mRNA immunomodulators

Abstract

The present disclosure relates to nucleic acid constructs and compositions thereof for expression of an inducer of inflammation in host cells, alone or in combination with expression of a cyclic GMP-AMP synthase. The compositions of the present disclosure are useful for inducing expression of interferon-stimulated genes and / or NF-kB-responsive genes, alone or in combination with cGAMP production.

Description

Docket No.: 16553-20014.40MRNA IMMUNOMODULATORSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U. S. Provisional Patent Application No. 63 / 778,869, filed March 27, 2025, which is incorporated by reference herein in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (165532001440seqlist.xml; Size: 68,653 bytes; and Date of Creation: March 12, 2026) is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates to nucleic acid constructs and compositions thereof for expression of an inducer of inflammation in host cells, alone or in combination with expression of a cyclic GMP-AMP synthase. The compositions of the present disclosure are useful for inducing expression of interferon-stimulated genes and / or NF-kB -responsive genes, alone or in combination with cGAMP production.BACKGROUND

[0004] Multiple types of COVID- 19 vaccines have been approved for use in humans, since the pandemic began in 2020. Specifically, the World Health Organization has approved the use of protein subunit vaccines, non-replicating viral vector vaccines, inactivated virus vaccines, and mRNA vaccines including SPIKEVAX® (Moderna) and COMIRNATY® (Pfizer and BioNTech). However, differences in types of immune responses elicited and efficacy have been reported. In particular, a COVID-19 mRNA vaccine was found to elicit higher antibody titers than did a DNA vaccine, an adenovirus-vectored vaccine, a protein subunit vaccine and an inactivated viral vaccine (Han et al., Front Immunol, 15:1455730, 2024). Conversely, the cellular immune response induced by the COVID- 19 mRNA vaccine was found to be weaker than that induced by an adenovirus-vectored vaccine (Han et al., supra, 2024).1MF-367050437Docket No.: 16553-20014.40

[0005] The induction of potent cellular immune responses by immunotherapeutics for cancer is critical. Additionally, the induction of robust cellular immune responses by prophylactic vaccines against infectious diseases is desirable. As such, formulations for diversifying the immune responses elicited by mRNA vaccines are needed in the art.BRIEF SUMMARY

[0006] The present disclosure relates to nucleic acid constructs and compositions thereof for expression of an inducer of inflammation in host cells, alone or in combination with expression of a cyclic GMP-AMP synthase. The compositions of the present disclosure are useful for inducing expression of interferon-stimulated genes and / or NF-kB -responsive genes, alone or in combination with cGAMP production.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1A depicts a bicistronic mRNA for expression of a cyclic GAMP-AMP synthase (cGAS) devoid of an amino-terminal phosphoinositide-binding domain (cGASAN) and a truncated gasdermin D (GSDMD) devoid of a carboxy-terminal auto-inhibitory domain (referred to as GSDMDAC, NT-GSDMD, or GsdmD(n)). In some preferred embodiments, the GSDMDAC comprises an I104N substitution.

[0008] FIG. IB depicts a bicistronic mRNA for expression of cGASAN and a stimulator of interferon genes (STING)-interleukin-l receptor-associated kinase 1 (IRAKI) fusion protein. In some preferred embodiments, the IRAKI is a truncated IRAKI devoid of a death domain, a proST (proline, serine, threonine) domain and a kinase domain (referred to as IRAKI AN or IRAKI (c)).

[0009] FIG. 2 shows relative viability of a GSDMD null human monocytic cell line (THP1-KO-GSDMD from InvivoGen) after contact with cGAMP, DiABZI, or lipid nanoparticles (LNPs) encapsulating an mRNA encoding an immunomodulator.

[0010] FIG. 3A and FIG.3B show levels of expression of a marker of interferon-stimulated genes (ISG) by a human monocytic reporter cell line (THP1-Blue™ ISG from InvivoGen) after contact with cGAMP, DiABZI, or lipid nanoparticles (LNPs) encapsulating an mRNA encoding an immunomodulator.2MF-367050437Docket No.: 16553-20014.40

[0011] FIG. 4A and FIG.4B show levels of expression of a marker of NF-kB-responsive genes by a human monocytic reporter cell line (THP1 -Lucia™ NF-kB from InvivoGen) after contact with cGAMP, DiABZI, or lipid nanoparticles (LNPs) encapsulating an mRNA encoding an immunomodulator.

[0012] FIG. 5 shows relative viability of a human monocytic cell line (THP1-Null2 from InvivoGen) after contact with cGAMP, DiABZI, or lipid nanoparticles (LNPs) encapsulating an mRNA encoding an immunomodulator.

[0013] FIG. 6 shows cGAMP production by a human monocytic cell line (THP1-Null2 from InvivoGen) after contact with cGAMP, DiABZI, or lipid nanoparticles (LNPs) encapsulating an mRNA encoding an immunomodulator.

[0014] FIG. 7 is a summary of responses of human monocytic cell lines after contact with cGAMP, DiABZI, or lipid nanoparticles (LNPs) encapsulating an mRNA encoding an immunomodulator.

[0015] FIG. 8 is a summary of chemokine and cytokine expression by a human monocytic cell line (THP1-Null2 from InvivoGen) after contact with cGAMP, DiABZI, or lipid nanoparticles (LNPs) encapsulating an mRNA encoding an immunomodulator.

[0016] FIG. 9 shows expression of IL-12B (IL-12p40) by a mouse conventional dendritic cell-1 line (MutuDC) after contact with lipid nanoparticles (LNPs) encapsulating an mRNA encoding an immunomodulator, in the presence or absence of an anti-CD40 antibody.

[0017] FIG. 10 is an alignment of amino acid sequences of primate cGASAN proteins including: human (Homo sapiens, SEQ ID NO:1); chimpanzee (Pan troglodytes, SEQ ID NO:7); western gorilla (Gorilla gorilla, SEQ ID NO: 8); and a cGASAN consensus sequence (SEQ ID NO:9).

[0018] FIG. 11 shows that luciferase mRNA-containing LNPs target myeloid cells resulting in expression of luciferase in human moDCs and monocytes, but not in T-cells, B-cells and NK-cells.

[0019] FIG. 12 is an illustration of the T-cell / moDC coculture system used to evaluate mRNA encoded adjuvants (cGASAN mRNA LNPs or Bicistronic mRNA LNPs).3MF-367050437Docket No.: 16553-20014.40

[0020] FIG. 13 shows that cGASAN mRNA LNPs and cGASAN-STING-IRAK mRNA LNPs induce expression of IFN-beta in T-cell / moDC cocultures in the presence and absence of peptide antigen.

[0021] FIG. 14 shows that cGASAN-STING-IRAK mRNA LNPs and STING-IRAK LNPs induce expression of IL-6 in T-cell / moDC cocultures in the presence and absence of peptide antigen.

[0022] FIG. 15 shows that cGASAN mRNA LNPs and other mRNA-encoded adjuvants induce expression of IFN-gamma in T-cell / moDC cocultures in the presence of peptide antigen.

[0023] FIG. 16 shows that treatment of human PBMCs with STING-IRAK mRNA-containing LNPs induce expression of IFN-gamma and TNF-alpha.

[0024] FIG. 17 shows that treatment of human PBMCs with mRNA-encoded adjuvants in LNPs does not induce proliferation.

[0025] FIG. 18 shows antigen-specific T-cell responses in blood of mice immunized with the indicated mRNA-LNP formulations as assessed by ex vivo restimulation with OVA peptide pool (PepTivator) followed by IFNy readouts.

[0026] FIG. 19 shows antigen-specific T-cell responses in spleens of mice immunized with the indicated mRNA-LNP formulations as assessed by ex vivo restimulation with OVA peptide pool (PepTivator) followed by IFNy readouts.

[0027] FIG. 20 shows antigen-specific T-cell responses in lungs of mice immunized with the indicated mRNA-LNP formulations as assessed by ex vivo restimulation with OVA peptide pool (PepTivator) followed by IFNy readouts.

[0028] FIG. 21A-21C show the effects of intravaginal booster immunization on cervical CD8+ T-cells, tissue-resident memory T-cells (TRM), and antigen-specific CD8+ T-cell responses. Mice were previously primed on Day 0 and Boosted on Day 14 by intramuscular administration and then Boosted again on Day 100 by intravaginal administration of OVA mRNA-LNP (0.05 pg / dose) combined with either luciferase mRNA LNPs, cGASAN mRNA LNPs, cGASAN STING-IRAKI mRNA LNPs, or STING-IRAKI mRNA LNPs (all at 1 pg / dose), or with high-dose control luciferase mRNA LNPs (1 pg / mouse) plus OVA mRNA 4MF-367050437Docket No.: 16553-20014.40 LNPs (1 pg / mouse). Five days post-boost, the cervix was collected from study mice and cells within were analyzed by flow cytometry. FIG.21A shows the absolute numbers of cervical CD3+CD8+T-cells. FIG.21B shows the absolute numbers of cervical CD8+tissue-resident memory T cells (TRM). FIG.21C shows the absolute numbers of OVA-specific, CD8+T-cells identified by SIINFEKL(SEQ ID NO: 16) tetramer staining.DETAILED DESCRIPTION

[0029] The present disclosure relates to nucleic acid constructs and compositions thereof for expression of an inducer of inflammation in host cells, alone or in combination with expression of a cyclic GMP-AMP synthase. The compositions of the present disclosure are useful for inducing expression of interferon-stimulated genes and / or NF-kB -responsive genes, alone or in combination with cGAMP production.General Techniques and Definitions

[0030] 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.

[0031] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless indicated otherwise. For example, “an” excipient includes one or more excipients.

[0032] The phrase “comprising” as used herein is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of’ is closed, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase “consisting essentially of’ is partially closed, indicating that such embodiments may further comprise elements that do not materially change the basic characteristics of such embodiments.

[0033] The term “about” as used herein in reference to a value, encompasses from 90% to 110% of that value (e.g., a molecular weight of about 900 daltons, refers to a molecular weight of from 810 daltons to 990 daltons).

[0034] An “effective amount” or a “sufficient amount” of a substance is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an5MF-367050437Docket No.: 16553-20014.40 “effective amount” depends upon the context in which it is being applied. For instance, in the context of administering an immunogenic composition comprising one or more mRNAs encoding an antigen and a constitutively-active cGAS, an effective amount contains sufficient mRNA, to stimulate an immune response against the antigen (e.g., antigen-reactive antibody and / or cellular immune response).

[0035] The terms “individual” and “subject” refer to mammals. “Mammals” include, but are not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats). In some embodiments, the subject is a human patient, such as a human patient suffering from cancer and / or an infectious disease.

[0036] The term “dose” as used herein in reference to an immunogenic composition refers to a measured portion of the immunogenic composition taken by (administered to or received by) a subject at any one time.

[0037] The terms “isolated” and “purified” as used herein refers to a 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 in reference 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 in reference to a recombinant protein, an isolated protein refers to a protein that has been removed from the culture medium of the host cell that produced the protein.

[0038] The terms “pharmaceutical formulation” and “pharmaceutical composition” refer to preparations that are in such form as to permit the biological activity of the active ingredient to be effective, and that contain no additional components that are unacceptably toxic to an individual to which the formulation or composition would be administered. Such formulations or compositions are intended to be sterile.

[0039] “Excipients” as used herein include pharmaceutically acceptable excipients, carriers, vehicles or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable excipient is an aqueous pH buffered solution.6MF-367050437Docket No.: 16553-20014.40

[0040] The terms “polypeptide” and “protein” are used interchangeably herein in reference to peptide chains that are at least 8, 9 or 10 amino acids in length.

[0041] The term “antigen” refers to a substance that is recognized and bound specifically by an antibody or by a T cell antigen receptor. Antigens can include peptides, polypeptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids and phospholipids; portions 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 a mRNA or a DNA. Polypeptide antigens are preferably at least eight amino acid residues in length, and may comprise one or more post-translational modifications.

[0042] The term “agonist” is used in the broadest sense and includes any molecule that activates signaling through a receptor. In some embodiments, the agonist binds to the receptor. For instance, a TLR8 agonist binds to a TLR8 and activates a TLR8-signaling pathway.

[0043] “Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groups. Cx alkyl refers to an alkyl group having x number of carbon atoms. Cx-Cy alkyl or Cx-y alkyl refers to an alkyl group having between x number and y number of carbon atoms, inclusive.

[0044] “Alkylene” refers to divalent saturated aliphatic hydrocarbyl groups.

[0045] “Alkenyl” refers to monovalent hydrocarbyl groups having at least one double bond (> C=C<). Cx alkenyl refers to an alkenyl group having x number of carbon atoms. Cx-Cy alkenyl or Cx-y alkenyl refers to an alkenyl group having between x number and y number of carbon atoms, inclusive.

[0046] “Stimulation” or “induction” of a response or parameter includes eliciting and / or enhancing that response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition (e.g., increase in TLR-signaling in the presence of a TLR agonist as compared to the absence of the TLR agonist). For example, “stimulation” of an immune response means an increase in the response. Depending upon the parameter measured, the increase may be from 2-fold to 2,000-fold, or from 5-fold to 500-fold or over, or from 2, 5, 10, 50, or 100-fold to 500, 1,000, 2,000, 5,000, or 10,000-fold.7MF-367050437Docket No.: 16553-20014.40

[0047] Conversely, “inhibition” of a response or parameter includes reducing and / or repressing that response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition (e.g., decrease in abnormal cell proliferation after administration of a composition of the present disclosure as compared to the administration of a placebo composition or no treatment). For example, “inhibition” of an immune response means a decrease in the response. Depending upon the parameter measured, the decrease may be from 2-fold to 2,000-fold, or from 5-fold to 500-fold or over, or from 2, 5, 10, 50, or 100-fold to 500, 1,000, 2,000, 5,000, or 10,000-fold.

[0048] The relative terms “higher” and “lower” refer to a measurable increase or decrease, respectively, in a response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition. For instance, a “higher level of DC hyperactivation” refers to a level of DC hyperactivation as a consequence of a treatment condition that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold above a level of DC hyperactivation as a consequence of a control condition. Likewise, a “lower level of DC hyperactivation” refers to a level of DC hyperactivation as a consequence of a treatment condition that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold below a level of DC hyperactivation as a consequence of a control condition.

[0049] As used herein the term “immunization” refers to a process that increases a mammalian subject’s immune response to an antigen and therefore improves its ability to resist or overcome infection and / or resist disease.

[0050] The term “vaccination” as used herein refers to the introduction of vaccine into a body of a mammalian subject.

[0051] “Adjuvant” refers to a substance which, when added to a composition comprising an antigen or a nucleic acid encoding an antigen, enhances or potentiates an immune response to the antigen in the mammalian recipient upon exposure.

[0052] The terms “treating” or “treatment” of a disease refer to executing a protocol, which may include administering one or more therapeutic agents to an individual (human or otherwise), in an effort to obtain beneficial or desired results in the individual, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or8MF-367050437Docket No.: 16553-20014.40 amelioration of one or more signs or symptoms of a disease, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total). “Treatment” also can mean prolonging survival as compared to expected survival of an individual not receiving treatment. Further, “treating” and “treatment” may occur by administration of one dose of a therapeutic agent or therapeutic agents, or may occur upon administration of a series of doses of a therapeutic agent or therapeutic agents. “Treating” or “treatment” does not require complete alleviation of signs or symptoms, and does not require a cure, and specifically includes protocols that have only a palliative effect on the individual. “Palliating” a disease or disorder means that the extent and / or undesirable clinical manifestations of the disease or disorder are lessened and / or time course of progression of the disease or disorder is slowed, as compared to the expected untreated outcome.

[0053] The term “constitutively-active” as used herein in reference to cGAS refers to a cGAS variant that displays self-DNA reactivity, leading to cGAMP synthesis under conditions in which native cGAS (e.g., full length human cGAS) has little to no enzymatic activity. In some preferred embodiments, the “constitutively-active cGAS” is a “truncated cGAS”, such as cGAS N comprising a C-terminal DNA-binding, enzymatic domain in the absence of a N-terminal disordered domain. That is, cGASAN is a constitutively-active cGAS devoid of regulation of enzymatic activity imparted by the N-terminal disordered domain of full length cGAS.

[0054] “Percent (%) sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example,9MF-367050437Docket No.: 16553-20014.40 the % sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated 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 equal the % sequence identity of B to A.I. Cyclic GMP-AMP Synthase (cGAS)

[0055] Compositions and methods of the present disclosure may comprise a nucleic acid encoding a cyclic GMP-AMP synthase, also referred to as cGAMP synthase or cGAS, which is an enzymatic sensor of cytosolic DNA. cGAS recognizes double-stranded DNA independent of its sequence resulting in dimerization, formation of liquid-like droplets and production of the secondary messenger 2’3’cyclic GMP-AMP (cGAMP), which binds to and activates STING resulting in expression of interferons and other inflammatory mediators.

[0056] Human cGAS is 522 amino acids in length, including a N-terminal phosphoinositide-binding domain (residues 1-59) and a C-terminal DNA-binding and enzymatic domain (residues 160-522) (Barnett et al., Cell, 176:1432-1446, 2019). Importantly, expression of cGAS N in a human leukemia monocytic cell line was found to result in higher levels of expression of interferon and interferon-stimulated genes (Barnett, supra, 2019).

[0057] The amino acid sequence of human cGAS is:MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQE RPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWD VPSPGLPVSAPILVRRDAAPGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLN TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKF RKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRK QLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQL KERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNL FSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF (SEQ ID NO: 10).

[0058] The amino acid sequence of the N-terminal domain of cGAS is:MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQE10MF-367050437Docket No.: 16553-20014.40 RPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWD VP SPGLPVSAP ILVRRDAA (SEQ ID NO: 11 ).

[0059] The amino acid sequence of the C-terminal domain of cGAS (cGASAN) is: PGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLNTGSYYEHVKISAPNEFDVM FKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVIMK RKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKEGNG FQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVK TAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQIE YERNNEFPVFDEF (SEQ ID NO: 1 ).

[0060] The nucleotide sequence encoding human cGASAN, which was codon-optimized for expression in mouse cells is set forth as SEQ ID NO: 17. A DNA template with this nucleotide sequence was used to prepare mRNA encoding human cGASAN, which was subsequently loaded into LNPs. The nucleotide sequence encoding human cGASAN, which was codon-optimized for expression in human cells is set forth as SEQ ID NO: 18.

[0061] Some compositions and methods of the present disclosure comprise a nucleic acid encoding a constitutively- active cGAS as a catalytic adjuvant for improving adaptive immune responses elicited by mRNA vaccines. In some preferred embodiments, the constitutively-active cGAS is a truncated cGAS devoid of the N-terminal phosphoinositidebinding domain (cGASAN). In some preferred embodiments, the constitutively-active cGAS is a truncated cGAS comprising the C-terminal DNA-binding and enzymatic domain (cGASAN). In some embodiments, the constitutively-active cGAS comprises at least one substitution.

[0062] In some embodiments, the cGAS is a variant of a full length or truncated human cGAS. In some embodiments, the variant cGAS comprises at least one substitution as described by Barber (US-2021-0017541-A1). In some embodiments, the variant cGAS comprises at least one substitution as described by Kranzusch et al. (US-2021-0324351-A1). In some embodiments, the variant cGAS comprises at least one substitution as described by Borra-Garske et al. (US-2022-0325285-A1). In some embodiments, the variant cGAS comprises at least one substitution as described by Dowling et al. (US-2024-0252598-A1). In some embodiments, the variant cGAS comprises at least one substitution as described by Ablasser et11MF-367050437Docket No.: 16553-20014.40 al. (WO 2024 / 261102). In some embodiments, the variant cGAS comprises at least one substitution as described by Manel et al. (WO 2025 / 027116).

[0063] In some embodiments, the variant cGAS has an improved property in comparison with the full length human cGAS (SEQ ID NO: 10) or the truncated human cGAS (SEQ ID NO:1). In some embodiments, the improved property is “constitutive activity”. In some embodiments, the improved property is “increased enzymatic activity”, which can be represented by an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of enzyme) as compared to the reference cGAS. That is, “increased enzymatic activity” by a variant cGAS comprises increased cGAMP production.

[0064] Homologs of cGAS are expressed in species across the animal kingdom, and cGAS amino acid sequences are conserved in higher primates. An alignment of the amino acid sequence of the c-terminal domain of human cGAS with the amino acid sequences of the c-terminal domains of chimp and gorilla cGAS proteins is shown in FIG. 10. A percent identity matrix for primate cGAS N proteins is shown in Table I.Table I. Percent Identity Matrix Created by Clustal 12.1A# Species 1 2 3 4 5 6 7 8 1 M._mulatta 100.00 96.42 87.53 85.95 88.71 87.26 86.78 86.50 2 P._anubis 96.42 100.00 87.53 86.23 88.98 88.09 87.88 87.33 3 N. Jeucogenys 87.53 87.53 100.00 96.14 93.37 89.97 90.03 90.30 4 H. Jar 85.95 86.23 96.14 100.00 92.31 88.92 88.98 88.71 5 P._abelii 88.71 88.98 93.37 92.31 100.00 93.07 93.39 92.84 6 P._troglodytes 87.26 88.09 89.97 88.92 93.07 100.00 97.51 96.95 7 H._sapiens 86.78 87.88 90.03 88.98 93.39 97.51 100.00 98.358 G._gorilla 86.50 87.33 90.30 88.71 92.84 96.95 98.35 100.00ANumbers in this table are not SEQ ID NOS.

[0065] In some preferred embodiments, the cGAS is a truncated human cGAS devoid of the N-terminal domain (SEQ ID NO: 11). In some preferred embodiments, the cGAS is a truncated human cGAS comprising the C-terminal domain (SEQ ID NO:1). In some preferred embodiments, cGASAN comprises the amino acid sequence of SEQ ID NO:1 or the amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1. In12MF-367050437Docket No.: 16553-20014.40 some preferred embodiments, cGASAN 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 the amino acid sequence 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, cGASAN comprises the consensus amino acid sequence of SEQ ID NO:9. For expression in transfected cells, the nucleic acid encoding cGASAN is in operable combination with a start codon (e.g., AUG, CUG, GUG or ACG, preferably AUG).II. Inducers of Inflammation

[0066] Compositions and methods of the present disclosure comprise a nucleic acid encoding one or more inducers of inflammation, alone or in combination with cGAS.Particularly desirable inducers of inflammation include but at not limited to gasdermin, STING and IRAK.A. Gasdermin (GSDM)

[0067] The gasdermin (GSDM) family of proteins is involved in the release of inflammatory cytokines and induction of pyroptosis, which is an inflammatory form of programmed cell death. Five of the six gasdermin family members have a conserved structure including a N-terminal lipid-binding domain and a C-terminal auto-inhibitory domain separated by a linker (Kaczor et al., Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 1880(2): 189283, 2025). Full-length human gasdermin D (GSDMD) is 484 amino acids in length (UniProt No. P57764). Upon cleavage of GSDMD between residues 275 and 276 by caspase 1, 4, 5, 8 or 11, the N-terminal domain (GSDMD-N or GSDMDAC residues 1-275) is liberated from the C-terminal domain (residues 276-484) and subsequently localizes to the plasma membrane where it homo-oligomerizes to form transmembrane pores (Devant and Kagan, Nature Immunology, 24:1064-1075, 2023). Compositions and methods of the present disclosure may comprise a nucleic acid encoding a truncated GSDMD devoid of a carboxy-terminal auto-inhibitory domain (GSDMDAC).

[0068] Particularly desirable GSDMDAC variants for use in the compositions and methods of the present disclosure comprise an I104N amino acid substitution, which has been found to induce pore formation while delaying pyroptosis (Kayagaki et al., Nature 526:666-671, 2015). The amino acid sequence of human GSDMDAC without an N-terminal methionine is set13MF-367050437Docket No.: 16553-20014.40 forth SEQ ID NO: 19 (comprising the I104N amino acid substitution, which is reflected by an asparagine at position 103). The amino acid sequence of GSDMDAC is conserved in higher primates, with the chimpanzee (P. troglodytes') and the gorilla (G. gorilla) homologs having at least 98% identity with the human sequence. The amino acid sequence of chimpanzee GSDMDAC comprising the I104N amino acid substitution without an N-terminal methionine is set forth SEQ ID NO:25. The amino acid sequence of gorilla GSDMDAC comprising the I104N amino acid substitution without an N-terminal methionine is set forth SEQ ID NO:26. Lastly, a consensus amino acid sequence comprising the I104N amino acid substitution without an N-terminal methionine, which was defined after aligning the human, chimpanzee and gorilla GSDMDAC sequences using CLUSTAL 2.1, is set forth as SEQ ID NO:27.

[0069] In some embodiments, the GSDMDAC comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the GSDMDAC comprises the consensus amino acid sequence of SEQ ID NO:27. In some embodiments, the GSDMDAC comprises the amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19, such as the chimpanzee GSDMDAC of SEQ ID NO:25 or the gorilla GSDMDAC of SEQ ID NO: 26. The nucleotide sequence encoding the human GSDMDAC mutant (I104N), which was codon-optimized for expression in human cells, is set forth as SEQ ID NO:34.B. Stimulator of Interferon Genes (STING)

[0070] The stimulator of interferon genes (STING) protein also known as transmembrane protein 173 (TMEM173) is involved in production of interferons and other inflammatory mediators. Specifically, STING activation upon binding of cGAMP results in a signaling cascade that ultimately activates IRF3 and NF-kB transcription factors (Chauvin et al., J Biol Chem, 299(7): 104866, 2023). IRF3 activation leads to the expression of genes such as IP-10 and Type I interferons that induce antiviral immune responses. NF-kB activation induces the expression of inflammatory cytokines such as IL-6 that enhance inflammatory immune responses. Full-length STING is 379 amino acids in length (UniProt No. Q86WV6) and includes four transmembrane domains and a C-terminal domain containing a dimerization domain and a carboxy -terminal tail.14MF-367050437Docket No.: 16553-20014.40

[0071] The amino acid sequence of human STING is set forth as SEQ ID NO:21. The amino acid sequence of STING is conserved in higher primates, with the chimpanzee (P. troglodytes') and the gorilla (G. gorilla) homologs having at least 98% identity with the human sequence. The amino acid sequence of chimpanzee STING is set forth SEQ ID NO:28. The amino acid sequence of gorilla STING is set forth SEQ ID NO:29. Lastly, a consensus amino acid sequence, which was defined after aligning the human, chimpanzee and gorilla STING sequences using CLUSTAL 2.1, is set forth as SEQ ID NO:30.

[0072] In some embodiments, the STING comprises the amino acid sequence of SEQ ID NO:21. In some embodiments, the STING comprises the consensus amino acid sequence of SEQ ID NO:30. In some embodiments, the STING comprises the amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21, such as the chimpanzee STING of SEQ ID NO:28 or the gorilla STING of SEQ ID NO: 29. The nucleotide sequence encoding the human STING, which was codon-optimized for expression in human cells, is set forth as SEQ ID NO:36.

[0073] Nucleic acids encoding STING are contemplated to provide particular benefit for expression of STING in cells in which function of endogenous STING is downregulated, such as in cancerous cells (Miglietta et al., Br J Cancer, 131:1567-1575, 2024; and Jiang et al., J Hematol Oncol, 13:81, 2020), or in cells expressing a STING allele comprising a loss-of-function mutation (Patel and Jin, Genes Immun, 20:82-89, 2019; and Motedayen Aval et al., J Clin Med, 9(10):3323, 2020).C. Interleukin-1 Receptor-Associated Kinase (IRAK)

[0074] The Interleukin- 1 Receptor- Associated Kinase (IRAK) family of proteins is involved in the release of inflammatory cytokines. Specifically, IRAK proteins are signal transducers and involved in the response to activation of some Toll-Like Receptor (TLR) signaling pathways and in activation of interleukin 1 receptor signaling pathway. Four IRAK family members have been described in humans, and all regulate the NF-kB and mitogen activated protein MAPK pathways. IRAK family proteins are conserved in primates, and contain a N-terminal death domain, a proline, serine, and threonine rich domain (pro ST), a kinase domain, and a C-terminal domain containing TNF receptor-associated factor 6 (TRAF6) binding sites (Pereira and Gazzinelli, Front Immunol, 14:1133354, 2023; and Gosu et al., PLoS One,15MF-367050437Docket No.: 16553-20014.40 7(ll):e49771, 2012). Full length human IRAKI is 712 amino acids in length (UniProt No.P51617), including three TRAF6 binding domains in its C-terminal domain (Ye et al., Nature, 418(6896):443-447, 2002).

[0075] The C-terminal domain of human IRAKI, referred to herein as IRAKI AN or IRAKI (c), is 190 amino acids in length, and consists of the amino acid sequence of SEQ ID NO:22. The amino acid sequence of IRAKI AN is conserved in higher primates, with the chimpanzee (P. troglodytes') and the gorilla (G. gorilla) homologs having at least 98% identity with the human sequence. The amino acid sequence of chimpanzee IRAKI AN is set forth SEQ ID NO:31. The amino acid sequence of gorilla IRAKI AN is set forth SEQ ID NO:32. Lastly, a consensus amino acid sequence, which was defined after aligning the human, chimpanzee and gorilla IRAK1 N sequences using CLUSTAL 2.1, is set forth as SEQ ID NO:33.

[0076] In some embodiments, the IRAKI AN comprises the amino acid sequence of SEQ ID NO:22. In some embodiments, the IRAK1 N comprises the consensus amino acid sequence of SEQ ID NO: 33. In some embodiments, the IRAKI AN comprises the amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:33, such as the chimpanzee IRAKI AN of SEQ ID NO:31 or the gorilla IRAKI AN of SEQ ID NO: 32. The nucleotide sequence encoding the human IRAKI AN, which was codon-optimized for expression in human cells, is set forth as SEQ ID NO:37.III. mRNA Components

[0077] The mRNA of the present disclosure may comprise elements in addition to a coding region of at least one HPV antigen, alone or in combination with a coding region of a cGAS. In some 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.

[0078] Preferably, the mRNA of the present disclosure comprises a modified nucleoside. Modified nucleosides for use in connection with the mRNAs of the present disclosure include but are not limited to modified uridine, modified cytidine, modified adenine and modified guanine. In some embodiments, the modified nucleoside comprises a modified16MF-367050437Docket No.: 16553-20014.40 uridine. In some embodiments, the modified uridine comprises Nl-methyl-pseudouridine. In some embodiments, the modified nucleoside comprises one or more of the group consisting of Nl-methyl-pseudouridine, 5 -methylcytidine (m5C), 5 -methyluridine (m5U), N6-methyladenosine (m6A), 2-thiouridine (s2U), pseudouridine, 2’-O-methyluridine (um), and 5-methoxyuridine (mo5U). Modified nucleosides for use in the mRNAs of the present disclosure are known in the art (see, e.g., paragraphs

[0049] and

[0050] of US 2025 -0019719- Al of Weissman and Kariko, which are incorporated herein by reference).

[0079] In some embodiments, the mRNA comprises a coding region of both a cyclic GMP-AMP synthase (cGAS) and a coding region of an inducer of inflammation (e.g., GSDM, STING, IRAK or a combination thereof), which are separated by an intervening sequence. In some embodiments comprising two or more inducers of inflammation, the coding regions of the inducers of inflammation are separated by an intervening sequence. Intervening sequences for use in the constructs of the present disclosure encode a 2A-like peptide sequences, encode a protease cleavage sites, or comprises an internal ribosome entry sites (IRES). In some embodiments, the intervening sequence encodes a T2A (SEQ ID NO: 12), P2A (SEQ ID NO: 13), E2A (SEQ ID NO: 14), or F2A (SEQ ID NO: 15) peptide sequence. Additional 2A-like peptide sequences are known in the art (see, e.g., Luke et al., J. Gen. Virol, 89:1036-1042, 2008, 2AL sequences of Figure 2 are incorporated herein by reference). In some embodiments, the intervening sequence encodes a protease cleavage site, such as a furin cleavage site (SEQ ID NO:40). In some embodiments, intervening sequence is an IRES.IV. Delivery Vehicles

[0080] Compositions and methods of the present disclosure may comprise mRNA encapsulated in a nanoparticle or as part of a complex. For instance, the compositions and methods of the present disclosure may comprise a lipid-based delivery vehicle for an mRNA vaccine. In some embodiments, the vehicle is a lipid nanoparticle (LNP). In other embodiments, the vehicle is a lipid that forms a complex with the mRNA (RNA-Lipoplex). In some embodiments, the nanoparticle is polymeric nanoparticle (PNP) of approximately 1 nanometer to 1000 nanometers in diameter formed from one or more polymers. Polymeric nanoparticles can be in the form of micelles, metal-organic frameworks, dendrimeric assemblies, hydrogels, liposomes, or polymerosomes (see, e.g., Xiao et al., Front Bioeng Biotechnol, 10:1024143,17MF-367050437Docket No.: 16553-20014.40 2022). In some embodiments, the polymeric nanoparticle comprises a non-lipid polymer. In some embodiments, the nanoparticle is a non-lipid polymeric nanoparticle formed primarily or exclusively from non-lipid polymers. In some embodiments, the nanoparticle is a protein nanoparticle. In some embodiments, the nanoparticle is a viral particle. In other embodiments, the nanoparticle is a virus-like particle. In some embodiments, the nanoparticle is in the form of a cationic nanoemulsion.

[0081] In some embodiments, the LNP comprises at least one lipid selected from the group consisting of an ionizable lipid, a cationic lipid, a phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the at least one lipid comprises an ionizable lipid. In some embodiments, the at least one lipid comprises a cationic lipid. In some embodiments, the at least one lipid comprises a phospholipid. In some embodiments, the at least one lipid comprises a pegylated lipid. In some embodiments, the at least one lipid comprises a structural lipid. In some embodiments, the at least one lipid comprise an ionizable lipid, a phospholipid, a pegylated lipid, and a structural lipid.

[0082] 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 distinct from the second lipid. In some embodiments, the first lipid is a cationic lipid and the second lipid is a neutral or anionic lipid.

[0083] Structures of lipids suitable for use in the lipid-based mRNA delivery vehicles of the present disclosure are known in the art (see, Figure 2 of Hou et al., Nature Review Materials, 6:1078-1094, 2021, herein incorporated by reference).

[0084] A wide variety of polymers can be used to form non-lipid PNPs, such as a poly(P-amino ester) (PBAE), a cyclodextrin (CD), a polyethyleneimine (PEI), a poly(lactic-co-glycolic acid) (PLGA), a poly(lactic acid) (PLA), a polyamidoamine (PAMAM) dendrimer or an ionizable amphiphilic Janus dendrimer. Poly(beta-amino ester) polymers have a reversible positive charge, which promotes binding to nucleic acids and imparts high buffering capacity (Karlsson et al., Expert Opin Drug Deliv, 17(10): 1395-1410, 2020). Cyclodextrins are cyclic oligosaccharides composed of glucose units connected by alpha- 1,4 linkages. Cyclodextrin nanoparticles (Gadade et al., Adv Pharm Bull, 10(2): 166-183, 2020) have been used for encapsulation of a wide variety of therapeutics, including nucleic acids. Polyethylenimine (PEI)18MF-367050437Docket No.: 16553-20014.40 is a cationic polymer, which condenses with nucleic acids to form nanoparticles. Nanoparticles formed from polyesters include but are not limited to poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA). PNPs formed from dendrimers are called dendrimersomes or dendrimeric nanoparticles. Exemplary dendrimers include but are not limited to PAM AM dendrimers (Tarach et al., Int J Mol Sci, 13;22(6):2912, 2021) and ionizable amphiphilic Janus dendrimers (Lu et al., J Am Chem Soc, 145(34):18760-18766, 2023).V. Pharmaceutical Formulations

[0085] Some compositions of the present disclosure are pharmaceutical formulations comprising a pharmaceutically acceptable excipient. Pharmaceutical formulations of the present disclosure may be in the form of a solution or a suspension. Alternatively, the pharmaceutical formulations may be a dehydrated solid (e.g., freeze dried or spray dried solid). The pharmaceutical formulations of the present disclosure are preferably sterile, and preferably essentially endotoxin-free. The term “pharmaceutical formulations” is used interchangeably herein with the terms “medicinal product” and “medicament”. In some embodiments, the pharmaceutical formation comprises specific ratios of the various components based on the intended purpose of the formulation.

[0086] Pharmaceutically acceptable excipients of the present disclosure include for instance, solvents, buffering agents, tonicity adjusting agents, bulking agents, and preservatives (See, e.g., Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the pharmaceutical formulations may comprise an excipient that functions as one or more of a solvent, a buffering agent, a tonicity adjusting agent, and a bulking agent (e.g., sodium chloride in saline may serve as both an aqueous vehicle and a tonicity adjusting agent).

[0087] In some embodiments, the pharmaceutical formulations comprise an aqueous vehicle as a solvent. Suitable vehicles include for instance sterile water, saline solution, phosphate buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0088] The pharmaceutical formulations may comprise a buffering agent. Buffering agents control pH to inhibit degradation of the active agent during processing, storage and optionally reconstitution. Suitable buffers include for instance salts comprising acetate, citrate,19MF-367050437Docket No.: 16553-20014.40 phosphate or sulfate. Other suitable buffers include for instance amino acids such as arginine, glycine, histidine, and lysine. The buffering agent may further comprise hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition within a 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 in which the lower limit is less than the upper limit.

[0089] The pharmaceutical compositions may comprise a tonicity adjusting agent. Suitable tonicity adjusting agents include for instance dextrose, glycerol, sodium chloride, glycerin and mannitol.

[0090] The pharmaceutical formulations may comprise a bulking agent. Bulking agents are particularly useful when the pharmaceutical composition is to be lyophilized before administration. In some embodiments, the bulking agent is a protectant that aids in the stabilization and prevention of degradation of the active agents during freeze or spray drying and / or during storage. Suitable bulking agents are sugars (mono-, di- and polysaccharides) such as sucrose, lactose, trehalose, mannitol, sorbital, glucose and raffinose.

[0091] The pharmaceutical formulations may comprise a preservative. Suitable preservatives include for instance antioxidants and antimicrobial agents. However, in preferred embodiments, the pharmaceutical formulation is prepared under sterile conditions and is in a single use container, and thus does not necessitate inclusion of a preservative.

[0092] 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 (e.g., not by orally, gastrically, or rectally).VI. Methods of Use

[0093] In some aspects, the present disclosure relates to methods of use of any one of the compositions or formulations described herein. The methods of use are suitable for a plurality of uses involving stimulating an immune response. In some embodiments, the methods of use comprise methods of treating cancer. In some embodiments, the methods of use comprise methods of inhibiting abnormal cell proliferation. In some embodiments, the methods of use comprise methods of treating or preventing an infectious disease. The methods comprise20MF-367050437Docket No.: 16553-20014.40 administering an effective amount of a formulation or a composition described herein to an individual in need thereof to achieve a specific outcome. The individual is a mammalian subject, such as a human patient. In other embodiments, the individual a non-human patient. In some embodiments, the methods of use involve clinical uses, while in other embodiments the methods of use involve pre-clinical and / or veterinary uses. For preclinical uses, the mammalian subject may be a non-human primate (e.g., monkey or ape) or a rodent (e.g., mouse or rat). For veterinary uses the mammalian subject may be a farm animal (e.g., cow), a sport animal (e.g., horse), or a pet (e.g., companion animal such as a dog or cat).

[0094] In brief, the present disclosure provides methods of stimulating an immune response in an individual, comprising administering to the individual a composition or formulation described herein in an amount sufficient to stimulate an immune response in the individual. “Stimulating” an immune response (used interchangeably with “eliciting” and immune response), means increasing the immune response, which can arise from eliciting a de novo immune response (e.g., as a consequence of an initial vaccination regimen) or enhancing an existing immune response (e.g., as a consequence of a booster vaccination regimen). In some embodiments, stimulating an immune response comprises one or more of the group consisting of: stimulating cytokine production; stimulating B lymphocyte proliferation; stimulating interferon pathway-associated gene expression; stimulating chemoattractant-associated gene expression; and stimulating dendritic cell DC maturation. Methods for measuring stimulation of an immune response are known in the art.

[0095] In some instances, the present disclosure provides methods of inducing an antigen-specific immune response in an individual by administering to the individual a composition or formulation described herein in an amount sufficient to induce an antigenspecific immune response in the individual. In preferred embodiments, the composition or formulation comprises the antigen. In some embodiments, the composition or formulation is administered to a tissue of the individual comprising the antigen. The immune response may comprise 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 titer of the antigen-specific antibodies above a threshold level such as a pre-administration baseline titer or a seroprotective level.21MF-367050437Docket No.: 16553-20014.40 “Inducing” an antigen-specific CTL response means increasing frequency of antigen-specific CTL found in peripheral blood above a pre-administration baseline frequency. “Inducing” an antigen-specific Th cell response means increasing frequency of antigen-specific Th cells found in peripheral blood above a pre-administration baseline frequency.

[0096] Analysis (both qualitative and quantitative) of the immune response can be 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 populations of lymphocytes such as B cells and helper T cells, production of cytokines such as IFN-alpha, IFN-gamma, IL-6, IL-12 and / or release of histamine. Methods for measuring antigen-specific antibody responses include enzyme-linked immunosorbent assay (ELISA). Activation of specific populations of lymphocytes can be measured by proliferation assays, and with fluorescence-activated cell sorting (FACS). Production of cytokines can also be measured by ELISA. In some embodiments, methods of stimulating an immune response comprise stimulation of interleukin-1 beta (IL-1β) secretion, interferon-gamma (IFN-y) secretion, and / or tumor necrosis factor-alpha (TNF-a) secretion by monocyte-derived dendritic cells or peripheral blood mononuclear cells. In some preferred embodiments, at least 50%, 55%, 60%, 65%, 70% or 75% of the cells contacted with a composition of the present disclosure remain viable at 40-56 hours (or about 48 hours) post-contact.

[0097] In some embodiments, the methods are suitable for stimulating an anti-tumor immune response. In other embodiments, the methods are suitable for stimulating an antimicrobe immune response. In some embodiments, the anti-microbe response is an anti-bacterial immune response. In some embodiments, the anti-microbe response is an anti-fungal immune response. In some embodiments, the anti-microbe response is an anti-viral immune response. In some embodiments, the anti-microbe response is an anti-protozoan immune response.

[0098] The nucleic acid constructs of the present disclosure and compositions thereof are suitable for diversifying immune activities of antigen presenting cells including monocytes, macrophages and dendritic cells. The constructs described herein are also suitable for promoting inflammation when present in other types of cells. In particular, the constructs described herein are suitable for increasing cGAMP production, interferon-stimulated gene (ISG) expression, NF-22MF-367050437Docket No.: 16553-20014.40 kB-responsive gene expression, and / or antigen release. Table II provides a summary of the activities contemplated to be promoted by the nucleic acid constructs of the present disclosure.Table II. Induction of Immune ActivitiesNucleic Acid Construct cGAMP IFN NF-kB Antigen Production Response Response Release cGASAN-dbl mut - - - - cGASAN + + - - cGASAN-STING-IRAKl(c) + + + - STING-IRAKI (c) - + / - + - cGASAN-GsdmAC + + - +GsdmAC - - - +A. cGAMP Production

[0099] The present disclosure provides methods of inducing cGAMP production by antigen presenting cells (APCs), comprising contacting the APCs with the composition or formulation as described herein comprising the coding region of a cyclic GMP-AMP synthase (cGAS) under conditions suitable for expressing cGAS in the APCs resulting in production of cGAMP by the APCs. One method of analyzing the immune response is by quantifying cGAMP production by lysing the APCs to release intracellular cGAMP and measuring cGAMP by enzyme-linked immunosorbent assay (ELISA).B. Interferon-Stimulated Gene Expression (“IFN Response”)

[0100] The present disclosure provides methods of inducing expression of one or more interferon-stimulated genes by antigen presenting cells (APCs), comprising contacting the APCs with the composition or formulation described herein comprising the coding region of a cyclic GMP-AMP synthase (cGAS) and / or the coding regions of stimulator of interferon genes (STING) and interleukin- 1 receptor-associated kinase (IRAK) under conditions suitable for expressing the cGAS and / or the STING and IRAK in the APCs resulting in expression of the one or more interferon-stimulated genes. One method of evaluating interferon-stimulated gene (ISG) expression is by quantifying SEAP production by a human monocytic cell line with a reporter controlled by ISG activation (THP1-Blue™ ISG). Another method of evaluating ISG expression is by measuring interferon-stimulated cytokine production by APCs using an immunoassay. In some embodiments, the interferon-stimulated cytokine comprises but is not limited to IP-1023MF-367050437Docket No.: 16553-20014.40 (CXCL10), IFN-P, RANTES (CCL5), IFN-X1, and combinations thereof. In some embodiments, the interferon-stimulated cytokine comprises monocyte chemoattractant protein 1 (MCP1 or CCL2).C. NF-kB-Responsive Gene Expression (“NF-kB Response”)

[0101] The present disclosure provides methods of inducing expression of one or more NF-kB -responsive genes by antigen presenting cells (APCs), comprising contacting the APCs with the composition or formulation described herein comprising the coding regions of stimulator of interferon genes (STING) and interleukin- 1 receptor- associated kinase (IRAK) under conditions suitable for expressing the STING and the IRAK in the APCs resulting in expression of the one or more NF-kB -responsive genes. One method of evaluating NF-kB responsive gene expression is by quantifying luciferase activity by a human monocytic cell line with a reporter controlled by NF-kB activation (THP1-Lucia™ NF-kB). Another method of evaluating NF-kB-responsive gene expression is by measuring NF-kB-responsive cytokine production by APCs using an immunoassay. In some embodiments, the NF-kB-responsive cytokine comprises but is not limited to TNF-a, IL-6, IL-8 (CXCL8), IL-10, IL-10, IL-12, and combinations thereof. In some embodiments, the NF-kB-responsive cytokine comprises macrophage inflammatory protein la (MIP-la or CCL3) and macrophage inflammatory protein 1P (MIP-1β or CCL4).D. Antigen Release

[0102] The present disclosure provides methods of inducing formation of pores in plasma membranes of cells, comprising contacting the cells with the composition or formulation described herein comprising the coding region of a gasdermin (GSDM) under conditions suitable for expressing the GSDM in the cells resulting in formation of the pores in the plasma membranes of the cells. One method of evaluating pore formation is by assessing gasdermin D oligomerization is by western blot, involving probing cell lysates with an antibody reactive with gasdermin D. In this way, assessing gasdermin D oligomerization is measured as a surrogate for assessing antigen release.24MF-367050437Docket No.: 16553-20014.40 E. Cytokine Secretion

[0103] Additionally, the present disclosure provides methods of inducing expression of one or more cytokines in mammalian cells, comprising contacting the mammalian cells with the composition or formulation described herein comprising the coding regions of cGASAN-STING-IRAKl(c), STING-IRAKI (c), cGASAN-GsdmAC, and / or GsdmAC under conditions suitable for expressing one or more of the cGAS N, STING, IRAKI (c) and GsdmAC in the mammalian cells resulting in expression of the one or more cytokines. In some embodiments, the mammalian cells are immune cells such as APCs and / or lymphocytes. Production of cytokines can be measured by immunoassay. As used herein, the term “cytokine” encompasses interleukins, lymphokines, monokines, interferons, chemokines and some growth factors (colony stimulating factors).Enumerated Embodiments1. A nucleic acid comprising a coding region of a cyclic GMP-AMP synthase (cGAS) and a coding region of an inducer of inflammation, optionally wherein the nucleic acid is isolated.2. The nucleic acid of embodiment 1, wherein the inducer of inflammation comprises a gasdermin (GSDM), a stimulator of interferon genes (STING), an interleukin- 1 receptor-associated kinase (IRAK), or a combination thereof.3. A nucleic acid comprising a coding region of a cyclic GMP-AMP synthase (cGAS) and a coding region of a gasdermin (GSDM).4. The nucleic acid of embodiment 2 or embodiment 3, wherein the GSDM is a truncated gasdermin D devoid of a carboxy -terminal auto-inhibitory domain (GSDMDAC).5. The nucleic acid of embodiment 4, wherein the GSDMDAC comprises:i) the amino acid sequence of SEQ ID NO: 19;ii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19; oriii) the consensus amino acid sequence of SEQ ID NO:27.25MF-367050437Docket No.: 16553-20014.40 6. The nucleic acid of embodiment 5, wherein the GSDMDAC comprises an I104N substitution, which corresponds to N at position 103 of SEQ ID NO: 19.7. A nucleic acid comprising a coding region of a cyclic GMP-AMP synthase (cGAS) and a coding region of one or both of a stimulator of interferon genes (STING) and an interleukin-1 receptor-associated kinase (IRAK).8. The nucleic acid of embodiment 2 or embodiment 7, comprising the coding region of STING, wherein the STING comprises:i) the amino acid sequence of SEQ ID NO:21;ii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21; oriii) the consensus amino acid sequence of SEQ ID NO:30.9. The nucleic acid of embodiment 2 or embodiment 7, comprising the coding region of IRAK, wherein the IRAK is a truncated IRAKI devoid of a death domain, a proST (proline, serine, threonine) domain and a kinase domain (IRAKI AN), or wherein the IRAK is a truncated IRAKI consisting of a carboxy -terminal domain comprising one or more tumor necrosis factor (TNF) receptor associated factor-6 (TRAF6)-binding sites (IRAK1C).10. The nucleic acid of embodiment 9, wherein the IRAKI AN comprises:i) the amino acid sequence of SEQ ID NO:22;ii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:22; oriii) the consensus amino acid sequence of SEQ ID NO:33.11. The nucleic acid of embodiment 2 or embodiment 7, comprising the coding region of STING and the coding region of IRAK, wherein the IRAK is a truncated IRAKI devoid of a death domain, a proST (proline, serine, threonine) domain and a kinase domain (IRAKI AN).12. The nucleic acid of embodiment 11, wherein the STING and the IRAKI AN are expressed as a STING-IRAKI AN fusion protein.26MF-367050437Docket No.: 16553-20014.40 13. The nucleic acid of embodiment 12, wherein the STING-IRAKI AN fusion protein comprises:i) the amino acid sequence of SEQ ID NO:24; orii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:24.14. The nucleic acid of any one of embodiments 1-13, wherein the cGAS is a constitutively-active cGAS that has a greater propensity to self-DNA reactivity than its wild-type counterpart.15. The nucleic acid of any one of embodiments 1-14, wherein the cGAS is a truncated cGAS devoid of an amino-terminal phosphoinositide-binding domain (cGASAN).16. The nucleic acid of embodiment 15, wherein the cGASAN comprises:i) the amino acid sequence of SEQ ID NO: 1;the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1; oriii) the consensus amino acid sequence of SEQ ID NO:9.17. The nucleic acid of any one of embodiments 1-16, wherein the coding region of the cGASAN is in operable combination with a start codon, optionally wherein the start codon is AUG, CUG, GUG or ACG, optionally wherein the start codon is AUG.18. The nucleic acid of embodiment 17, wherein the cGASAN is encoded by the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 17.19. The nucleic acid of any one of embodiments 2-6 or any one of embodiments 14-18, wherein the coding region of the cGAS is separated from the coding region of the GSDM by an intervening sequence, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES).20. The nucleic acid of embodiment 19, wherein the intervening sequence encodes a 2A-like peptide sequence such that the coding regions of the cGAS and the GSDM are configured as cGAS-2A-GSDMcomprising the amino acid sequence of SEQ ID NO:20 or the amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:20.27MF-367050437Docket No.: 16553-20014.40 21. The nucleic acid of embodiment 20, wherein the cGAS-2A-GSDM is encoded by the nucleotide sequence of SEQ ID NO:35.22. The nucleic acid of embodiment 1, embodiment 2 or any one of embodiments 12-18, wherein the coding region of the cGAS is separated from the coding region of the STING-IRAK fusion protein by an intervening sequence, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES).23. The nucleic acid of embodiment 22, wherein the intervening sequence encodes a 2A-like peptide sequence such that the coding regions of the cGAS and the STING-IRAK are configured as cGAS-2A-STING-IRAKcomprising the amino acid sequence of SEQ ID NO:23 or the amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:23.24. The nucleic acid of embodiment 23, wherein the cGAS-2A-STING-IRAK is encoded by the nucleotide sequence of SEQ ID NO:38.25. A nucleic acid comprising a coding region of a stimulator of interferon genes (STING) and a coding region of interleukin- 1 receptor-associated kinase (IRAK).26. The nucleic acid of embodiment 25, wherein the STING comprises:i) the amino acid sequence of SEQ ID NO:21;ii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21; oriii) the consensus amino acid sequence of SEQ ID NO:30.27. The nucleic acid of embodiment 25 or embodiment 26, wherein the IRAK is a truncated IRAKI devoid of a death domain, a proST (proline, serine, threonine) domain and a kinase domain (IRAKI AN) or wherein the IRAK is a truncated IRAKI consisting of a carboxyterminal domain comprising one or more tumor necrosis factor (TNF) receptor associated factor-6 (TRAF6)-binding sites (IRAK1C).28. The nucleic acid of embodiment 27, wherein the IRAK1 N comprises:i) the amino acid sequence of SEQ ID NO:22;ii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or28MF-367050437Docket No.: 16553-20014.40 99% identical to SEQ ID NO:22; oriii) the consensus amino acid sequence of SEQ ID NO:33.29. The nucleic acid of embodiment 28, wherein the STING and the (IRAKI AN) are expressed as a STING-IRAKI AN fusion protein.30. The nucleic acid of embodiment 29, wherein the STING-IRAKI AN fusion protein comprises:i) the amino acid sequence of SEQ ID NO:24; orii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:24.31. The nucleic acid of embodiment 30, wherein the STING-IRAK is encoded by the nucleotide sequence of SEQ ID NO:39.32. The nucleic acid of any one of embodiments 1-31, wherein the nucleic acid is an mRNA.33. The nucleic acid of embodiment 32, wherein the mRNA comprises a 5’ untranslated region (5’UTR) and a 3’ untranslated region (3’UTR).34. The nucleic acid of embodiment 33, wherein the mRNA further comprises one or both of a 5' cap structure and a poly A tail.35. The nucleic acid of any one of embodiments 32-34, wherein the mRNA comprises a modified nucleoside, optionally wherein the modified nucleoside comprises one or more of the group consisting of Nl-methyl-pseudouridine, 5 -methylcytidine (m5C), 5-methyluridine (m5U), N6-methyladenosine (m6A), 2-thiouridine (s2U), pseudouridine, 2’-O-methyluridine (um), and 5 -methoxyuridine (mo5U), optionally wherein the modified nucleoside comprises N 1 -methyl-pseudouridine.36. The nucleic acid of any one of embodiments 32-35, wherein the mRNA is less than about 5000, 4000 or 3000 nucleotides in length.37. The nucleic acid of any one of embodiments 1-31, wherein the nucleic acid is a DNA.29MF-367050437Docket No.: 16553-20014.40 38. The nucleic acid of embodiment 37, wherein the DNA comprises an expression cassette in which the coding region of the cGAS or the STING is in operable combination with a promoter.39. The nucleic acid of embodiment 38, wherein the expression cassette is present within a plasmid, wherein the plasmid comprises an origin of replication and a selectable marker.40. The nucleic acid of embodiment 39, wherein the plasmid DNA is less than about 15,000, 14,000, 13,000, 12,000, 11,000, 10,000, 9,000 or 8,000 nucleotides in length.41. A composition comprising the mRNA of any one of embodiments 32-36 encapsulated in a nanoparticle.42. The composition of embodiment 41, wherein the nanoparticle is a lipid nanoparticle (LNP) comprising an ionizable lipid, a pegylated lipid, a structural lipid, a phospholipid, or a combination thereof, optionally wherein the LNP comprises an ionizable lipid.43. The composition of embodiment 42, wherein the LNP comprises the phospholipid, the ionizable lipid, the pegylated lipid, and the structural lipid.44. The composition of embodiment 42 or embodiment 43, wherein the ionizable lipid comprises:i) 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester (SM-102) or analogs or derivatives thereof; and / orii) 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium (ALC-0315) or analogs or derivatives thereof; and / oriii) (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) or analogs or derivatives thereof.45. The composition of any one of embodiments 42-44, wherein the pegylated lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatide acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglyerol, and combinations thereof, optionally wherein the pegylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG],30MF-367050437Docket No.: 16553-20014.40 46. The composition of any one of embodiments 42-45, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and combinations thereof, optionally wherein the structural lipid comprises cholesterol.47. The composition of any one of embodiments 42-46, wherein the phospholipid comprises:i) a hydrophilic head moiety selected from the group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin; andii) one or more fatty acid tail moieties selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanoic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.48. The composition of any one of embodiments 42-46, wherein the phospholipid is selected from the group consisting of: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,l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine,1.2-dilinolenoyl-sn-glycero-3-phosphocholine,1.2-diarachidonoyl-sn-glycero-3-phosphocholine,1.2-didocosahexaenoyl-sn-glycero-3 -phosphocholine,1.2-dioleoyl-sn-glycero-3-phosphoethanola mine (DOPE),1.2-diphytanoyl-sn-glycero-3-phosphoethanolamine,1.2-distearoyl-sn-glycero-3 -phosphoethanolamine,31MF-367050437Docket No.: 16553-20014.40 1.2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine,1.2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1.2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1.2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine,1.2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), sphingomyelin, andcombinations thereof,optionally wherein the phospholipid comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).49. The composition of embodiment 41, wherein the nanoparticle is a polymeric nanoparticle comprising a non-lipid polymer.50. The composition of embodiment 49, wherein the non-lipid polymer comprises a poly(P-amino ester) (PBAE), a cyclodextrin (CD), a polyethyleneimine (PEI), a poly(lactic-co-glycolic acid) (PLGA), a polyamidoamine (PAMAM) dendrimer or an ionizable amphiphilic Janus dendrimer.51. The composition of embodiment 41, wherein the nanoparticle is a protein nanoparticle, a viral particle, a virus-like particle (VLP) or a cationic nanoemulsion.52. A composition comprising the mRNA of any one of embodiments 32-36 complexed with one or more lipids (RNA-lipoplex), wherein the one or more lipids comprise a first lipid and a second lipid.53. The composition of embodiment 52, wherein the first lipid is a cationic lipid, and the second lipid is a neutral or anionic lipid.54. The composition of embodiment 53, wherein the cationic lipid comprises one or both of:i) l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) or analogs or derivatives thereof; andii) l,2-dioleoyl-3-trimethylammonium propane (DOTAP) or analogs or derivatives thereof.55. The composition of embodiment 53 or embodiment 54, wherein the neutral or anionic lipid comprises:32MF-367050437Docket No.: 16553-20014.40 i) l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or analogs or derivatives thereof; and / orii) cholesterol or analogs or derivatives thereof; and / oriii) 1,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC) or analogs or derivatives thereof.56. The composition of any one of embodiments 41-55, further comprising a further mRNA encapsulated in the nanoparticle or complexed with the one or more lipids, wherein the further mRNA comprises a coding region of an antigen.57. The composition of any one of embodiments 41-55, wherein the mRNA further comprises a coding region of an antigen, and wherein the coding region of the cGAS, the STING or the IRAK is separated from the coding region of the antigen by an intervening sequence, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES).58. The composition of any one of embodiments 41-57, wherein the composition does not comprise a TLR7 / 8 agonist.59. The composition of any one of embodiments 41-58, wherein the composition does not comprise a lysophosphatidylcholine (LPC).60. A pharmaceutical formulation comprising the composition of any one of embodiments 41-59 and a pharmaceutically acceptable excipient.61. A method of inducing cGAMP production by antigen presenting cells (APCs), the method comprising contacting the APCs with the composition or formulation of any of the preceding embodiments comprising the coding region of a cyclic GMP-AMP synthase (cGAS) under conditions suitable for expressing cGAS in the APCs resulting in production of cGAMP by the APCs.62. A method of inducing expression of one or more interferon-stimulated genes by antigen presenting cells (APCs), the method comprising contacting the APCs with the composition or formulation of any of the preceding embodiments comprising the coding region of a cyclic GMP-AMP synthase (cGAS) and / or the coding regions of stimulator of interferon genes (STING) and interleukin- 1 receptor- associated kinase (IRAK) under conditions suitable33MF-367050437Docket No.: 16553-20014.40 for expressing the cGAS and / or the STING and IRAK in the APCs resulting in expression of the one or more interferon- stimulated genes.63. The method of embodiment 62, wherein the one or more interferon- stimulated genes comprise IP-10 (CXCL10), IFN-0, RANTES (CCL5), IFN- / J, or a combination thereof.64. A method of inducing expression of one or more NF-kB-responsive genes by antigen presenting cells (APCs), the method comprising contacting the APCs with the composition or formulation of any of the preceding embodiments comprising the coding regions of stimulator of interferon genes (STING) and interleukin- 1 receptor-associated kinase (IRAK) under conditions suitable for expressing the STING and the IRAK in the APCs resulting in expression of the one or more NF-kB-responsive genes.65. The method of embodiment 64, wherein the one or more NF-kB-responsive genes comprise TNF-a, IL-6, IL-8, IL-10, IL-10, IL-12, or a combination thereof.66. A method of inducing expression of one or more interferon-stimulated genes and one or more NF-kB-responsive genes by antigen presenting cells (APCs), the method comprising contacting the APCs with the composition or formulation of any of the preceding embodiments comprising the coding region of a cyclic GMP-AMP synthase (cGAS) and the coding regions of stimulator of interferon genes (STING) and interleukin- 1 receptor- associated kinase (IRAK) under conditions suitable for expressing the cGAS, the STING and the IRAK in the APCs resulting in expression of the one or more interferon-stimulated genes and the one or more NF-kB-responsive genes.67. The method of embodiment 66, wherein the one or more interferon-stimulated genes comprise IP-10 (CXCL10), IFN-0, RANTES (CCL5), IFN-X1, or a combination thereof, and the one or more NF-kB-responsive genes comprise TNF-a, IL-6, IL-8, IL-10, IL-10, IL-12, or a combination thereof.68. The method of any one of embodiments 61-67, wherein the APCs comprise dendritic cells.69. The method of any one of embodiments 61-68, wherein the APCs comprise monocytes and / or macrophages.34MF-367050437Docket No.: 16553-20014.40 70. The method of any one of embodiments 61-69, wherein the APCs are contacted in vivo with the composition.71. The method of any one of embodiments 61-69, wherein the APCs are contacted ex vivo with the composition72. A method of inducing formation of pores in plasma membranes of cells, the method comprising contacting the cells with the composition or formulation of any of the preceding embodiments comprising the coding region of a gasdermin (GSDM) under conditions suitable for expressing the GSDM in the cells resulting in formation of the pores in the plasma membranes of the cells.73. The method of embodiment 72, wherein the formation of the pores results in release of antigens by the cells.74. The method of embodiment 72, wherein the formation of the pores results in a reduction of viability of the cells (as compared to control cells that had not been contacted with the composition or formulation).75. The method of embodiment 72, wherein the formation of the pores results in pyroptosis of the cells.76. A method of preparing the composition of any one of embodiments 41-51 or any one of embodiments 56-59, comprising encapsulating the mRNA in the particle.77. A method of preparing the composition of any one of embodiments 52-59, comprising forming a complex between the mRNA and the one or more lipids.78. A method of treating cancer, comprising administering an effective amount of the pharmaceutical formulation of embodiment 60 to a cancer patient to treat the cancer.79. A method of treating or preventing an infectious disease, comprising administering an effective amount of the pharmaceutical formulation of embodiment 60 to a subject in need thereof to treat or prevent the infectious disease.35MF-367050437Docket No.: 16553-20014.40 80. A method of stimulating an immune response against an antigen, comprising administering an effective amount of the composition of any one of embodiments 56-59 to a subject in need thereof to stimulate an immune response against the antigen.81. The method of embodiment 80, wherein stimulating the immune response comprises increasing the numbers of antigen-specific T-cells in blood of the subject.82. The method of embodiment 80 or embodiments 81, wherein stimulating the immune response comprises increasing the antigen-specific interferon-gamma response in PBMC of the subject.83. Use of the composition of any one of embodiments 41-59 in the manufacture of a medicament for treating cancer in a subject in need thereof.84. Use of the composition of any one of embodiments 41-59 in the manufacture of a medicament for treating or preventing an infectious disease in a subject in need thereof.85. Use of the composition of any one of embodiments 56-59 in the manufacture of a medicament for stimulating an immune response against the antigen in a subject in need thereof.36MF-367050437Docket No.: 16553-20014.40EXAMPLES

[0104] Abbreviations: BMDC (bone marrow-derived dendritic cell); carboxyfluorescein succinimidyl ester (CFSE); cyclic GMP-AMP synthase (cGAS); DAMP (damage-associated molecular pattern); DC (dendritic cell); diABZI (amidobenzimidazole compound 3); dLN (draining lymph node); DLS (dynamic light scattering); DMG-PEG-2000 (polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG]; DSPC (1,2-distearoyl-sn-glycero-3 -phosphocholine); GFP (green fluorescent protein); GSDM, Gsdm, or Gasdm (gasdermin); GSDMD, GsdmD or GasdmD (gasdermin D); IFN0 (interferon-beta); IFNy (interferon-gamma); IRAK (interleukin- 1 receptor- associated kinase); KO (knockout); LNP (lipid nanoparticle); LPC / Lyso PC (lysophosphatidylcholine); Luci (luciferase); Lyso PC(22:0) (l-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine); MC3 ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, also referred to as DLin-MC3-DMA); mcg or pg (microgram); moDC (monocyte-derived dendritic cell); MW (molecular weight); NA (not applicable); NF-kB, NFkB or NF-KB (Nuclear factor kappa-light-chain-enhancer of activated B cells); OVA (ovalbumin); PAMP (pathogen-associated molecular pattern); PBMCs (peripheral blood mononuclear cells); PRR (pathogen recognition receptor); PBS (phosphate buffered saline); RANTES (regulated on activation, normal T-cell expressed and secreted); R848 (resiquimod); SD (standard deviation); SFC (spot-forming cells); STING (stimulator of interferon genes); TNFa (tumor necrosis factor- alpha); TLR (toll-like receptor) and TRM (tissue resident memory).

[0105] Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the following examples should not be construed as limiting the scope of the present disclosure, which is delineated by the appended claims.37MF-367050437Docket No.: 16553-20014.40 Example 1: Responses of Human Cell Lines to Expression of an Inducer of Inflammation Alone or in Combination with cGASMaterials & Methods

[0106] LNP Production and Characterization. Lipids for LNP synthesis were acquired from Cayman Chemical Co. { 8- [(2-hydroxy ethyl) [6-oxo-6-(undecyloxy)hexyl] amino] -octanoic acid, 1 -octylnonyl ester(SM-102)} and Avanti (distearoyl-phosphatidylcholine[DSPC]; l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 [DMG-PEG2000]). Cholesterol was purchased from Sigma. mRNAs for LNP synthesis were purchased from TriLink BioTechnologies. CleanCap mRNA encoding firefly luciferase modified with 5 -methoxyuridine (5moU) was purchased off-the-shelf (TriLink). cGASAN, cGASAN dblmutant (also known as cGASAN mut, containing E225A, D227A, C396A and C397A amino acid mutations), cGASAN-STING-IRAKl (c) also referred to herein as cGASAN-STING-IRAKl, STING-IRAKI (c) also referred to herein as STING-IRAKI, cGASAN-GsdmD, and GsdmD mRNAs were custom-ordered from TriLink and synthesized via in vitro transcription from linearized template DNA. The mRNA sequences were capped using Trilink’s CleanCap mRNA technology, with an Nl-methylpseudouridine base modification, and a 120 residue polyA tail. The amino acid sequences of the coding regions of the mRNAs are set forth in SEQ ID NOs as listed in Table 1-1.Table 1-1. Amino Acid Sequences Encoded by mRNA ConstructsConstruct SequencecGASAN dblmut (negative control) SEQ ID NO:41cGASAN SEQ ID NO:1 plus N-terminal methionine cGASAN-STING-IRAKl (c) SEQ ID NO:23STING-IRAKI (c) SEQ ID NO:24cGASAN-GsdmD SEQ ID NO:20GsdmD SEQ ID NO: 19 plus N-terminal methionine

[0107] The lipid mix was prepared with 50% SM-102 as ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG2000. The total lipid concentration used for synthesis was 12.5 mM in ethanol. mRNAs were each prepared in pH 4 sodium citrate buffer, and mixed with the lipids at an N / P (ratio of amine groups (N) of the ionizable lipid to the nucleic acid phosphate group (P)) ratio of 4 or 6. LNPs were synthesized using the NanoAssemblr Ignite38MF-367050437Docket No.: 16553-20014.40 instrument (Precision Nanosystems). Lipids in ethanol were combined with the mRNA solutions individually at a 1:3 volumetric ratio, using a flow rate of 12 mL / min. LNPs were washed in 10 volumes of Tris buffer to remove residual ethanol, and then concentrated using Amicon 50K molecular weight cut off (MWCO) centrifugal filters. LNPs were filtered through a 0.2- m filter. Sucrose was added to 15% as a cryoprotectant, and LNPs were stored at -80°C until use.

[0108] Loading of mRNA into LNPs was quantified using a RiboGreen assay (ThermoFisher) following the manufacturer’s protocol. Samples were diluted to fall within the range of the standard curve. LNPs were lysed using Triton X-100 to assess encapsulation of mRNA into LNPs. Both total mRNA and encapsulated mRNA were quantified. The size of the LNPs was assessed using dynamic light scattering (DLS) on the NanoBrook Omni (Brookhaven). LNPs were diluted 1:10 in PBS before running DLS. The zeta potential of the LNPs was assessed using mixed-mode measurement phase analysis light scattering (M3-PALS) on the Zetasizer Lab (Malvern Panalytical). LNPs were diluted 1:1000 in water before running M3-PALS.

[0109] Protein Expression in vitro in HEK293T, THP1-Null2, and THPl-KO-GsdmD cells. Protein expression from the constructs was evaluated in three different cell lines: HEK-293T (which lack cGAS and STING signaling), THP1-Null2 (all signaling pathways intact), and THPl-KO-GsdmD cells (lacking Gasdermin D signaling). HEK-293T cells (ATCC) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 100 U / mL penicillin / streptomycin, 10% heat inactivated Fetal Bovine Serum (FBS), and 2 mM L-glutamine. THP1-Null2 cells (InvivoGen) and THPl-KO-GsdmD cells (InvivoGen) were grown in Roswell Park Memorial Institute 1640 (RPMI) medium containing 100 U / mL penicillin / streptomycin, 10% heat inactivated FBS, 25 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), and 100 pg / mL normocin. Cells were harvested and plated at either 300,000 cells per well for HEK-293T cells, or 500,000 cells per well for THP1-Null2 cells in a flat-bottom 96-well tissue culture-treated plate in the same growth medium minus the addition of normocin. Cells were treated with LNPs containing mRNA constructs at 1 pg / mL encapsulated mRNA.

[0110] After 24 hours, cell culture supernatant was removed and cells were lysed using lx Triton-X Buffer. Protein content was measured via the Pierce Dilution-Free Rapid39MF-367050437Docket No.: 16553-20014.40 Gold bicinchoninic acid (BCA) Protein Assay Kit (ThermoScientific) using the manufacturer’s protocol. Lysates were mixed with 3X concentrated sample loading buffer + 30X dithiothreitol (DTT) (Cell Signaling Technology) and boiled for 5 minutes at 100°C. Ten pg protein was loaded per lane of a Mini-PROTEAN TGX precast gel (Bio-Rad) for electrophoresis. Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked using 3% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST) which contained 20 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween 20. Rabbit anti-cGAS antibody (CST-79978) was diluted 1,000-fold in blocking buffer. Rabbit anti-STING antibody (CST- 13647) was diluted 1,000-fold in blocking buffer. Rabbit anti-IRAK antibody (CST 4504) was diluted 1,000-fold in blocking buffer. Rabbit anti-GsdmD antibody (CST 39754) was diluted 1,000-fold in blocking buffer. Rabbit anti-vinculin (Cell Signaling Technology 13901) was diluted 1,000-fold in blocking buffer.Membranes were incubated overnight at 4°C on a rocker for the primary antibody incubation. Anti-rabbit HRP secondary antibody (Cell Signaling Technology 7074S) was diluted 5,000-fold in blocking buffer. Samples were incubated at room temperature on a shaker for one hour with the secondary antibody. Three five-minute wash incubations using TBST were done after primary antibody and secondary antibody incubations. The Clarity Western ECL substrate (BioRad) was used to detect luminescing protein bands on the ChemiDoc Go Imaging System (BioRad).

[0111] THP1-Null2, THPl-KO-GasdmD, THPl-NFkB, and THP1-Blue™ ISG stimulation. THP1-Null2 cells (InvivoGen), THPl-KO-GsdmD cells (InvivoGen), THP1-Lucia™ NFkB Lucia™ cells (InvivoGen), and THP1-Blue™ ISG cells (InvivoGen) were grown in Roswell Park Memorial Institute 1640 (RPMI) medium containing 100 U / mL penicillin / streptomycin, 10% heat inactivated FBS, 25mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), and 100 pg / mL normocin. On the day of stimulation, cells were harvested and plated at 100,000 cells per well in a flat-bottom 96-well tissue culture-treated plate in the same growth medium minus the addition of normocin. THP1-Null2, THPl-KO-GasdmD, and THP1 -Lucia™ NFkB cells were treated with LNPs containing mRNA constructs at 0.1-1 pg / mL encapsulated mRNA for 21-24 hours at 37°C. THP1-Blue™ ISG cells were treated with LNPs containing mRNA constructs at 2-250 ng / mL encapsulated mRNA for 21-24 hours at 37°C. Cells were also left unstimulated, or were stimulated with other STING agonists such as 40MF-367050437Docket No.: 16553-20014.40 diABZI or cGAMP as positive controls for assay readouts. After an overnight incubation, cells and culture supernatant were used for downstream readouts.

[0112] Viability Assessments. THPl-KO-GasdmD cells and THP1-Null2 cells were stimulated for 24 hours as indicated above. After an overnight stimulation, supernatants were collected and the presence of lactate dehydrogenase (LDH) was assessed as an indicator of cytotoxicity. LDH was quantified using the CyQuant LDH Cytotoxicity Assay Kit (Invitrogen) according to manufacturer’s protocol. Viability was normalized to unstimulated cells.

[0113] Interferon Stimulated Gene (ISG) Activity. THP1-Blue™ ISG cells were stimulated for 21 hours as indicated above. After an overnight stimulation, supernatants were collected and the presence of secreted embryonic alkaline phosphatase (SEAP) was assessed as an indicator of interferon regulatory factor activation and interferon signaling. SEAP was quantified using the Quanti-Blue Solution Kit (InvivoGen) according to manufacturer’s protocol.

[0114] NF-KB Activity. THPl-Lucia™ NF-KB cells were stimulated for 24 hours as indicated above. After an overnight stimulation, supernatants were collected and the presence of secreted luciferase was assessed as an indicator of NF-KB activation. Luciferase was quantified using the Quanti-Luc 4 Lucia Reagent (InvivoGen) according to manufacturer’s protocol.

[0115] cGAMP Quantification. THP1-Null2 cells were stimulated for 24 hours as indicated above. After an overnight stimulation, cells were lysed to collect cGAMP using a Triton X-100-based extraction buffer for 10 minutes at room temperature. Cell debris was pelleted by spinning at 400 x g for 4 minutes. Supernatants were then collected to perform a competitive ELISA for cGAMP (Cayman Chemical Co.) according to the manufacturer’s instructions.

[0116] Cytokine and Chemokine Production. THP1-Null2 cells were stimulated for 24 hours as indicated above. After an overnight stimulation, supernatants were collected and the presence of cytokines was assessed using a custom LEGENDplex™ multi-analyte flow assay kit (BioLegend®) including detection of secreted cytokines and chemokines specific to ISG and NF-KB gene activation: IP-10, IL-8, IFN-X1, IL-10, IFN-0, IL-6, TNF-a, IL-10. LEGENDplex™ cytokine quantification was performed according to manufacturer’s instructions and analyzed on a NovoCyte Quanteon (Agilent). Data was analyzed using BioLegend’s cloud-based software.41MF-367050437Docket No.: 16553-20014.40 Results

[0117] To determine if the addition of GsdmD(n) or STING-IRAKI (c) activities to cGASAN activities allow for a stimulation of multiple immune pathways, LNPs were synthesized to contain mRNAs for cGASAN, cGASAN-STING-IRAKl(c), STING-IRAKI (c), cGASAN-GsdmD(n), or GsdmD(n). See Table 1-1 for SEQ ID NOs for amino acid sequences. As experimental controls, LNPs were made with mRNA encoding a mutated version cGASAN where the mutation renders the enzyme inactive. These LNPs were tested on THP-1 cells (a human myeloid cell line) as well as THP1 reporters for ISG and NFkB activities, and a THP1 KO for Gasdermin D.

[0118] LNPs exhibit similar sizing and loading profiles with cGASAN, cGASAN-GsdmD(n), and cGASAN -STING-IRAKI (c) mRNAs. All LNPs synthesized had effective diameters less than 120 nm, with a relatively uniform size profile, exhibited by poly dispersity indexes less than 0.2. All mRNAs loaded into LNPs with greater than 80% of mRNA encapsulated in the LNPs.

[0119] Protein Expression in vitro in HEK293T, THP1-Null2, and THPl-KO-GsdmD cells. Protein expression was detected in vitro in HEK293T cells, THP1-Null2 cells, and THPl-KO-GsdmD cells for all constructs by Western Blot. cGASAN, cGASAN-STING-IRAKl(c), and STING-IRAKI (c) expression was evaluated in HEK293T cells, which have cGAS and STING knocked out, so the only detectable cGAS or STING would be due to mRNA delivered via LNPs. After HEK293T cells were stimulated for 24 hours with 1 pg / mL mRNA encapsulated in LNPs, bands were detected with an antibody against cGAS: ~40 kDa band for cGASAN, and ~45 kDa band for cGASAN containing part of the T2A ribosomal skipping sequence used to separate cGASAN from STING-IRAKI (c) in the polycistronic mRNA. Bands were also detected with antibodies against STING, and against IRAKI at ~60 kDa, which was the size of the fusion protein. In THP1-Null2 and THPl-KO-GsdmD cells, after 24 hours of stimulation with 1 pg / mL mRNA encapsulated in LNPs, bands were detected: ~40 kDa for cGASAN, and ~45 kDa for cGASAN containing part of the T2A ribosomal skipping sequence used to separate cGASAN from GsdmD(n) in the bicistronic mRNA. Bands were also detected ~30 kDa for the GsdmD(n).

[0120] LNPs made with GsdmD(n)-containing constructs cause cell death in a dosedependent manner in THPl-KO-GasdmD cells. After stimulation for 24 hours in the presence of42MF-367050437Docket No.: 16553-20014.40 0.1-1 g / mL mRNA-LNPs, the viability of THPl-KO-GsdmD cells was assessed by quantifying the presence of extracellular LDH. Cells that were treated with LNPs containing cGASAN-mut mRNA, cGASAN mRNA, cGASAN-STING-IRAKl(c), and STING-IRAKI (c) did not show any drop in viability. However, constructs that contained the mutated Gasdermin D, both with cGASAN or as GsdmD alone, did show decreased viability, suggesting these LNPs facilitated the generation of GsdmD pores and when GsdmD mRNA was delivered (FIG.2).

[0121] LNPs made with cGAS N -containing constructs activate Interferon Stimulated Gene (ISG) Activity. After stimulation for 24 hours in the presence of 2-250 ng / mL mRNA-LNPs, 50 pg / mL cGAMP, or 10 g / mL diABZI, activation of THP1-Blue™ ISG cells was assessed. Cells that were treated with LNPs containing cGASAN, cGASAN-STING-IRAKl(c), or cGASAN-GsdmD(n) mRNAs, with LNPs containing high doses of STING-IRAKI (c) mRNA, or with soluble STING-agonists cGAMP or diABZI showed ISG activity (FIG. 3A and 3B). Cells left untreated, or treated with LNPs containing mRNAs encoding for proteins that are irrelevant to ISG stimulation: cGASAN-mut or GsdmD mRNAs, did not show any activity.

[0122] LNPs made with STING-IRAKl(c) and cGASAN -containing constructs activate NF-kB Activity. After stimulation for 24 hours in the presence of 0.1-1 pg / mL mRNA-LNPs, 50 pg / mL cGAMP, or 10 pg / mL diABZI, activation of THPl-Lucia™ NF-kB cells was assessed. Cells that were treated with LNPs containing the IRAKI (c) construct, which had been added to the mRNAs to engage TLR activation, such as the STING-IRAKI (c) or cGASAN cGASAN-STING-IRAKl(c) mRNAs - showed high levels of NF-kB activation (FIGs.4A and 4B). Cells that were treated with LNPs containing cGASAN constructs, cGASAN or cGASAN-GsdmD(n) mRNAs, also showed lower levels of NF-kB activation. No NF-kB activation was seen when cells were left untreated, or treated with soluble STING agonists cGAMP or diABZI, or treated with LNPs containing cGASAN-mut or GsdmD(n) mRNA.

[0123] LNPs containing immunomodulatory mRNAs activate multiple pathways in monocytic THP1-Null2 cells. After stimulation for 24 hours in the presence of 0.1-1 pg / mL mRNA-LNPs, 50 pg / mL cGAMP, or 10 pg / mL diABZI, viability of THP1-Null2 cells was assessed. While THP1-Null2 cells left untreated, treated with cGAMP or diABZI, or treated with any dose of cGASAN maintained high cell viability, cells treated with any of the mRNAs that43MF-367050437Docket No.: 16553-20014.40 were intended to diversify immunomodulatory activities showed decreased cell viabilities (FIG. 5). Only the 0.1 |ig / mL dose of cGASAN-STING-IRAKl(c), STING-IRAKI (c), cGASAN-GsdmD(n), or GsdmD(n) LNPs did not cause toxicity in the THP1-Null2 cells.

[0124] After the 24 hours stimulation, cells were lysed to detect intracellular cGAMP production, which is produced by the constitutively active cGASAN after the protein has been translated from the mRNA delivered by LNPs. All constructs containing cGASAN allowed for the production of cGAMP, though to varying levels (FIG.6). It is possible that the additional immunostimulatory activities provided by the addition of STING-IRAKI (c) and GsdmD(n) used some of the cGAMP produced by the cGASAN delivered to cells.

[0125] The activities of THP1 monocytic cells in response to stimulation with the various constructs is summarized in the heat map in FIG. 7 and in Table 1-2. In particular, this map shows ISG pathway responses when cGASAN is present, NF-kB activities when IRAKI (c) or cGASAN are included, GsdmD activities in response to LNPs containing GsdmD in cells where GsdmD is knocked out.Table 1-2. Relative Activity of Monocyte Cell Lines Under Various ConditionsTHPl-NFkB THP1-ISG THP1-NU112 Condition / Activity Activity Activity cGAMP AVG SD AVG SD AVG SDMedia 0.00 0.00 0.08 0.01 0.00 0.00 cGAMP 0.01 0.00 0.94 0.03 NA NA diABZI 0.02 0.00 0.85 0.06 0.00 0.00 cGASAN-mut LNP 0.00 0.00 0.07 0.00 0.00 0.00 cGASAN LNP 0.14 0.01 0.88 0.11 0.97 0.10 cGASAN-STING-IRAKl(c) LNP 0.37 0.02 0.24 0.02 0.01 0.00 STING-IRAKI (c) LNP 0.99 0.02 0.07 0.00 0.00 0.00 cGASAN-GsdmD(n) LNP 0.23 0.01 0.58 0.03 0.11 0.01GsdmD(n) LNP 0.01 0.00 0.07 0.00 0.00 0.00

[0126] LNPs containing immunomodulatory mRNAs induce cytokine and chemokine production in monocytic THP1-Null2 cells. After stimulation for 24 hours in the presence of 0.1 pg / mL mRNA LNPs - a dose that did not cause toxicity in THP1-Null2 cells, 50 pg / mL cGAMP, or 10 pg / mL diABZI, supernatants were collected to assess cytokine and chemokine production. cGASAN-LNP treatment allowed for the production of cytokines and chemokines44MF-367050437Docket No.: 16553-20014.40 stimulated by the cGAS-STING pathway, including IP-10, IFN-X1, and IFN-0 (FIG.8). The inclusion of STING-IRAKI (c) activities allowed for the production of cytokines and chemokines stimulated by TLR activation, including IL-6, TNF-a, IL- 10. IL-8 and IL-slO secretion also increased in the presence of STING-IRAKI (c). These changes in cytokine and chemokine production profiles shown in Table 1-3 and Table 1-4 demonstrate the successful broadening of immune response by combining cGASAN activities with STING-IRAKI (c) activities.Table 1-3. Relative Secretion by Monocyte Cell Line Under Various Conditions Condition / Secretion IP- 10 IL-8 IFN-kl IL-10AVG SD AVG SD AVG SD AVG SDMedia 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 cGAMP 0.09 0.01 0.00 0.00 0.01 0.00 0.00 0.00 diABZI 0.18 0.01 0.00 0.00 0.17 0.00 0.00 0.00 cGASAN-mut LNP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 cGASAN LNP 0.16 0.02 0.00 0.00 0.15 0.03 0.00 0.00 cGASAN-STING-IRAKl(c) LNP 0.97 0.03 0.51 0.06 0.94 0.08 0.02 0.00 STING-IRAKI (c) LNP 0.42 0.15 1.00 0.00 0.00 0.00 0.77 0.33 cGASAN-Gsdm(n) LNP 0.69 0.08 0.00 0.00 0.62 0.07 0.01 0.00Gsdm(n) LNP 0.01 0.00 0.03 0.01 0.00 0.00 0.00 0.00Table 1-4. Relative Secretion by Monocyte Cell Line Under Various Conditions Condition / Secretion IFb l-P IL-6 TNF-a IL-ip AVG SD AVG SD AVG SD AVG SDMedia 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 cGAMP 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 diABZI 0.04 0.01 0.00 0.00 0.00 0.00 0.00 0.00 cGASAN-mut LNP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 cGASAN LNP 0.05 0.01 0.00 0.00 0.00 0.00 0.00 0.00 cGASAN-STING-IRAKl(c) LNP 0.89 0.10 0.31 0.01 0.02 0.00 0.00 0.00 STING-IRAKI (c) LNP 0.01 0.00 0.88 0.17 0.83 0.25 0.84 0.26 cGASAN-Gsdm(n) LNP 0.43 0.10 0.01 0.00 0.00 0.00 0.00 0.00Gsdm(n) LNP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0045MF-367050437Docket No.: 16553-20014.40 Example 2: Responses of Murine Cell Lines to Expression of an Inducer of Inflammation Alone or in Combination with cGASMaterials & Methods

[0127] MuTu Dendritic Cell Culture and Stimulation. Immortalized Mouse Dendritic Cells (MuTu DCs) (Applied Biological Materials, Inc) were grown in Iscove’s Modified Dulbecco’s Medium (IMDM) containing 10% FBS, 100 U / mL penicillin / streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, and 55 pM P-mercaptoethanol. On the day of stimulation, cells were harvested and plated at 100,000 cells per well in a flat-bottom 96-well tissue culture-treated plate in the same growth medium. MuTu DCs cells were treated with LNPs containing mRNA constructs at 0.1-0.5 pg / mL encapsulated mRNA for 21-24 hours at 37°C. Cells were also left unstimulated, or were stimulated with other STING agonists such as diABZI or cGAMP as positive controls for assay readouts. For some stimulations, MuTu DCs were also treated with anti-CD40 to stimulate CD40 ligation and allow for production of IL-12p40 following TLR stimulation provided by IRAKI -containing constructs. After an overnight incubation, cells and culture supernatant were used for downstream readouts.

[0128] Viability Assessments. MuTu DCs were stimulated for 24 hours as indicated above. After an overnight stimulation, supernatants were collected and the presence of lactate dehydrogenase (LDH) was assessed as an indicator of cytotoxicity. LDH was quantified using the CyQuant LDH Cytotoxicity Assay Kit (Invitrogen) according to manufacturer’s protocol. Viability was normalized to unstimulated cells.

[0129] cGAMP Quantification. MuTu DCs were stimulated for 24 hours as indicated above. After an overnight stimulation, cells were lysed to collect cGAMP using a Triton X- 100-based extraction buffer for 10 minutes at room temperature. Cell debris was pelleted by spinning at 400 x g for 4 minutes. Supernatants were then collected to perform a competitive ELISA for cGAMP (Cayman Chemical Co.) according to the manufacturer’s instructions.

[0130] Cytokine and Chemokine Production. MuTu DCs were stimulated for 24 hours as indicated above. After an overnight stimulation, supernatants were collected and the presence of cytokines was assessed using the Mouse Anti-Virus Response LEGENDplex™ (BioLegend) kit including detection of secreted cytokines and chemokines specific to ISG and46MF-367050437Docket No.: 16553-20014.40 NF-KB gene activation: IP-10, IL-8, IFN-X1, IL-10, IFN-0, IL-6, TNF-a, IL-10. LEGENDplex™ cytokine quantification was performed according to manufacturer’s instructions and analyzed on a NovoCyte Quanteon (Agilent). Data was analyzed using BioLegend’s cloud-based software. IL-12p40 detection was completed by ELISA (Invitrogen) following manufacturer’s instructions.Results

[0131] LNPs containing immunomodulatory mRNAs activate multiple pathways in MuTu Dendritic cells. After stimulation for 24 hours in the presence of 0.1 -0.5 pg / mL mRNA-LNPs, 50 g / mL cGAMP, or 10 pg / mL diABZI, supernatants were collected to assess viability of MuTu DCs. MuTu DCs left untreated, treated with soluble cGAMP or diABZI, or treated with LNPs containing cGASAN cGASAN-STING-IRAKl(c), STING-IRAKI (c), cGASAN-GsdmD(n), or GsdmD(n) LNPs did not cause toxicity in the MuTu DCs. Cells were lysed to detect intracellular cGAMP production, which is produced by the constitutively active cGASAN after the protein has been translated from the mRNA delivered by LNPs. All constructs containing cGASAN allowed for the production of cGAMP, though to varying levels. It is possible that the additional immunostimulatory activities provided by the addition of STING-IRAKI (c) and GsdmD(n) used some of the cGAMP produced by the cGASAN delivered to cells.

[0132] After stimulation for 24 hours in the presence of 0.1 -0.5 pg / mL mRNA LNPs, 50 pg / mL cGAMP, or 10 pg / mL diABZI, supernatants were collected to assess cytokine and chemokine production. cGASAN-LNP treatment allowed for the production of cytokines and chemokines stimulated by the cGAS-STING pathway, including IP-10, IFN-0, and RANTES. The inclusion of STING-IRAKI (c) activities allowed for the production of cytokines and chemokines stimulated by TLR activation, including IL-6 and TNF-a. When cultured in the presence of anti-CD40, IL-12p40 was detected at low levels when MuTu DCs were treated cGASAN containing constructs, but at high levels treated with constructs containing STING-IRAKI (c) (FIG. 9). The expression of IL12p40 is regulated by the combination of TLR signaling and CD40 ligation. As such, these changes in cytokine and chemokine production profiles suggest the successful broadening of immune response by combining cGASAN activities with STING-IRAKI (c) activities.47MF-367050437Docket No.: 16553-20014.40 Example 3: Expression by Antigen Presenting Cells of Protein Encoded by mRNA in LNPs Materials & Methods

[0133] Monocytes were thawed into RPMI1640 containing 10% FBS, 100 U / mL penicillin / streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 1% Non-Essential Amino Acids (v / v), and 55 M P-mercaptoethanol. To differentiate monocytes into monocyte-derived dendritic cells (moDCs), cells were seeded at 5xlOA7 in a T75 flask with 20mL growth medium and 50ng / mL GM-CSF and 25ng / mL IL-4. Three days later, lOmL of growth medium supplemented with 50ng / mL GM-CSF and 25ng / mL IL-4 was added to the flasks and cells were harvested on day 6. Autologous PBMCs were thawed in the same growth medium as moDCs. PBMCs were seeded in a 96-well U-bottom plate at a density of 200,000 cells per well and moDCs were seeded in a 96-well U-bottom at a density of 100,000 cells per well in the growth media. Cells were left unstimulated or treated with LNPs containing firefly luciferase mRNA at 1 g / mL for 24 hours in triplicate. Additional cells were seeded for staining controls. After stimulation, cells were stained with fluorophore-conjugated antibodies for lineage specific cell markers. Cells were fixed and permeabilized to intracellularly stain for luciferase protein expression. Flow cytometry was used to analyze cell populations and their luciferase production.Results

[0134] Human PBMCs contain multiple immune cell types with differing functions. To understand what cell types are likely targeted for mRNA delivery and eventual translation by LNPs, PBMCs were incubated with LNPs containing the reporter firefly luciferase mRNA. Additionally, moDCs were similarly treated and investigated because dendritic cells are important mediators of antigen presentation to T-cells. After stimulation for 24 hours in the presence of 1 pg / mL luciferase LNPs, cells were collected for flow cytometry to identify moDCs (CDllc+CD209+), monocytes (CDllc+CD14+), T-cells (CD3+), B-cells (CD19+), and NK-cells (CDllc-CD3-CD56+ or CDllc-CD3-CD16+) and stained for luciferase protein. Luciferase was not detected in untreated conditions. When cells were treated with luciferase LNPs, luciferase was detected in moDC cultures and among monocytes in wells seeded with PBMCs (FIG. 11). In contrast, luciferase was not detected in T, B, or NK cells after LNP treatment (FIG. 11). These results indicate that myeloid cells are the main targets for LNPs.48MF-367050437Docket No.: 16553-20014.40Example 4: Immunostimulatory Activity of mRNA Encoded AdjuvantsMaterials & Methods

[0135] Human monocytes were thawed into RPMI 1640 containing 10% FBS, 100 U / mL penicillin / streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 1% Non-Essential Amino Acids (v / v), and 55 pM P-mercaptoethanol. Monocytes were repurified with CD14 Ultrapure Beads (Miltenyi Biotec) according to manufacturer’s protocol and seeded at 5xlOA7 in a T75 flask with 20mL growth medium and 50ng / mL GM-CSF and 25ng / mL IL-4. Three days later, lOmL of growth medium supplemented with 50ng / mL GM-CSF and 25ng / mL IL-4 was added to the flasks and cells were harvested on day 6.

[0136] On the day of stimulation, moDCs were harvested and plated at 20,000 cells per well in a flat-bottom 96-well tissue culture-treated plate in RPMI growth media. HPV16 E711 19-specific CD8+ T-cells (Charles River Laboratories) were thawed into the same medium and plated at 60,000 cells per well in the 96-well flat bottom containing moDCs. Monoculture conditions for each cell type were also plated as control conditions. Cells were left unstimulated, or treated with LNPs containing mRNA constructs at 1 pg / mL in the presence or absence of E711 19peptide (Charles River Laboratories) at lOOng / mL for 24 hours at 37°C. After an overnight incubation, culture supernatants were collected to assess cytokine production.Cytokines were assessed using Lumit (Promega) kits for detection of IFN-beta, IL-6 and IFN-gamma. Cytokine quantification was performed according to manufacturer’s instructions, and luminescent signal was measured on the GloMax Explorer (Promega). Data were analyzed using Graphpad Prism software. Statistical comparisons were made using two-way ANOVA followed by Tukey’s multiple comparisons test with a single pooled variance..Results

[0137] To compare the relative immunostimulatory capacity of the mRNA-encoded adjuvants, in vitro cocultures of moDC and HPV16 E7-specific CD8+ T-cells were set up as illustrated in FIG. 12. Cells were incubated for 24 hours in the presence of Ipg / mL LNPs, with or without E711 19peptide. To ensure the functionality of cGASAN-containing constructs, IFN-beta was measured in the supernatant. Media and cGASANdblmut LNP treatment did not induce49MF-367050437Docket No.: 16553-20014.40 IFN-beta secretion in cocultures (FIG. 13). STING-IRAK and GSDMD were not designed to activate the STING signaling pathway, and no IFN-beta was produced from stimulations with these mRNA-encoded adjuvants. In contrast, LNPs containing cGASAN or cGASAN-STING-IRAK mRNA induced IFN-beta production, indicative of activation of the STING signaling pathway (FIG. 13). Interestingly, IFN-beta was not detected in cocultures treated with cGASAN-GSDMD, potentially because cells expressing this construct underwent pyroptosis. The presence or absence of the E7 peptide would not be predicted to affect IFN-beta production, and in fact no effect was observed.

[0138] STING-IRAK and cGASAN-STING-IRAK constructs were designed to activate NF-KB signaling, and IL-6 would be expected to be produced downstream from the activation of this transcription factor. When IL-6 was measured in the cell culture supernatant, STING-IRAK and cGASAN-STING-IRAK mRNA-containing LNP treatments resulted in IL-6 production, whereas other cell treatments did not (FIG. 14). Additionally, presence or absence of the E7 minimal epitope did not affect IL-6 production, as expected.

[0139] Based on IFN-beta and IL-6 production, the LNP treatments activated the intended innate immune signaling pathways. These responses were derived from LNP activities on the moDCs, based on moDC monocultures (data not shown). moDC cannot produce IFN-gamma directly, and this response is dependent upon APC-mediated activation of T-cells. IFN-gamma was measured to determine how the various LNP treatments affected T-cell activity in the presence or absence of antigen. In the absence of E7 peptide, minimal IFN-gamma responses were detected (FIG. 15). In the presence of peptide, an increase in IFN-gamma was observed in media and cGASANdblmut compared to conditions without E7 peptide, indicating that the presence of antigen triggered a basal level of T-cell activity (FIG. 15). In comparison to these negative controls, use of the cGASAN mRNA-containing LNPs in the presence of peptide increased IFN-gamma production significantly, confirming that cGASAN enhances the ability of moDCs to activate T-cells. Treatment with STING-IRAK, cGASAN-STING-IRAK, and cGASAN-GSDMD mRNA-containing LNPs further increased IFN-gamma production compared to cGASAN mRNA-containing LNPs. (FIG. 15). These data indicate that STING-IRAK, cGASAN-STING-IRAK, and cGASAN-GSDMD were more potent at inducing IFN-gamma from T-cells compared to cGASAN. Notably, the bicistronic mRNAs are longer in length50MF-367050437Docket No.: 16553-20014.40 compared to cGASAN so when equally dosed at Ipg / mL, fewer copies of bicistronic mRNAs were added to wells compared to cGASAN. If the cultures were treated with equimolar concentrations, a larger difference in IFN-gamma is likely to have been observed. Finally, the GSDMD construct which induces pyroptosis was not found to enhance IFN-gamma responses, but when pyroptosis was combined with cGASAN (cGASAN-GSDMD), T-cell activity was enhanced compared to cGASAN treatment alone (FIG. 15).Example 5: Effects of mRNA-Encoded Adjuvants on Responses of Human PBMCs Materials & Methods

[0140] PBMCs from one human donor were thawed into RPMI 1640 containing 10% FBS, 100 U / mL penicillin / streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 1% Non-Essential Amino Acids (v / v), and 55 pM P-mercaptoethanol. For cytokine readouts, unlabeled PBMCs were seeded in triplicate in a 96-well U-bottom at 200,000 cells per well. For proliferation readout, PBMCs were set to a concentration of 1x107cells / mL and labeled with IpM carboxyfluorescein succinimidyl ester (CFSE) dye at 37°C for 5 minutes. Postlabeling, PBMCs were seeded in triplicate in a 96-well U-bottom plate at 200,000 cells per well in the growth media described above. Cells were left unstimulated or treated with LNPs containing mRNA constructs at 1 pg / mL or with control stimuli: lOpg / mL cGAMP (Invivogen), 0.5pM diABZI (Invivogen), 0.01% (v / v) Cell Activation Cocktail (Biolegend), O.lpg / mL lipopolysaccharide (LPS) (Enzo), or anti-human CD3 and anti-human CD28 antibodies (Biolegend) for 24 hours for cytokine readouts and 72 hours for proliferation readout. Wells were coated with anti-CD3 and anti-CD28 antibodies at lOpg / mL for 2 hours, and excess antibodies were washed off of tissue culture plates with PBS prior to culturing cells.

[0141] After an overnight stimulation, supernatants were collected and the presence of cytokines was assessed using Lumit (Promega) kits for detection of IFN-gamma and TNF-alpha. Cytokine quantification was performed according to manufacturer’s instructions and read on the GloMax Explorer (Promega). Data were analyzed using Graphpad Prism software.

[0142] In order to assess proliferation, CFSE-labeled PBMCs that had been stimulated for 72 hours were stained for lineage specific markers using fluorophore-conjugated51MF-367050437Docket No.: 16553-20014.40 antibodies. CD3+ T-cells were analyzed on the Agilent Novocyte flow cytometer to assess proliferation based on dilution of CFSE dye. Data were analyzed using Flowjo software.Results

[0143] Several of the new mRNA constructs were designed as an improvement on the original cGASAN construct, with the intention of adding functionality by engaging additional innate immune signaling pathways. To better understand the inflammatory response elicited from the mRNA-encoded adjuvants, LNPs carrying these constructs were tested on human PBMCs in vitro. After stimulation for 24 hours in the presence of I pg / mL LNPs or positive controls, supernatants were collected to assess cytokine production. IFN-gamma and TNF-alpha production were observed from PBMCs treated with STING-IRAK and cGASAN-STING-IRAK mRNA-containing LNPs, whereas these cytokines were not detected from cells treated with cGASAN mRNA-containing LNPs (FIG. 16). These cytokines were also produced when cells were treated with LPS and anti-CD3 / anti-CD28 agonist antibodies. PMA / ionomycin induced a low level of TNF-alpha production but not IFN-gamma production. In contrast, STING pathway small molecule agonists such as diABZI and cGAMP did not induce IFN-gamma and TNF-alpha production (FIG. 16). These results indicate that addition of the STING-IRAK coding region to mRNA encoding cGASAN initiates immune signaling distinct from what can be achieved using cGASAN mRNA on its own.

[0144] A portion of PBMCs were stained with CFSE prior to LNP treatment to evaluate effects of mRNA-encoded adjuvants on T-cell proliferation. After stimulation for 72 hours in the presence of I pg / mL LNPs or positive controls, cells were collected to assess T-cell proliferation based on dilution of CFSE fluorescent signal. Similar to cGASAN, STING-IRAK and cGASAN-STING-IRAK treatment did not induce proliferation of T-cells (FIG. 17). In contrast, T-cells were observed to proliferate when treated with anti-CD3 / anti-CD28 antibodies (FIG. 17). These results demonstrate that while LNPs containing the new mRNA constructs induced potent cytokine responses, they did not induce T-cell proliferation.Example 6: Effects of mRNA-Encoded Adjuvants on Responses to mRNA-Encoded Antigen in Mice52MF-367050437Docket No.: 16553-20014.40 Materials & Methods

[0145] Immunizations. C57B6 / J mice (n= 5 mice / group) were immunized intramuscularly with mRNA-encoding OVA antigen (0.05 pg) in LNPs in combination with mRNA-encoded adjuvants in LNPs on Day 0 and Day 14. As a control, mRNA encoding luciferase (Luci) in LNPs was used. Adjuvant or control LNPs were used at a concentration 1 pg mRNA / dose. Control groups received PBS and some mice received a high dose of mRNA-encoding the OVA antigen (1 pg) in combination with mRNA encoding the Luci control (1 pg). To assess mucosal immunity, some immunized mice received a second boost intravaginally on Day 100.

[0146] Tissue Processing. Spleens and lungs were collected at Days 56 and 98 postimmunization. Spleens were processed using Mouse-Spleen Dissociation Kit (Miltenyi) with mechanical and enzymatic dissociation via gentleMACS, followed by ACK lysis for RBC removal. Lungs were processed using Mouse-Lung Dissociation Kit (Miltenyi) with enzymatic dissociation, and CD45+ cells were enriched using MACS microbeads. The cervical tissue digestion protocol begins with aseptic dissection of vaginal tissue followed by a brief rinse in cold RPMI. The tissue was then minced into approximately 1 mm pieces using sterile scissors or scalpel. For digestion, the minced tissue was incubated in DisCozyme 2 (2.5 mg / mL) with DNase I (100 pg / mL) at 37°C for 40 minutes with gentle rocking or rotation, which helped loosen epithelial cells and release immune cells while minimizing damage to the immune cells. After digestion, the mixture was filtered through a 70 pm cell strainer and rinsed with cold FACS buffer to maximize cell recovery. Finally, the filtered cells were centrifuged at 400 x g for 5 minutes at 4°C and resuspended in FACS buffer.

[0147] Blood was collected in EDTA-coated tubes to prevent coagulation. The blood was mixed with wash buffer 1 to adjust volume (approximately 500 pL for cardiac bleeds). After a brief spin to collect the sample, ACK lysis buffer was added at a 1: 10 ratio (5 mL) and incubated at room temperature for exactly 5 minutes. The lysis was quenched by adding 45 mL of wash buffer 2 (9x volume). The sample was then centrifuged at 350 x g for 10 minutes at room temperature (acceleration 5, deceleration 4). The supernatant was carefully removed leaving 500 pL to avoid disturbing the cell pellet. The pellet was resuspended in 10 mL wash buffer 2 and centrifuged at 400 x g for 5 minutes. This wash step was repeated until a clear53MF-367050437Docket No.: 16553-20014.40 supernatant was obtained. Finally, the cell pellet (approximately 0.25 mL) was resuspended in RPMI by gentle pipetting and transferred to a 96-well U-bottom plate for subsequent analysis.

[0148] Flow Cytometry. For cervical tissue analysis, cells were surface stained with a panel of antibodies including CD45-BV785 (clone 30-F11), CD3 / TCRb-BV421 (clone 17A2), CD8-FITC (clone KT15), CD4-PerCD Cy5.5 (clone GK1.5), CD103-APC-Cy7, and CD69-BV605. Prior to antibody staining, cells were stained with Live / Dead Aqua dye to assess viability. The staining protocol involved blocking with Fc block, followed by surface antibody staining for 20 minutes at room temperature, fixation with 4% PFA, and final resuspension in FACS buffer with counting beads (1:4 dilution) before flow cytometry analysis. All antibodies were used at 1:100 dilution in FACS buffer (1% BSA + 2.5 mM EDTA in PBS). For tetramer staining, cells were stained with a SIINFEKL (SEQ ID NO: 16) tetramer (MBL) for 30min at 37 degrees.

[0149] Cytokine Measurement. For analysis of T-cell responses, single cell suspensions (5x106cells / mL for splenocytes, 1x106cells / mL for lung, and CD45+ cells for blood) were restimulated with OVA peptivator (0.5 pg / mL) for 72 hours. Cytokine production was assessed by IFNy Lumit assay following the manufacturer’s protocol (Promega).Results

[0150] cGASAN is a variant of the DNA-sensing pattern recognition receptor (PRR) cyclic GMP-AMP synthase (cGAS), which lacks the N-terminal amino acids normally present in the wild-type protein. Unlike its wild-type counterpart, the truncated N-terminus of human cGASAN contains a mitochondrial targeting signal that affords this enzyme access to intra-mitochondrial DNA. Upon delivery of an mRNA-lipid nanoparticle (LNP) to cells, cGASAN is expressed and becomes self-DNA reactive resulting in the production of the STING agonistic cyclic dinucleotide cGAMP (Zhivaki et al., mBIO, 14(6):e0250623, 2023). cGASAN has been shown to induce expression of type I interferons (IFNs) and upregulate genes that promote antigen presentation. However, combining cGASAN with other PRR agonists was contemplated to be desirable for diversifying immune responses and / or inducing long lived immunity. In addition, combining PRR agonists in an mRNA-encoded adjuvant is likely to be beneficial in reducing the dose of the antigen mRNA in mRNA-LNP vaccines.54MF-367050437Docket No.: 16553-20014.40

[0151] To test the effect of bicistronic mRNA-encoded adjuvants on T-cell responses in vivo, ovalbumin (OVA) mRNA was used as a source of model antigen. OVA mRNA was used at a suboptimal dose that typically does not induce a strong T cells responses in vivo to more clearly observe the effect of the mRNA-encoded adjuvants. BL6 mice were immunized on DO and D14 with either PBS, or with OVA mRNA LNPs at 0.05pg / mouse in combination with control luciferase mRNA LNPs at a concentration of I pg / mouse or with the bicistronic mRNA LNPs at a concentration of 1 pg / mouse. To compare these responses to T-cell responses generated by high doses of antigens, mice were immunized with control luciferase mRNA LNPs at a concentration of I pg / mouse in combination with OVA mRNA LNPs at a concentration of Ipg / mouse. Day 56 and Day 98 post first immunization, blood, spleen, and lungs were collected from the immunized mice. Single cell suspensions from each tissue were prepared and CD45+ cells were enriched using magnetic beads from the lung tissue. Cells were then left either unstimulated or were restimulated with OVA peptivator containing peptides spanning the full length of the ovalbumin protein. About 72 hours post restimulation, the supernatants were collected and tested for IFN-gamma production.

[0152] 56 days post first immunization, when mice were immunized with luciferase mRNA (Ipg) LNPs + OVA mRNA (0.05pg) LNPs (condition 2), no T-cell responses were observed in blood, spleen and lungs (FIGs. 18-20, left panels), much like PBS immunized mice (condition 1). However, when mice were immunized with cGASAN mRNA LNPs + OVA mRNA LNPs (0.05pg) (condition 3) T-cell responses were strongly enhanced in the blood, spleen and lungs (FIGs. 18-20, left panels), which was observed as increased IFN-gamma secretion after OVA peptivator restimulation as compared to condition 2. These data confirmed our previous observations that cGASAN LNPs strongly enhance antigen-specific T-cell responses in vivo (Zhivaki et al., supra, 2023). Surprisingly, when mice were immunized with cGASAN STING IRAKI mRNA LNPs + OVA mRNA LNPs (0.05pg) (condition 4), IFN-gamma responses after restimulation with OVA peptivators were even higher in the blood (FIG. 18, left panel) as compared to cGASAN mRNA LNPs + OVA mRNA LNPs (0.05pg) (condition 3) indicative of a higher frequency of circulating OVA antigen-specific T-cells. No difference was observed between condition 3 and condition 4 in the spleen and lungs. In addition, when mice were immunized with cGASAN-gasderminD mRNA LNP + OVA mRNA55MF-367050437Docket No.: 16553-20014.40 LNPs (0.05pg) (condition 5) or STING IRAKI mRNA LNPs (condition 6), no major advantages were observed compared to cGASAN mRNA LNPs + OVA mRNA LNPs (0.05pg) (condition 3). Interestingly, when mice were injected with a high dose of antigen mRNA in combination with control mRNA in LNPs (luciferase mRNA LNPs + OVA mRNA LNPs ( Ipg) (condition 7), the T cell responses generated were not as strong as when mice were immunized with suboptimal dose of OVA mRNA in combination with cGASAN mRNA LNPs or cGASAN STING-IRAKI mRNA LNPs (condition 3 and condition 4, respectively) (FIGs. 18-20). Overall, two take home messages are derivable from this data. First, the usage of an mRNA-encoded adjuvant such as cGASAN mRNA LNPs or cGASAN STING-IRAKI mRNA LNPs can strongly enhance antigen-specific T-cell responses even with suboptimal doses of antigen mRNA, which indicates that the total dose of mRNA can lowered if the right adjuvant is added to an mRNA vaccine. Second, cGASAN STING-IRAKI mRNA LNPs induce stronger circulating antigenspecific T-cell responses as compared to cGASAN mRNA LNPs.

[0153] 98 days post first immunization, the same general trends were observed. Interestingly, when mice were immunized with cGASAN STING-IRAKI mRNA LNPs + OVA mRNA LNPs (condition 4) the IFN-gamma responses after restimulation with the OVA peptivator were most strongly maintained in the blood, spleen and lungs (FIG. 18-20, right panels), as compared to all other conditions. These data indicate that cGASAN STING IRAKI mRNA LNPs induce durable, antigen-specific T-cell responses that can be maintained in tissues long after immunization.

[0154] To assess the ability of the bicistronic adjuvant cGASAN STING-IRAKI to induce mucosal immunity in the cervix, mice were given a second boost intravaginally, 100 days post first immunization. For the second boost, the following were administered: OVA mRNA LNPs (0.05pg / dose) in combination with either luciferase mRNA LNPs or with LNPs containing cGASAN mRNA, cGASAN STING-IRAKI mRNA or STING-IRAKI mRNA at a concentration of Ipg / dose. To compare these responses to T-cell responses generated by high doses of antigen, mice were immunized with control luciferase mRNA LNPs at a concentration of I pg / mouse in combination with OVA mRNA LNPs at a concentration of I pg / mouse. After five days, T-cells found in the cervix of study mice were monitored by flow cytometry. When mice were immunized intravaginally with cGASAN STING-IRAK mRNA LNPs + OVA mRNA LNPs, a56MF-367050437Docket No.: 16553-20014.40 higher abundance of CD3+CD8+ T-cells were observed in the cervix (FIG.21), and among these T-cells, higher levels of T resident memory cells (TRM) were detected in the cervix (FIG. 21B) In addition, the absolute number of antigen-specific CD8+ T-cells as determined tetramer staining (SHNFEKL+ CD 8+ T-cells) were highest in the mice immunized with cGASAN STING-IRAK mRNA LNPs + OVA mRNA LNPs (FIG.21C). Interestingly, when mice were injected with a high dose of antigen with control LNPs (luciferase mRNA LNPs + OVA mRNA LNPs (1 pg), the T-cell responses generated were not as strong as when mice were immunized with a suboptimal dose of OVA mRNA LNPs in combination with cGASAN STING-IRAKI mRNA LNPs. Overall, these data indicate that the use of cGASAN STING-IRAKI mRNA can strongly enhance antigen-specific circulating and mucosal T-cell responses even with a suboptimal dose of antigen mRNA. In addition, these data indicate that cGASAN STING-IRAKI mRNA LNPs + antigen mRNA LNPs induce durable mucosal T-cell responses compared to cGASAN mRNA LNPs + antigen mRNA LNPs.SEQUENCES SEQ ID NO: 1> H. SAPIENSPGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLN TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKF RKI IKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRK QLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQL KERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNL FSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO: 2> M._MU LATTAPGARKLRAVLEKLRLSHQDISKAAKWNGWGHLLRRLKCEPEFGGVEQLH TGSYYEHVKISAPNEFDVMFKLQVPRIQLEEYSDTGAYYFVKFKRNPEGNPLSLFLEDEILSASKMLSKF RKI IKEEINNIKDVQPDADVIMKRKRPGSPAVTLLINKEISVDITLALELKTSWPASTQEGLAIKNWLSA KVRQQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILSNHGKSKTCCENKEMKCCRKDCLKLMKYL LEQLKEKFKDKKHLDKFSSYHVKTAFFHLCTENPQDSQWDPKDLGLCFDNCVTYFLECLRTEQLKNYFIP GFNLFSSNLIDKTSKEFLSKQIEYERNNEFPVFGEF SEQ ID NO: 3> P. _ANUBISPGARKLRAVLEKLRLSHQDISKAAKWNGWGHLLHRLKCESEFEGVEQLH TGSYYEHVKISAPNEFDVMFKLQVPRIQLEEYSDTGAYYFVKFKRNPKGNPLSQFLEDEILSASKMLSQF RKI IKEEINNIKDTDVIMKRKRRGSPAVTLLINKEISVDITLALELKTSWPASTQEGLAIKNWLSAKVRQ QLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILSNHGKSKTCCENKEMKCCRKDCLKLMKYLLEQL57MF-367050437Docket No.: 16553-20014.40 KEKFKDKKHLDKFSSYHVKTAFFHLCTENPQDSQWDPKDLGLCFDNCVTHFLQCLRTEKLANYFIPGFNL FSSNLIDKSSKEFLSKQIEYERNNEFPVFGEF SEQ ID NO: 4> N._LEUCOGENYS PGARKLRAVLEKLKLSRQEISEAAEWNGWDHLLRRLQKCDSEFRGVGLLRTGSYYEH VKISAPNEFDVMFKLEVPRIQLEEYSNTGAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKI IKEE INNIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESNSNSWPASTQEGLPIKDWLSGKVRRQLRLKP FYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKTKTCCENKNVKCCRKDCLKLMKYLLEQLKERFKD KKHLDKFSSYHVKTAFFHVCTENPQDSQWDPKNLGFCFDKCVIYFLKCLRTEQLGNYFIPGFNLFSSNLI DKRSKEFLSKQIEYERNNEYPVFG SEQ ID NO: 5> H_LARPGARKLPAVLEKLKLSRQ EICEAAEWNGWDHLLRRLQKCDSEFRGVGLLRTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTG AYYFVKFKRNPKENPLCQFLEGEILSASKMLSKFRKI IKEEINNIKDTDVILKRKRGGSPAVTLLISEKI SVDITLALESKSSSWPASTQEGLPIKDWLSGKVRTQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKE ILNNHGETKTCCENKKVKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTENPKDSQW DPKDLGLCFDKCVIYFLQCLSTEQLGNYFIPGFNLFSSNLIDKRSKEFLSKQIEYERNNEYPVFGKC SEQ ID NO: 6> P,_ABELIIPGASKLRAVLEKLKLSRLEISKAAEWNRWDHLLRRLQKYNSEFRGVGLLRT GSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTGAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFR KI IKEEINNIKDTDVIMKRKRGGSPAVTLLISEEISVDITLALESKSSWPASTQEGLPIKNWLSAKVRKQ LRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCEDKEVKCCRKDCLKLMKYLLEQLK ERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDPKDLGLCFDNCVTYFLQCLRTEQLGNYFIPGFNLF SSNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO: 7> P._TROGLODYTESPGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLN TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKF RKI IKEEINNIKDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLPIKNWLSAKVRKQL RLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCEIKEAKCCRKDCLKLMKYLLEQLKE KFKDKKHLDKFSSYHVKTAFFHVCTENPQDSQWDRKDLGLCFDNCVTYFLQCLRIEKLENYFIPEFNLFS SDLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO: 8> G. _GORILLAPGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLN TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKF RKI IKEEINNIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRR QLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEAKCCRKDCLKLMKYLLEQL KERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKQLGLCFDNCVTYFLQCLRTERLENYFIPEFNL FSRNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO: 9> CGASAN_ CONSENSUS PGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLN58MF-367050437Docket No.: 16553-20014.40 TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKF RKIIKEEINX1IKDX2X3VIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLX4IX5NWLSAK VRXsQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCEXvKEXsKCCRKDCLKLMKY LLEQLKEX9FKDKKHLDKFSSYHVKTAFFHVCTX10NPQDSQWDRKX11LGLCFDNCVTYFLQCLRX12EX13L ENYFIPEFNLFSX14X15LIDKRSKEFLTKQIEYERNNEFPVFDEF, WhereinXi = N or D; X2= T or absent; X3= D or absent; X4= R or P; X5= Q or K; X6= K or R; X7= N or I; X8= E or A; X9= R or K; X10= Q or E; X41= D or Q; X12 = T or I; X43= K or R; X44= S or R; and X45= N or D SEQ ID NO: 10> HUMAN-CGAS_FULL-LENGTH MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQE RPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWD VPSPGLPVSAPILVRRDAAPGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLN TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKF RKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRK QLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQL KERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNL FSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO: 11> HUMAN-CGAS_N-TERMINUS MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQE RPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWD VP SP GLP VSAP I LVRRD AA SEQ ID NO: 12> THOSEA ASIGNA VIRUS T2A RIBOSOME-SKIPPING PEPTIDE GSGEGRGSLLTCGDVEENPGP SEQ ID NO: 13> PORCINE TESCHOVIRUS-1 P2A RIBOSOME-SKIPPING PEPTIDE GSGATNFSLLKQAGDVEENPGP SEQ ID NO: 14> EQUINE RHINITIS A VIRUS E2A RIBOSOME-SKIPPING PEPTIDEGS GQCTNYALLKLAGDVE SNP GP SEQ ID NO: 15> FOOT-AND-MOUTH DISEASE VIRUS F2A RIBOSOME-SKIPPING PEPTIDE GSGVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 16> GALLUS GALLUS OVALBUMIN PEPTIDESIINFEKL SEQ ID NO: 17> SYNTHETIC CGASAN MOUSE CODON-OPTIMIZED ATGCCCGGCGCCAGCAAGCTGAGGGCCGTGCTGGAGAAGCTGAAGCTGAGCAGGGACGACATCA GCACCGCCGCCGGCATGGTGAAGGGCGTGGTGGACCACCTGCTGCTGAGGCTGAAGTGCGACAG CGCCTTCAGGGGCGTGGGCCTGCTGAACACCGGCAGCTACTACGAGCACGTGAAGATCAGCGCC59MF-367050437Docket No.: 16553-20014.40 CCCAACGAGTTCGACGTGATGTTCAAGCTGGAGGTGCCCAGGATCCAGCTGGAGGAGTACAGCA ACACCAGGGCCTACTACTTCGTGAAGTTCAAGAGGAACCCCAAGGAGAACCCCCTGAGCCAGTT CCTGGAGGGCGAGATCCTGAGCGCCAGCAAGATGCTGAGCAAGTTCAGGAAGATCATCAAGGAG GAGATCAACGACATCAAGGACACCGACGTGATCATGAAGAGGAAGAGGGGCGGCAGCCCCGCCG TGACCCTGCTGATCAGCGAGAAGATCAGCGTGGACATCACCCTGGCCCTGGAGAGCAAGAGCAG CTGGCCCGCCAGCACCCAGGAGGGCCTGAGGATCCAGAACTGGCTGAGCGCCAAGGTGAGGAAG CAGCTGAGGCTGAAGCCCTTCTACCTGGTGCCCAAGCACGCCAAGGAGGGCAACGGCTTCCAGG AGGAGACCTGGAGGCTGAGCTTCAGCCACATCGAGAAGGAGATCCTGAACAACCACGGCAAGAG CAAGACCTGCTGCGAGAACAAGGAGGAGAAGTGCTGCAGGAAGGACTGCCTGAAGCTGATGAAG TACCTGCTGGAGCAGCTGAAGGAGAGGTTCAAGGACAAGAAGCACCTGGACAAGTTCAGCAGCT ACCACGTGAAGACCGCCTTCTTCCACGTGTGCACCCAGAACCCCCAGGACAGCCAGTGGGACAG GAAGGACCTGGGCCTGTGCTTCGACAACTGCGTGACCTACTTCCTGCAGTGCCTGAGGACCGAG AAGC T G GAGAAC T AC T T C AT C C C C GAG T T C AAC C T G T T C AGC AGC AAC C T GAT C GAC AAG AGGA GCAAGGAGTTCCTGACCAAGCAGATCGAGTACGAGAGGAACAACGAGTTCCCCGTGTTCGACGA GTTCTAATGA SEQ ID NO: 18>synthetic CGASAN_human_codon-optimized ATGCCCGGCGCCAGCAAGCTCCGGGCCGTGCTGGAGAAGCTGAAGCTCAGCAGGGACGACATCTCCACCG CCGCCGGCATGGTGAAGGGCGTGGTGGACCACCTGCTGCTCAGACTGAAGTGCGACTCCGCCTTCAGAGG CGTCGGCCTGCTGAACACCGGCAGCTACTACGAGCACGTGAAGATCAGCGCCCCCAATGAGTTCGACGTC ATGTTCAAGCTGGAGGTCCCCAGAATCCAGCTCGAGGAGTACTCCAACACTAGGGCCTACTACTTCGTGA AG T T C AAG AG AAAT C C G AAGG AG AT C C C C T G AG C C AG T T C C T G G AG G G T G AG AT AC T C T C AG C C AG C A GAT G C T G T C AAAG T T C AG G AAG AT C AT C AAGG AG GAG AT C AAC G AC AT C AAG G AC AC C G AC G T C AT C AT G AAG AGG AAGAG AG G AGG C AGC C C C G C C G T G AC C C T G C T GAT C AG C GAG AAG AT AAG C GT G GAC AT AAC C C TGGCCCTGGAGTCAAAGAGCAGCTGGCCCGCCAGCACCCAGGAGGGCCTGAGGATCCAGAACTGGCTGTC AGCCAAGGTGAGGAAGCAGCTGCGACTGAAGCCATTCTACCTGGTGCCCAAGCACGCCAAGGAGGGAAAT GGTTTCCAGGAGGAGACCTGGCGGCTGTCCTTCAGCCACATCGAGAAGGAGATCCTGAACAATCACGGAA AG AG C AAG AC C T G C T GT GAGAAC AAG GAG G AG AAGT G T T G C AGG AAG G AC T G T C T G AAG C T G AT GAAG TA C C T G C T G G AG C AG C T GAAG GAG AGG T T C AAGG AC AAG AAG C AC C T GG AC AAG T T C AG C AG C T AC C AC G T G AAGACTGCCTTCTTCCACGTGTGTACCCAGAACCCCCAGGACAGCCAGTGGGACAGGAAGGACCTGGGCC TCTGCTTCGACAACTGCGTGACCTACTTCCTGCAGTGCCTCAGGACCGAGAAGCTGGAGAATTACTTCAT CCCCGAGTT C AAT C T GT T C AG C AGC AAC C T C AT C GAC AAG AG AAG C AAG GAGTTCCTGAC C AAG C AG AT C GAG T AC G AGAG AAAC AAT GAG TTCCCAGTGTTC GAC G AGT T C T AAT GA SEQ ID NO: 19> H. SAPIENS GSDMDAC-Mutant_ (274aa) GSAFERWRRWQELDHGGEFIPVTSLQSSTGFQPYCLWRKPSSSWFWKPRYKCVNLSIKDILEPDAAE PDVQRGRSFHFYDAMDGQIQGSVELAAPGQAKNAGGAAVSDSSSTSMNVYSLSVDPNTWQTLLHERHLRQ PEHKVLQQLRSRGDNVYWTEVLQTQKEVEVTRTHKREGSGRFSLPGATCLQGEGQGHLSQKKTVTIPSG STLAFRVAQLVIDSDLDVLLFPDKKQRTFQPPATGHKRSTSEGAWPQLPSGLSMMRCLHNFLTD SEQ ID NO: 20>cGASdN-2A-GSDMDAC-Mutant_ ( 659aa)MPGASKLRAVLEKLKLSRDDI STAAGMVKGWDHLLLRLKCDSAFRGVGLLNTGSYYEHVKI SAPNEFDV MFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKI IKEE IND IKDTDVIM KRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKEGN GFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHV KTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQI EYERNNEFPVFDEFGSGEGRGSLLTCGDVEENPGPGSAFERWRRWQELDHGGEFIPVTSLQSSTGFQP60MF-367050437Docket No.: 16553-20014.40 YCLWRKPSSSWFWKPRYKCVNLSIKDILEPDAAEPDVQRGRSFHFYDAMDGQIQGSVELAAPGQAKNAG GAAVSDSSSTSMNVYSLSVDPNTWQTLLHERHLRQPEHKVLQQLRSRGDNVYWTEVLQTQKEVEVTRTH KREGSGRFSLPGATCLQGEGQGHLSQKKTVTIPSGSTLAFRVAQLVIDSDLDVLLFPDKKQRTFQPPATG HKRSTSEGAWPQLPSGLSMMRCLHNFLTD SEQ ID NO: 21> H. SAPIENS STING_ (378aa) PHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELR HIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAP AEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDN LSMADPNIRFLDKLPQQTGDRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSR EDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAV PSTSTMSQEPELLISGMEKPLPLRTDFS SEQ ID NO: 22> H. SAPIENS IRAK1-Cterminus_ ( 190aa) AWAGVPGHSEAASCIPPSPQENSYVSSTGRAHSGAAPWQPLAAPSGASAQAAEQLQRGPNQPVESDESL GGLSAALRSWHLTPSCPLDPAPLREAGCPQGDTAGESSWGSGPGSRPTAVEGLALGSSASSSSEPPQIII NPARQKMVQKLALYEDGALDSLQLLSSSSLPGLGLEQDRQGPEESDEFQS SEQ ID NO: 23>cGASdN-2A-STING-IRAKl_ (953aa) MPGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLNTGSYYEHVKISAPNEFDV MFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKI IKEEINDIKDTDVIM KRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKEGN GFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHV KTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQI EYERNNEFPVFDEFGSGEGRGSLLTCGDVEENPGPPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGL GEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFY YSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQ ARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDRAGIKDRVYSNSIYE LLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQE PADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSAWAGVP GHSEAASCIPPSPQENSYVSSTGRAHSGAAPWQPLAAPSGASAQAAEQLQRGPNQPVESDESLGGLSAAL RSWHLTPSCPLDPAPLREAGCPQGDTAGESSWGSGPGSRPTAVEGLALGSSASSSSEPPQIIINPARQKM VQKLALYEDGALDSLQLLSSSSLPGLGLEQDRQGPEESDEFQS SEQ ID NO: 24> STING-IRAK1_ ( 569aa) MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEEL RHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLA PAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPD NLSMADPNIRFLDKLPQQTGDRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFS REDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSA VPSTSTMSQEPELLISGMEKPLPLRTDFSAWAGVPGHSEAASCIPPSPQENSYVSSTGRAHSGAAPWQP LAAPSGASAQAAEQLQRGPNQPVESDESLGGLSAALRSWHLTPSCPLDPAPLREAGCPQGDTAGESSWGS GPGSRPTAVEGLALGSSASSSSEPPQI I INPARQKMVQKLALYEDGALDSLQLLSSSSLPGLGLEQDRQG PEESDEFQS SEQ ID NO: 19> H. SAPIENS GSDMDAC-Mutant_I / N SUBSTITUTION (274aa)61MF-367050437Docket No.: 16553-20014.40 GSAFERWRRWQELDHGGEFIPVTSLQSSTGFQPYCLWRKPSSSWFWKPRYKCVNLSIKDILEPDAAE PDVQRGRSFHFYDAMDGQIQGSVELAAPGQAKNAGGAAVSDSSSTSMNVYSLSVDPNTWQTLLHERHLRQ PEHKVLQQLRSRGDNVYWTEVLQTQKEVEVTRTHKREGSGRFSLPGATCLQGEGQGHLSQKKTVTIPSG STLAFRVAQLVIDSDLDVLLFPDKKQRTFQPPATGHKRSTSEGAWPQLPSGLSMMRCLHNFLTD SEQ ID NO: 25> P. TROGLODYTES GSDMDAC-Mutant I / N SUBSTITUTION— (274aa) GSAFERWRRWQELDHGGEFIPVTSLQSSTGFQPYCLWRKPSSSWFWKPRYKCVNLSIKDILEPDTPE PDVQRGRSFHFYDAMDGQIQGSVELAAPGQAKNAGGAAVSDSSSTSMNVYSLSVDPNTWQTLLHERHLRQ PEHKVLQQLRSRGDNVYWTEVLQTQKEVEVTRTHKREGSGRFSLPGAMCLQGEGQGHLSQKKTVTIPSG SILAFRVAQLVIDSDLDVLLFPDKKQRTFQPPPTGHKRSTSEGAWPQLPSGLSMMRCLHNFLTD SEQ ID NO: 26> G. GORILLA GSDMDAC-Mutant I / N SUBSTITUTION— (274aa) GSAFERWRRWQELDHGGEFIPVTSLQSSTGFQPYCLWRKPSSSWFWKPRYKCVNLSIKDILEPDAPE PDVQRGRSFHFYDAMDGQIQGSVELAAPGQAKNAGGAAVSDSSSTSMNVYSLSVDPNTWQTLLHERHLRQ PEHKVLQQLRSRGDNVYWTEVLQTQKEVEVTRTHKREGSGRFSLPGAMCLQGEGQGHLSQKKTVTIPSG SILAFRVAQLVIDSDLDVLLFPDKKQRTFQPPPTGHKRSTSEGAWPQLPSGLSMMRCLHNFLTD SEQ ID NO: 27> CONSENSUS GSDMDAC-Mutant I / N SUBSTITUTION GSAFERWRRWQELDHGGEFIPVTSLQSSTGFQPYCLWRKPSSSWFWKPRYKCVNLSI KDILEPDX1X2EPDVQRGRSFHFYDAMDGQIQGSVELAAPGQAKNAGGAAVSDSSSTSMNVY SLSVDPNTWQTLLHERHLRQPEHKVLQQLRSRGDNVYWTEVLQTQKEVEVTRTHKREGS GRFSLPGAX3CLQGEGQGHLSQKKTVTIPSGSX4LAFRVAQLVIDSDLDVLLFPDKKQRTFQ PPX5TGHKRSTSEGAWPQLPSGLSMMRCLHNFLTDXi = A or TX2= A or PX3= T or MX4= T or IX5= A or PSEQ ID NO: 28> P. TROGLODYTES STING PHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELR HIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAP AEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDN LSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSR EDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAV PSTSTMSQEPELLISGMEKPLPLRTDFS SEQ ID NO: 29> G. GORILLA STING PHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRCLVLHLASLQLGLLLNGLCSLAEELR HIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAP AEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRDAVSQRLYILLPLDCGVPDN LSMADPNIRFLDKLPQQTADRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQCSQAGFSR EDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAV PSTSTMSQEPELLISGMEKPLPLRTDFS SEQ ID NO: 3062MF-367050437Docket No.: 16553-20014.40 > STING CONSENSUS PHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRXiLVLHLASLQLGLLLN GX2CSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWML ALLGLSQALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRT YNQHYNNLLRX3AVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTX4DX5AGIKDRVYS NSIYELLENGQRAGTCVLEYATPLQTLFAMSQXgSQAGFSREDRLEQAKLFCRTLEDILAD APESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEP ELLISGMEKPLPLRTDFSXi = Y or CX2= V or LX3= G or DX4= G or AX5= R or HX6= Y or CSEQ ID NO: 31> P. TROGLODYTES IRAKI (C) _ ( 190AA) AWAGVPGHSEAASCIPPSPQENSYVSSTGRAHSGAAPWQPLAAPSGASAQAAEQLQRGPNQPVESDESL GGLSAALRSWHLTPSCPLDPAPLREAGCPQGDTAGESSWGSGPGSRPTAVEGLALGSSASSSSEPPQIII NPARQKMVQKLALYEDGALDSLQLLSSSSLPGLGLEQDRQGPEESDEFQS SEQ ID NO: 32> G. GORILLA IRAKI (C) _ (190AA) AWAGVPGHSEAASCIPPSPQENSYVSSTGSAHSGAAPWQPLAAPSGASAQAAEQLQRGPNQPVESDESL GGLSAALRSWHLTPSCPLDPAPLREAGCPQGATAGESSWGSGPGSRPTAVEGLALGSSASSSSEPPQIII NPARQKMVQKLALYEDGALDSLQLLSSSSLPGLGLEQDRQGPEESDEFQS SEQ ID NO: 33> IRAK1 (C) CONSENSUS AWAGVPGHSEAASCIPPSPQENSYVSSTGXiAHSGAAPWQPLAAPSGASAQAAEQLQRGP NQPVESDESLGGLSAALRSWHLTPSCPLDPAPLREAGCPQGX2TAGESSWGSGPGSRPTAV EGLALGSSASSSSEPPQI I INPARQKMVQKLALYEDGALDSLQLLSSSSLPGLGLEQDRQ GPEESDEFQSXi = R or SX2= D or ASEQ ID NO: 34> H. SAPIENS GSDMDAC-Mutant_human_codon-optimized GGGAGTGCCTTTGAGAGGGTAGTGAGGAGAGTGGTGCAGGAGCTGGACCATGGTGGGGAGTTCATCCCTG TGACCAGCCTGCAGAGCTCCACTGGCTTCCAGCCCTACTGCCTGGTGGTTAGGAAGCCCAGCAGCAGCTG GTTCTGGAAACCCAGGTATAAGTGTGTGAACCTGTCTATCAAGGACATCCTGGAGCCCGATGCCGCCGAA CCCGACGTGCAGCGAGGCAGGAGCTTCCACTTCTACGATGCCATGGATGGGCAGATACAGGGCAGCGTGG AGCTGGCCGCCCCTGGACAGGCCAAGAACGCTGGCGGGGCCGCCGTGTCTGACAGCTCCAGCACCAGCAT GAATGTGTACAGCCTGAGTGTGGACCCTAACACCTGGCAGACTCTGCTCCATGAGAGGCACCTGAGGCAG CCCGAACACAAAGTGCTGCAGCAGCTGCGCAGCCGCGGGGACAACGTGTACGTGGTGACTGAGGTGCTGC AGACACAGAAGGAGGTGGAAGTGACCCGCACCCACAAGAGGGAGGGCAGTGGCAGGTTTTCCCTGCCCGG AGCCACCTGCTTGCAGGGTGAGGGCCAGGGCCATCTGAGCCAGAAGAAGACCGTGACCATCCCCAGCGGC AGCACCCTCGCCTTCAGGGTGGCCCAGCTGGTTATTGACTCTGACTTGGACGTGCTTCTCTTCCCCGATA AGAAGCAGAGGACCTTCCAGCCCCCCGCCACAGGCCACAAGAGATCCACCAGCGAAGGCGCCTGGCCCCA GCTGCCCTCTGGCCTCTCCATGATGAGGTGCCTCCACAACTTCCTGACAGATTAATGA63MF-367050437Docket No.: 16553-20014.40 SEQ ID NO: 35>cGASdN-2A-GSDMDAC_human_codon-optimized ATGCCCGGCGCCAGCAAGCTCCGGGCCGTGCTGGAGAAGCTGAAGCTCAGCAGGGACGACATCTCCACCG CCGCCGGCATGGTGAAGGGCGTGGTGGACCACCTGCTGCTCAGACTGAAGTGCGACTCCGCCTTCAGAGG CGTCGGCCTGCTGAACACCGGCAGCTACTACGAGCACGTGAAGATCAGCGCCCCCAATGAGTTCGACGTC ATGTTCAAGCTGGAGGTCCCCAGAATCCAGCTCGAGGAGTACTCCAACACTAGGGCCTACTACTTCGTGA AG T T C AAG AG AAAT C C G AAGG AG AAT C C C C T G AG C C AG T T C C T G G AG G G T G AG AT AC T C T C AG C C AG C AA GAT G C T G T C AAAG T T C AG G AAG AT C AT C AAGG AG GAG AT C AAC G AC AT C AAG G AC AC C G AC G T C AT C AT G AAG AGG AAGAG AG G AGG C AGC C C C G C C G T G AC C C T G C T GAT C AG C GAG AAG AT AAG C GT G GAC AT AAC C C TGGCCCTGGAGTCAAAGAGCAGCTGGCCCGCCAGCACCCAGGAGGGCCTGAGGATCCAGAACTGGCTGTC AGCCAAGGTGAGGAAGCAGCTGCGACTGAAGCCATTCTACCTGGTGCCCAAGCACGCCAAGGAGGGAAAT GGTTTCCAGGAGGAGACCTGGCGGCTGTCCTTCAGCCACATCGAGAAGGAGATCCTGAACAATCACGGAA AG AG C AAG AC C T G C T GT G AGAAC AAG GAG G AG AAGT G T T G C AGG AAG G AC T G T C T G AAG C T G AT GAAG TA C C T G C T G G AG C AG C T GAAG GAG AGG T T C AAGG AC AAG AAG C AC C T GG AC AAG T T C AG C AG C T AC C AC G T G AAGACTGCCTTCTTCCACGTGTGTACCCAGAACCCCCAGGACAGCCAGTGGGACAGGAAGGACCTGGGCC TCTGCTTCGACAACTGCGTGACCTACTTCCTGCAGTGCCTCAGGACCGAGAAGCTGGAGAATTACTTCAT CCCCGAGTT C AAT C T GT T C AG C AGC AAC C T C AT C GAC AAG AG AAG C AAG GAGTTCCTGAC C AAG C AG AT C GAGTACGAGAGAAACAATGAGTTCCCAGTGTTCGACGAGTTCGGCTCTGGAGAGGGCAGAGGCTCTCTGC TGACATGCGGAGACGTGGAAGAGAATCCCGGCCCTGGGAGTGCCTTTGAGAGGGTAGTGAGGAGAGTGGT GCAGGAGCTGGACCATGGTGGGGAGTTCATCCCTGTGACCAGCCTGCAGAGCTCCACTGGCTTCCAGCCC TACTGCCTGGTGGTTAGGAAGCCCAGCAGCAGCTGGTTCTGGAAACCCAGGTATAAGTGTGTGAACCTGT CTATCAAGGACATCCTGGAGCCCGATGCCGCCGAACCCGACGTGCAGCGAGGCAGGAGCTTCCACTTCTA CGATGCCATGGATGGGCAGATACAGGGCAGCGTGGAGCTGGCCGCCCCTGGACAGGCCAAGAACGCTGGC GGGGCCGCCGTGTCTGACAGCTCCAGCACCAGCATGAATGTGTACAGCCTGAGTGTGGACCCTAACACCT GGCAGACTCTGCTCCATGAGAGGCACCTGAGGCAGCCCGAACACAAAGTGCTGCAGCAGCTGCGCAGCCG CGGGGACAACGTGTACGTGGTGACTGAGGTGCTGCAGACACAGAAGGAGGTGGAAGTGACCCGCACCCAC AAGAGGGAGGGCAGTGGCAGGTTTTCCCTGCCCGGAGCCACCTGCTTGCAGGGTGAGGGCCAGGGCCATC TGAGCCAGAAGAAGACCGTGACCATCCCCAGCGGCAGCACCCTCGCCTTCAGGGTGGCCCAGCTGGTTAT TGACTCTGACTTGGACGTGCTTCTCTTCCCCGATAAGAAGCAGAGGACCTTCCAGCCCCCCGCCACAGGC CACAAGAGATCCACCAGCGAAGGCGCCTGGCCCCAGCTGCCCTCTGGCCTCTCCATGATGAGGTGCCTCC ACAACTTCCT GAC AG AT T AAT GA SEQ ID NO: 36> H. SAPIENS STING_human_codon-optimized ATGCCCCACAGCAGCCTGCATCCCAGCATCCCGTGTCCCAGGGGCCACGGGGCCCAGAAGGCAGCCTTGG TGCTGCTGAGTGCCTGCCTGGTGACCCTTTGGGGGTTGGGAGAGCCCCCCGAGCACACCCTCAGGTACCT GGTCCTCCACCTGGCCAGCCTGCAGCTGGGACTGCTGCTTAACGGGGTCTGCAGCCTGGCTGAGGAGCTG AGGCACATCCACAGCAGGTACAGGGGCAGCTACTGGCGAACAGTGAGGGCCTGCCTGGGCTGCCCCCTTA GGAGGGGGGCCCTGTTGCTGCTGAGCATCTATTTCTACTACAGCCTCCCCAATGCGGTCGGCCCGCCCTT CACCTGGATGCTTGCCCTCCTGGGCTTGAGCCAGGCACTGAACATCCTCCTGGGCCTCAAGGGCCTGGCC CCCGCTGAGATCTCTGCAGTGTGTGAAAAAGGGAATTTCAACGTGGCCCATGGGCTGGCATGGAGCTATT ACATCGGATATCTGAGGCTGATCCTGCCCGAGCTCCAGGCCAGGATTCGAACCTACAATCAGCATTACAA CAACCTGCTTAGGGGCGCAGTGAGCCAGAGACTGTATATTCTCCTCCCCTTGGACTGTGGGGTGCCCGAC AACCTGAGTATGGCTGACCCCAACATTAGGTTCCTGGACAAACTGCCCCAGCAGACCGGCGACAGGGCTG GCATCAAGGACAGGGTGTACAGCAACAGCATCTATGAGCTTCTGGAGAACGGGCAGAGGGCGGGCACCTG TGTCCTGGAGTACGCCACCCCCTTGCAGACCTTGTTTGCCATGAGCCAATACAGTCAAGCTGGCTTTAGC AGGGAGGACAGGCTTGAGCAGGCCAAACTCTTCTGCAGGACACTTGAGGACATCCTGGCAGACGCCCCCG AGTCTCAGAACAACTGCAGGCTCATTGCCTACCAGGAACCCGCAGACGACAGCAGCTTCAGCCTGAGCCA GGAGGTGCTCAGGCACCTGAGGCAGGAGGAAAAGGAAGAGGTGACCGTGGGCAGCTTGAAGACCAGCGCG GTGCCCAGTACCAGCACGATGAGCCAAGAGCCCGAGCTCCTCATCAGTGGAATGGAAAAGCCCCTCCCCC TCAGGACGGACTTCTCTTAATGA64MF-367050437Docket No.: 16553-20014.40SEQ ID NO: 37> H. SAPIENS IRAKl-Cterminus_human_codon-optimized GCCGTGGTGGCTGGGGTGCCCGGACACAGCGAGGCTGCCAGCTGCATCCCCCCCTCTCCCCAGGAGAACT CTTACGTGAGCAGCACCGGCAGAGCACACAGTGGCGCCGCCCCCTGGCAGCCTCTGGCTGCTCCCTCTGG CGCCTCTGCCCAGGCCGCCGAGCAACTGCAGAGGGGACCCAATCAGCCCGTGGAGTCCGACGAAAGCTTG GGCGGACTGTCTGCAGCCCTTAGGAGCTGGCATCTGACCCCCAGCTGCCCTCTGGACCCCGCCCCTCTGC GAGAGGCCGGCTGTCCCCAGGGCGACACAGCTGGCGAGAGCAGCTGGGGCTCTGGCCCCGGGAGCAGACC CACCGCCGTGGAAGGCCTGGCACTGGGCTCTAGTGCATCCAGCAGCAGCGAGCCCCCCCAGATCATTATT AATCCCGCCAGGCAGAAAATGGTGCAGAAGCTGGCACTGTATGAGGACGGGGCCCTCGACAGCCTGCAAC TGCTGAGCAGCAGCAGCCTGCCCGGACTGGGACTGGAGCAAGACAGACAGGGCCCCGAGGAGAGCGACGA GT T T CAGAGC T AAT GA SEQ ID NO: 38>cGASdN-2A-STING-IRAKl_human_codon-optimized ATGCCCGGCGCCAGCAAGCTCCGGGCCGTGCTGGAGAAGCTGAAGCTCAGCAGGGACGACATCTCCACCG CCGCCGGCATGGTGAAGGGCGTGGTGGACCACCTGCTGCTCAGACTGAAGTGCGACTCCGCCTTCAGAGG CGTCGGCCTGCTGAACACCGGCAGCTACTACGAGCACGTGAAGATCAGCGCCCCCAATGAGTTCGACGTC ATGTTCAAGCTGGAGGTCCCCAGAATCCAGCTCGAGGAGTACTCCAACACTAGGGCCTACTACTTCGTGA AG T T C AAG AG AAAT C C G AAGG AG AAT C C C C T G AG C C AG T T C C T G G AGG G T G AG AT AC T C T C AG C C AG C AA GAT G C T G T C AAAG T T C AG G AAG AT C AT C AAGG AG GAG AT C AAC G AC AT C AAG G AC AC C G AC GT C AT C AT G AAGAGGAAGAGAGGAGGCAGCCCCGCCGTGACCCTGCTGATCAGCGAGAAGATAAGCGTGGACATAACCC TGGCCCTGGAGTCAAAGAGCAGCTGGCCCGCCAGCACCCAGGAGGGCCTGAGGATCCAGAACTGGCTGTC AGCCAAGGTGAGGAAGCAGCTGCGACTGAAGCCATTCTACCTGGTGCCCAAGCACGCCAAGGAGGGAAAT GGTTTCCAGGAGGAGACCTGGCGGCTGTCCTTCAGCCACATCGAGAAGGAGATCCTGAACAATCACGGAA AG AG C AAG AC C T G C T GT G AGAAC AAG GAG G AG AAGT G T T G C AGG AAGG AC T G T C T G AAG C T GAT GAAG TA C C T G C T G G AG C AG C T GAAG GAG AGG T T C AAGG AC AAG AAG C AC C T G GAC AAG T T C AG C AG C T AC C AC G T G AAGACTGCCTTCTTCCACGTGTGTACCCAGAACCCCCAGGACAGCCAGTGGGACAGGAAGGACCTGGGCC TCTGCTTCGACAACTGCGTGACCTACTTCCTGCAGTGCCTCAGGACCGAGAAGCTGGAGAATTACTTCAT CCCCGAGTTCAATCTGTTCAGCAGCAACCTCATCGACAAGAGAAGCAAGGAGTTCCTGACCAAGCAGATC GAGTACGAGAGAAACAATGAGTTCCCAGTGTTCGACGAGTTCGGCTCTGGAGAGGGCAGAGGCTCTCTGC TGACATGCGGAGACGTGGAAGAGAATCCCGGCCCTCCCCACAGCAGCCTGCATCCCAGCATCCCGTGTCC CAGGGGCCACGGGGCCCAGAAGGCAGCCTTGGTGCTGCTGAGTGCCTGCCTGGTGACCCTTTGGGGGTTG GGAGAGCCCCCCGAGCACACCCTCAGGTACCTGGTCCTCCACCTGGCCAGCCTGCAGCTGGGACTGCTGC TTAACGGGGTCTGCAGCCTGGCTGAGGAGCTGAGGCACATCCACAGCAGGTACAGGGGCAGCTACTGGCG AACAGTGAGGGCCTGCCTGGGCTGCCCCCTTAGGAGGGGGGCCCTGTTGCTGCTGAGCATCTATTTCTAC TACAGCCTCCCCAATGCGGTCGGCCCGCCCTTCACCTGGATGCTTGCCCTCCTGGGCTTGAGCCAGGCAC TGAACATCCTCCTGGGCCTCAAGGGCCTGGCCCCCGCTGAGATCTCTGCAGTGTGTGAAAAAGGGAATTT CAACGTGGCCCATGGGCTGGCATGGAGCTATTACATCGGATATCTGAGGCTGATCCTGCCCGAGCTCCAG GCCAGGATTCGAACCTACAATCAGCATTACAACAACCTGCTTAGGGGCGCAGTGAGCCAGAGACTGTATA TTCTCCTCCCCTTGGACTGTGGGGTGCCCGACAACCTGAGTATGGCTGACCCCAACATTAGGTTCCTGGA CAAACTGCCCCAGCAGACCGGCGACAGGGCTGGCATCAAGGACAGGGTGTACAGCAACAGCATCTATGAG CTTCTGGAGAACGGGCAGAGGGCGGGCACCTGTGTCCTGGAGTACGCCACCCCCTTGCAGACCTTGTTTG CCATGAGCCAATACAGTCAAGCTGGCTTTAGCAGGGAGGACAGGCTTGAGCAGGCCAAACTCTTCTGCAG GACACTTGAGGACATCCTGGCAGACGCCCCCGAGTCTCAGAACAACTGCAGGCTCATTGCCTACCAGGAA CCCGCAGACGACAGCAGCTTCAGCCT GAG C C AG GAG GTGCTCAGGCACCTGAGGCAGGAG G AAAAG GAAG AGGTGACCGTGGGCAGCTTGAAGACCAGCGCGGTGCCCAGTACCAGCACGATGAGCCAAGAGCCCGAGCT CCTCATCAGTGGAATGGAAAAGCCCCTCCCCCTCAGGACGGACTTCTCTGCCGTGGTGGCTGGGGTGCCC GGACACAGCGAGGCTGCCAGCTGCATCCCCCCCTCTCCCCAGGAGAACTCTTACGTGAGCAGCACCGGCA GAGCACACAGTGGCGCCGCCCCCTGGCAGCCTCTGGCTGCTCCCTCTGGCGCCTCTGCCCAGGCCGCCGA GCAACTGCAGAGGGGACCCAATCAGCCCGTGGAGTCCGACGAAAGCTTGGGCGGACTGTCTGCAGCCCTT65MF-367050437Docket No.: 16553-20014.40 AGGAGCTGGCATCTGACCCCCAGCTGCCCTCTGGACCCCGCCCCTCTGCGAGAGGCCGGCTGTCCCCAGG GCGACACAGCTGGCGAGAGCAGCTGGGGCTCTGGCCCCGGGAGCAGACCCACCGCCGTGGAAGGCCTGGC ACTGGGCTCTAGTGCATCCAGCAGCAGCGAGCCCCCCCAGATCATTATTAATCCCGCCAGGCAGAAAATG GTGCAGAAGCTGGCACTGTATGAGGACGGGGCCCTCGACAGCCTGCAACTGCTGAGCAGCAGCAGCCTGC CCGGACTGGGACTGGAGCAAGACAGACAGGGCCCCGAGGAGAGCGACGAGTTTCAGAGCTAATGA SEQ ID NO: 39> STING-IRAKl_human_codon-optimized ATGCCCCACAGCAGCCTGCATCCCAGCATCCCGTGTCCCAGGGGCCACGGGGCCCAGAAGGCAGCCTTGG TGCTGCTGAGTGCCTGCCTGGTGACCCTTTGGGGGTTGGGAGAGCCCCCCGAGCACACCCTCAGGTACCT GGTCCTCCACCTGGCCAGCCTGCAGCTGGGACTGCTGCTTAACGGGGTCTGCAGCCTGGCTGAGGAGCTG AGGCACATCCACAGCAGGTACAGGGGCAGCTACTGGCGAACAGTGAGGGCCTGCCTGGGCTGCCCCCTTA GGAGGGGGGCCCTGTTGCTGCTGAGCATCTATTTCTACTACAGCCTCCCCAATGCGGTCGGCCCGCCCTT CACCTGGATGCTTGCCCTCCTGGGCTTGAGCCAGGCACTGAACATCCTCCTGGGCCTCAAGGGCCTGGCC CCCGCTGAGATCTCTGCAGTGTGTGAAAAAGGGAATTTCAACGTGGCCCATGGGCTGGCATGGAGCTATT ACATCGGATATCTGAGGCTGATCCTGCCCGAGCTCCAGGCCAGGATTCGAACCTACAATCAGCATTACAA CAACCTGCTTAGGGGCGCAGTGAGCCAGAGACTGTATATTCTCCTCCCCTTGGACTGTGGGGTGCCCGAC AACCTGAGTATGGCTGACCCCAACATTAGGTTCCTGGACAAACTGCCCCAGCAGACCGGCGACAGGGCTG GCATCAAGGACAGGGTGTACAGCAACAGCATCTATGAGCTTCTGGAGAACGGGCAGAGGGCGGGCACCTG TGTCCTGGAGTACGCCACCCCCTTGCAGACCTTGTTTGCCATGAGCCAATACAGTCAAGCTGGCTTTAGC AGGGAGGACAGGCTTGAGCAGGCCAAACTCTTCTGCAGGACACTTGAGGACATCCTGGCAGACGCCCCCG AGTCTCAGAACAACTGCAGGCTCATTGCCTACCAGGAACCCGCAGACGACAGCAGCTTCAGCCTGAGCCA GGAGGTGCTCAGGCACCTGAGGCAGGAGGAAAAGGAAGAGGTGACCGTGGGCAGCTTGAAGACCAGCGCG GTGCCCAGTACCAGCACGATGAGCCAAGAGCCCGAGCTCCTCATCAGTGGAATGGAAAAGCCCCTCCCCC TCAGGACGGACTTCTCTGCCGTGGTGGCTGGGGTGCCCGGACACAGCGAGGCTGCCAGCTGCATCCCCCC CTCTCCCCAGGAGAACTCTTACGTGAGCAGCACCGGCAGAGCACACAGTGGCGCCGCCCCCTGGCAGCCT CTGGCTGCTCCCTCTGGCGCCTCTGCCCAGGCCGCCGAGCAACTGCAGAGGGGACCCAATCAGCCCGTGG AGTCCGACGAAAGCTTGGGCGGACTGTCTGCAGCCCTTAGGAGCTGGCATCTGACCCCCAGCTGCCCTCT GGACCCCGCCCCTCTGCGAGAGGCCGGCTGTCCCCAGGGCGACACAGCTGGCGAGAGCAGCTGGGGCTCT GGCCCCGGGAGCAGACCCACCGCCGTGGAAGGCCTGGCACTGGGCTCTAGTGCATCCAGCAGCAGCGAGC CCCCCCAGATCATTATTAATCCCGCCAGGCAGAAAATGGTGCAGAAGCTGGCACTGTATGAGGACGGGGC CCTCGACAGCCTGCAACTGCTGAGCAGCAGCAGCCTGCCCGGACTGGGACTGGAGCAAGACAGACAGGGC C C C GAG G AGAG C G AC GAG T T T C AGAG C T AAT G A SEQ ID NO: 40>furin cleavable linkerRGRKRRS SEQ ID NO: 41> CGASAN Negative Control Sequence (dblmut )MPGASKLRAVLEKLKLSRDDI STAAGMVKGWDHLLLRLKCDSAFRGVGLLNTGSYYEHVKI SAPNAFAV MFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKI IKEE IND IKDTDVIM KRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKEGN GFQEETWRLSFSHIEKEILNNHGKSKTAAENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHV KTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQI EYERNNEFPVFDEF66MF-367050437

Claims

Docket No.: 16553-20014.40CLAIMSWe claim:

1. A nucleic acid comprising a coding region of a cyclic GMP-AMP synthase (cGAS) and a coding region of an inducer of inflammation.

2. The nucleic acid of claim 1, wherein the inducer of inflammation comprises a gasdermin (GSDM), a stimulator of interferon genes (STING), an interleukin-1 receptor-associated kinase (IRAK), or a combination thereof.

3. A nucleic acid comprising a coding region of a cyclic GMP-AMP synthase (cGAS) and a coding region of a gasdermin (GSDM).

4. The nucleic acid of claim 2 or claim 3, wherein the GSDM is a truncated gasdermin D devoid of a carboxy-terminal auto-inhibitory domain (GSDMDAC).

5. The nucleic acid of claim 4, wherein the GSDMDAC comprises:i) the amino acid sequence of SEQ ID NO: 19;ii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19; oriii) the consensus amino acid sequence of SEQ ID NO:27.

6. The nucleic acid of claim 5, wherein the GSDMDAC comprises an I104N substitution.

7. A nucleic acid comprising a coding region of a cyclic GMP-AMP synthase (cGAS) and a coding region of one or both of a stimulator of interferon genes (STING) and an interleukin-1 receptor-associated kinase (IRAK).

8. The nucleic acid of claim 2 or claim 7, comprising the coding region of STING, wherein the STING comprises:i) the amino acid sequence of SEQ ID NO:21;ii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or67MF-367050437Docket No.: 16553-20014.40 99% identical to SEQ ID NO:21; oriii) the consensus amino acid sequence of SEQ ID NO:30.

9. The nucleic acid of claim 2 or claim 7, comprising the coding region of IRAK, wherein the IRAK is a truncated IRAKI devoid of a death domain, a proST (proline, serine, threonine) domain and a kinase domain (IRAKI AN).

10. The nucleic acid of claim 9, wherein the IRAKI AN comprises:i) the amino acid sequence of SEQ ID NO:22;ii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:22; oriii) the consensus amino acid sequence of SEQ ID NO: 33.

11. The nucleic acid of claim 2 or claim 7, comprising the coding region of STING and the coding region of IRAK, wherein the IRAK is a truncated IRAKI devoid of a death domain, a proST (proline, serine, threonine) domain and a kinase domain (IRAKI AN).

12. The nucleic acid of claim 11, wherein the STING and the IRAKI AN are expressed as a STING-IRAKI AN fusion protein.

13. The nucleic acid of claim 12, wherein the STING-IRAKI AN fusion protein comprises:i) the amino acid sequence of SEQ ID NO:24; orii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:24.

14. The nucleic acid of any one of claims 1-13, wherein the cGAS is a constitutively-active cGAS that has a greater propensity to self-DNA reactivity than its wild-type counterpart.

15. The nucleic acid of any one of claims 1-14, wherein the cGAS is a truncated cGAS devoid of an amino-terminal phosphoinositide-binding domain (cGAS AN).

16. The nucleic acid of claim 15, wherein the cGASAN comprises:i) the amino acid sequence of SEQ ID NO:1;68MF-367050437Docket No.: 16553-20014.40 the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1; oriii) the consensus amino acid sequence of SEQ ID NO:9.

17. The nucleic acid of any one of claims 1-16, wherein the coding region of the cGASAN is in operable combination with a start codon, optionally wherein the start codon is AUG, CUG, GUG or ACG, optionally wherein the start codon is AUG.

18. The nucleic acid of claim 17, wherein the cGASAN is encoded by the nucleotide sequence of SEQ ID NO: 18.

19. The nucleic acid of any one of claims 2-6 or any one of claims 14-18, wherein the coding region of the cGAS is separated from the coding region of the GSDM by an intervening sequence, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES).

20. The nucleic acid of claim 19, wherein the intervening sequence encodes a 2A-like peptide sequence such that the coding regions of the cGAS and the GSDM are configured as cGAS-2A-GSDM comprising the amino acid sequence of SEQ ID NO:20 or the amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:20.

21. The nucleic acid of claim 20, wherein the cGAS-2 A-GSDM is encoded by the nucleotide sequence of SEQ ID NO: 35.

22. The nucleic acid of claim 1, claim 2 or any one of claims 12-18, wherein the coding region of the cGAS is separated from the coding region of the STING-IRAK fusion protein by an intervening sequence, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES).

23. The nucleic acid of claim 22, wherein the intervening sequence encodes a 2A-like peptide sequence such that the coding regions of the cGAS and the STING-IRAK are configured as cGAS-2A-STING-IRAK comprising the amino acid sequence of SEQ ID NO:23 or the amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:23.69MF-367050437Docket No.: 16553-20014.40 24. The nucleic acid of claim 23, wherein the cGAS-2A-STING-IRAK is encoded by the nucleotide sequence of SEQ ID NO:38.

25. A nucleic acid comprising a coding region of a stimulator of interferon genes (STING) and a coding region of interleukin-1 receptor-associated kinase (IRAK).

26. The nucleic acid of claim 25, wherein the STING comprises:i) the amino acid sequence of SEQ ID NO:21;ii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21; oriii) the consensus amino acid sequence of SEQ ID NO:30.

27. The nucleic acid of claim 25 or claim 26, wherein the IRAK is a truncated IRAKI devoid of a death domain, a proST (proline, serine, threonine) domain and a kinase domain (IRAKI AN).

28. The nucleic acid of claim 27, wherein the IRAKI AN comprises:i) the amino acid sequence of SEQ ID NO:22;ii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:22; oriii) the consensus amino acid sequence of SEQ ID NO: 33.

29. The nucleic acid of claim 28, wherein the STING and the (IRAKI AN) are expressed as a STING-IRAKI AN fusion protein.

30. The nucleic acid of claim 29, wherein the STING-IRAKI AN fusion protein comprises:i) the amino acid sequence of SEQ ID NO:24; orii) the amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:24.

31. The nucleic acid of claim 30, wherein the STING-IRAK is encoded by the nucleotide sequence of SEQ ID NO:39.70MF-367050437Docket No.: 16553-20014.40 32. The nucleic acid of any one of claims 1-31, wherein the nucleic acid is an mRNA.

33. The nucleic acid of claim 32, wherein the mRNA comprises a 5’ untranslated region (5’UTR) and a 3’ untranslated region (3’UTR).

34. The nucleic acid of claim 33, wherein the mRNA further comprises one or both of a 5' cap structure and a polyA tail.

35. The nucleic acid of any one of claims 32-34, wherein the mRNA comprises a modified nucleoside, optionally wherein the modified nucleoside comprises one or more of the group consisting of Nl-methyl-pseudouridine, 5 -methylcytidine (m5C), 5 -methyluridine (m5U), N6-methyladenosine (m6A), 2-thiouridine (s2U), pseudouridine, 2’-O-methyluridine (um), and 5 -methoxyuridine (mo5U), optionally wherein the modified nucleoside comprises Nl-methyl-pseudouridine.

36. The nucleic acid of any one of claims 32-35, wherein the mRNA is less than about 5000, 4000 or 3000 nucleotides in length.

37. The nucleic acid of any one of claims 1-31, wherein the nucleic acid is a DNA.

38. The nucleic acid of claim 37, wherein the DNA comprises an expression cassette in which the coding region of the cGAS or the STING is in operable combination with a promoter.

39. The nucleic acid of claim 38, wherein the expression cassette is present within a plasmid, wherein the plasmid comprises an origin of replication and a selectable marker.

40. The nucleic acid of claim 39, wherein the plasmid DNA is less than about 15,000, 14,000, 13,000, 12,000, 11,000, 10,000, 9,000 or 8,000 nucleotides in length.

41. A composition comprising the mRNA of any one of claims 32-36 encapsulated in a nanoparticle.71MF-367050437Docket No.: 16553-20014.40 42. The composition of claim 41, wherein the nanoparticle is a lipid nanoparticle (LNP) comprising an ionizable lipid, a pegylated lipid, a structural lipid, a phospholipid, or a combination thereof, optionally wherein the LNP comprises an ionizable lipid.

43. The composition of claim 42, wherein the LNP comprises the phospholipid, the ionizable lipid, the pegylated lipid, and the structural lipid.

44. The composition of claim 42 or claim 43, wherein the ionizable lipid comprises: i) 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester (SM-102) or analogs or derivatives thereof; and / orii) 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium (ALC-0315) or analogs or derivatives thereof; and / oriii) (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) or analogs or derivatives thereof.

45. The composition of any one of claims 42-44, wherein the pegylated lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatide acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglyerol, and combinations thereof, optionally wherein the pegylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG],46. The composition of any one of claims 42-45, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and combinations thereof, optionally wherein the structural lipid comprises cholesterol.

47. The composition of any one of claims 42-46, wherein the phospholipid comprises: i) a hydrophilic head moiety selected from the group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin; andii) one or more fatty acid tail moieties selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic72MF-367050437Docket No.: 16553-20014.40 acid, alpha-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanoic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

48. The composition of any one of claims 42-46, wherein the phospholipid is selected from the group consisting of: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,l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine,1.2-dilinolenoyl-sn-glycero-3-phosphocholine,1.2-diarachidonoyl-sn-glycero-3-phosphocholine,1.2-didocosahexaenoyl-sn-glycero-3 -phosphocholine,1.2-dioleoyl-sn-glycero-3-phosphoethanola mine (DOPE),1.2-diphytanoyl-sn-glycero-3-phosphoethanolamine,1.2-distearoyl-sn-glycero-3 -phosphoethanolamine,1.2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine,1.2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1.2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1.2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine,1.2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), sphingomyelin, andcombinations thereof,optionally wherein the phospholipid comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

49. The composition of claim 41, wherein the nanoparticle is a polymeric nanoparticle comprising a non-lipid polymer.73MF-367050437Docket No.: 16553-20014.40 50. The composition of claim 49, wherein the non-lipid polymer comprises a poly(β-amino ester) (PBAE), a cyclodextrin (CD), a polyethyleneimine (PEI), a poly(lactic-co-glycolic acid) (PLGA), a polyamidoamine (PAMAM) dendrimer or an ionizable amphiphilic Janus dendrimer.

51. The composition of claim 41, wherein the nanoparticle is a protein nanoparticle, a viral particle, a virus-like particle (VLP) or a cationic nanoemulsion.

52. A composition comprising the mRNA of any one of claims 32-36 complexed with one or more lipids (RNA-lipoplex), wherein the one or more lipids comprise a first lipid and a second lipid.

53. The composition of claim 52, wherein the first lipid is a cationic lipid, and the second lipid is a neutral or anionic lipid.

54. The composition of claim 53, wherein the cationic lipid comprises one or both of: i) l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) or analogs or derivatives thereof; andii) l,2-dioleoyl-3-trimethylammonium propane (DOTAP) or analogs or derivatives thereof.

55. The composition of claim 53 or claim 54, wherein the neutral or anionic lipid comprises:i) l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphoethanolamine (DOPE) or analogs or derivatives thereof; and / orii) cholesterol or analogs or derivatives thereof; and / oriii) 1,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC) or analogs or derivatives thereof.

56. The composition of any one of claims 41-55, further comprising a further mRNA encapsulated in the nanoparticle or complexed with the one or more lipids, wherein the further mRNA comprises a coding region of an antigen.

57. The composition of any one of claims 41-55, wherein the mRNA further comprises a coding region of an antigen, and wherein the coding region of the cGAS, the STING or the IRAK is separated from the coding region of the antigen by an intervening sequence,74MF-367050437Docket No.: 16553-20014.40 wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES).

58. The composition of any one of claims 41-57, wherein the composition does not comprise a TLR7 / 8 agonist.

59. The composition of any one of claims 41-58, wherein the composition does not comprise a lysophosphatidylcholine (LPC).

60. A pharmaceutical formulation comprising the composition of any one of claims 41-59 and a pharmaceutically acceptable excipient.

61. A method of inducing cGAMP production by antigen presenting cells (APCs), the method comprising contacting the APCs with the composition or formulation of any of the preceding claims comprising the coding region of a cyclic GMP-AMP synthase (cGAS) under conditions suitable for expressing cGAS in the APCs resulting in production of cGAMP by the APCs.

62. A method of inducing expression of one or more interferon-stimulated genes by antigen presenting cells (APCs), the method comprising contacting the APCs with the composition or formulation of any of the preceding claims comprising the coding region of a cyclic GMP-AMP synthase (cGAS) and / or the coding regions of stimulator of interferon genes (STING) and interleukin- 1 receptor-associated kinase (IRAK) under conditions suitable for expressing the cGAS and / or the STING and IRAK in the APCs resulting in expression of the one or more interferon-stimulated genes.

63. The method of claim 62, wherein the one or more interferon-stimulated genes comprise IP- 10 (CXCL10), IFN-0, RANTES (CCL5), IFN-kl, or a combination thereof.

64. A method of inducing expression of one or more NF-kB-responsive genes by antigen presenting cells (APCs), the method comprising contacting the APCs with the composition or formulation of any of the preceding claims comprising the coding regions of stimulator of interferon genes (STING) and interleukin- 1 receptor-associated kinase (IRAK)75MF-367050437Docket No.: 16553-20014.40 under conditions suitable for expressing the STING and the IRAK in the APCs resulting in expression of the one or more NF-kB -responsive genes.

65. The method of claim 64, wherein the one or more NF-kB -responsive genes comprise TNF-a, IL-6, IL-8, IL-10, IL-10, IL-12, or a combination thereof.

66. A method of inducing expression of one or more interferon-stimulated genes and one or more NF-kB-responsive genes by antigen presenting cells (APCs), the method comprising contacting the APCs with the composition or formulation of any of the preceding claims comprising the coding region of a cyclic GMP-AMP synthase (cGAS) and the coding regions of stimulator of interferon genes (STING) and interleukin- 1 receptor-associated kinase (IRAK) under conditions suitable for expressing the cGAS, the STING and the IRAK in the APCs resulting in expression of the one or more interferon-stimulated genes and the one or more NF-kB-responsive genes.

67. The method of claim 66, wherein the one or more interferon-stimulated genes comprise IP-10 (CXCL10), IFN-0, RANTES (CCL5), IFN-A.1, or a combination thereof, and the one or more NF-kB-responsive genes comprise TNF-a, IL-6, IL-8, IL-10, IL-10, IL-12, or a combination thereof.

68. The method of any one of claims 61-67, wherein the APCs comprise dendritic cells.

69. The method of any one of claims 61-68, wherein the APCs comprise monocytes and / or macrophages.

70. The method of any one of claims 61-69, wherein the APCs are contacted in vivo with the composition.

71. The method of any one of claims 61-69, wherein the APCs are contacted ex vivo with the composition72. A method of inducing formation of pores in plasma membranes of cells, the method comprising contacting the cells with the composition or formulation of any of the76MF-367050437Docket No.: 16553-20014.40 preceding claims comprising the coding region of a gasdermin (GSDM) under conditions suitable for expressing the GSDM in the cells resulting in formation of the pores in the plasma membranes of the cells.

73. The method of claim 72, wherein the formation of the pores results in release of antigens by the cells.

74. The method of claim 72, wherein the formation of the pores results in a reduction of viability of the cells as compared to control cells that had not been contacted with the composition or formulation.

75. The method of claim 72, wherein the formation of the pores results in pyroptosis of the cells.

76. A method of preparing the composition of any one of claims 41-51 or any one of claims 56-59, comprising encapsulating the mRNA in the particle.

77. A method of preparing the composition of any one of claims 52-59, comprising forming a complex between the mRNA and the one or more lipids.

78. A method of treating cancer, comprising administering an effective amount of the pharmaceutical formulation of claim 60 to a cancer patient to treat the cancer.

79. A method of treating or preventing an infectious disease, comprising administering an effective amount of the pharmaceutical formulation of claim 60 to a subject in need thereof to treat or prevent the infectious disease.

80. A method of stimulating an immune response against an antigen, comprising administering an effective amount of the composition of any one of claims 56-59 to a subject in need thereof to stimulate an immune response against the antigen.

81. The method of claim 80, wherein stimulating the immune response comprises increasing the numbers of antigen-specific T-cells in blood of the subject.77MF-367050437Docket No.: 16553-20014.40 82. The method of claim 80 or claim 81, wherein stimulating the immune response comprises increasing the antigen-specific interferon-gamma response in PBMC of the subject.

83. Use of the composition of any one of claims 41-59 in the manufacture of a medicament for treating cancer in a subject in need thereof.

84. Use of the composition of any one of claims 41-59 in the manufacture of a medicament for treating or preventing an infectious disease in a subject in need thereof.

85. Use of the composition of any one of claims 56-59 in the manufacture of a medicament for stimulating an immune response against the antigen in a subject in need thereof.78MF-367050437

Images