ID NO:

17.

34. The composition of any one of claims 30-33, wherein the coding region of cGASΔN is operably combined with a start codon (ATG).

35. The composition of any one of claims 1-34, wherein the antigen is a tumor antigen.

36. The composition of claim 35, wherein the tumor antigen is a tumor-associated antigen.

37. The composition of claim 35, wherein the tumor antigen is a neoantigen.

38. The composition of any one of claims 1-34, wherein the antigen comprises a microbial antigen.

39. The composition of claim 38, wherein the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoan antigen, or a fungal antigen.

40. The composition of any one of claims 1-34, wherein the antigen comprises a surface protein of a pathogen or a fragment thereof.

41. The composition of claim 40, wherein the pathogen is capable of causing disease in a human subject.

42. A composition according to claim 40 or claim 41, wherein the pathogen is a virus.

43. The composition of claim 42, wherein the virus is SARS-CoV-2.

44. A composition according to claim 43, wherein the antigen is the spike (S) glycoprotein of SARS-CoV-2, optionally wherein the spike glycoprotein is a pre-fusion stabilized variant.

45. The composition of any one of claims 1-44, wherein the composition does not comprise lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA).

46. ​​The composition of any one of claims 1-45, wherein the composition does not comprise oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) or species of oxPAPC.

47. The composition of claim 46, 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).

48. A pharmaceutical formulation comprising the composition of any one of claims 1-47 and a pharmaceutically acceptable excipient.

49. A method for producing superactivated dendritic cells, the method comprising contacting dendritic cells with an effective amount of any one of claims 1-47, the composition of any one of claims 21-47, or the formulation of claim 48 to produce superactivated dendritic cells, wherein the superactivated dendritic cells secrete IL-1β without undergoing cell death within about 48 hours of exposure.

50. The method of claim 49, wherein the dendritic cells are: (i) contacting with the composition in vivo; or (ii) contacting with said composition ex vivo.

51. The method of claim 49 or claim 50, wherein the hyperactivated dendritic cells: (i) secrete higher levels of one or more of RANTES, IP-10 and IFNα 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 CD40, CD86, CD69, MHC class II, MHC class I, CCR7, and combinations thereof.

52. 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 49-51 and a pharmaceutically acceptable excipient.

53. 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 48 or claim 52 to stimulate an immune response to the antigen.

54. A method of treating cancer, the method comprising administering to an individual in need thereof an effective amount of the pharmaceutical formulation of claim 48 or claim 52 to treat the cancer.

55. 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 48 or claim 52 to inhibit abnormal cell proliferation.

56. A method for treating or preventing an infectious disease, the method comprising administering an effective amount of the pharmaceutical formulation according to claim 48 to an individual in need thereof to treat or prevent the infectious disease.

57. The method of claim 56, wherein the infectious disease is a viral disease.

58. The method of claim 56, wherein the infectious disease is a bacterial disease.

59. The method or pharmaceutical formulation of any one of claims 49-55, wherein the dendritic cells are mammalian cells.

60. The method or pharmaceutical formulation of claim 59, wherein the mammalian cell is a human cell.

61. The method of any one of claims 53-59, wherein the individual is a mammal.

62. The method of claim 61, wherein the mammal is a human.

63. The method of claim 61, wherein the mammal is a dog or a cat.

64. The composition, formulation or method of any one of claims 1-63, wherein the 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.

65. The composition, formulation or method of claim 64, wherein the at least one lipid further comprises a PEGylated lipid, optionally wherein the PEGylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoylglycerol [DMG].

66. The composition, formulation or method of any one of claims 1-65, 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.

67. The composition, formulation or method of claim 66, wherein the LNP has an effective diameter of less than about 200 nanometers.

68. The composition, formulation or method of claim 67, wherein the LNP has an effective diameter of less than about 150 nanometers.

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