Recombinant production of cyclic dinucleotides

Abstract

Embodiments of the present disclosure pertain to a nucleic acid sequence that includes a promoter operatively linked to a heterologous nucleotide sequence encoding a cyclic GMP-AMP synthase (cGAS) polypeptide. The nucleotide sequence also encodes a stabilizing peptide, such as a small ubiquitin modifying protein (SUMO) polypeptide. Further embodiments of the present disclosure pertain to fusion proteins that include a cGAS polypeptide and a stabilizing peptide. Additional embodiments of the present disclosure pertain to a recombinant cell that expresses a heterologous nucleotide sequence of the present disclosure. Further embodiments of the present disclosure pertain to methods of utilizing the recombinant cells of the present disclosure to produce 2'3' cyclic GMP-AMP (2'3'-cGAMP).

Description

Attorney Docket No: AF23853.P206WO RECOMBINANT PRODUCTION OF CYCLIC DINUCLEOTIDES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 533,594, filed on August 18, 2023. The entirety of the aforementioned application is incorporated herein by reference. SEQUENCE DISCLOSURE STATEMENT

[0002] Pursuant to 37 C.F.R. § 1.834, Applicant has submitted a sequence listing in XML format (“Sequence Listing”). The name of the file containing the Sequence Listing is “AF23853.P206WO.xml”. The date of the creation of the Sequence Listing is August 18, 2024. The size of the Sequence Listing is 121,000 bytes. Applicant hereby incorporates by reference the material in the Sequence Listing. FIELD

[0003] Embodiments of the present disclosure relate to compositions and methods for expressing cyclic GMP-AMP Synthase (cGAS) in a recombinant cell and applications thereof in connection with the production of 2’,3’-cGAMP (cGAMP), in particular, the production of 2’3- cGAMP at a high yield and purity without chemical synthesis, or the need for 2’3-cGAMP purification and / or endotoxin removal. BACKGROUND

[0004] A need exists for more effective systems, reagents and methods to produce 2’3- cGAMP. Numerous embodiments of the present disclosure aim to address the aforementioned need. SUMMARY

[0005] Cyclic Dinucleotides (CDNs) are second messenger molecules widely present in bacterial and mammalian cells, including c-di-GMP, c-di-AMP, 3 '3'-cGAMP, and 2 '3'-cGAMP (the latter is referred to herein also as cGAMP), and the like, and play an important role in regulating the pathogenicity, morphology of pathogenic microorganisms, and innate immune responses in mammals. CDNs can be recognized by the immune system of a human body to resist attack of pathogenic microorganisms, and can kill tumor cells by stimulating the immune systemAttorney Docket No: AF23853.P206WO of a tumor patient, so that the cyclic dinucleotide has a good drug development prospect. The large scale preparation of natural cyclic dinucleotides has been limited by their high cost and relatively cumbersome procedures. Therefore, it is of great importance to develop a reliable, simple and efficient production methods for large-scale preparation of Cyclic Dinucleotides (CDNs), including 2 '3' -cGAMP.

[0006] In some embodiments, the present disclosure addresses the lack of suitable technologies for the bacterial production of 2-3’-GMP-AMP (cGAMP) at a high yield and / or without the need for a process for endotoxin removal. In some embodiments, the present disclosure pertains to a nucleic acid sequence that includes a promoter operatively linked to a heterologous nucleotide sequence encoding a cyclic GMP–AMP synthase (cGAS) polypeptide. In some embodiments, the nucleic acid sequence comprises a codon optimized cGAS nucleic acid sequence selected from SEQ ID NO: 1 (mcGAS), SEQ ID NO: 2 (hcGAS), SEQ ID NO: 23 (hcGAS), or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 23.

[0007] In some embodiments, the nucleotide sequence also encodes a stabilizing peptide. In some embodiments, the expressed stabilizing peptide forms a fusion protein with the expressed cGAS polypeptide in any order. In some embodiments, the stabilizing peptide is selected from the group consisting of a small ubiquitin modifying protein (SUMO) polypeptide, a maltose- binding protein (MBP), N-utilization substance (NusA), Thioredoxin (Trx), disulfide bond C (DsbC), glutathione S-transferase (GST), ubiquitin (Ub), fragments thereof, or combinations thereof.

[0008] Further embodiments of the present disclosure pertain to a fusion protein that includes a cyclic GMP–AMP synthase (cGAS) polypeptide and a stabilizing peptide. In some embodiments, the cGAS polypeptide comprises a sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 3, or SEQ ID NO: 4.

[0009] Additional embodiments of the present disclosure pertain to a recombinant cell that expresses a heterologous nucleotide sequence of the present disclosure. Further embodiments of the present disclosure pertain to methods of utilizing the recombinant cells of the present disclosure to produce 2’3’ cyclic GMP–AMP (2’3’-cGAMP). In some embodiments, such methods include: (a) culturing a recombinant cell of the present disclosure in a culture media suchAttorney Docket No: AF23853.P206WO that the culturing induces the expression and activation of the cGAS polypeptide; (b) collecting the culture media; (c) performing anion exchange chromatography (AEC) of the collected culture media; and (d) collecting the elutant from an AEC column, where the elutant comprises the 2’3’- cGAMP. In some embodiments, the methods of the present disclosure also include a step of (e) filtering the elutant.

[0010] In some embodiments, the present disclosure pertains to compositions, methods and systems for bacterial production of 2-3’-GMP-AMP (cGAMP) without the need for purification of 2’3-cGAMP, or the removal of endotoxins. In some embodiments, the present disclosure pertains to: (1) immunostimulatory sequences, (2) mcGAS mutations, and (3) optimal culture conditions on the yield of 2’3-cGAMP secreted from bacterial cells (e.g., E. coli BL21(DE3)) cells. Without wishing to be bound by theory, embodiments of the present disclosure pertain to a simplified purification protocol that relies only on anion exchange chromatography and yields a high yield of purified 2’3-cGAMP (e.g., with < 20 EU / ml endotoxin). For illustrative purposes only, embodiments of the present disclosure pertain to the production of 2’3-cGAMP at a yield of 58.8 ± 2.1 mg / L with < 20 EU / mL of endotoxin. The high yield of endotoxin-free 2’3-cGAMP with the aid of simple purification makes the process attractive for fermentation-based production of 2’3-cGAMP on large scale.

[0011] In some embodiments, the present disclosure pertains to compositions and methods for preparing 2-3’-GMP-AMP (cGAMP) by using recombinant cells, comprising a heterologous nucleic acid, where the expression of the heterologous nucleic acid in a suitable culture media enables the generation and production of 2-3’-GMP-AMP (cGAMP), which can be separated with high purity and with minimal endotoxin contamination. In some embodiments, the heterologous nucleic acid is codon-optimized for expression in the host recombinant cell, and where the heterologous nucleic acid encodes a fusion protein comprising a stabilizing peptide and a cGAS polypeptide. In some embodiments, the presence of one, or both of, (i) the stabilizing peptide, and / or (ii) the codon-optimized nucleic acid sequence promotes proper expression, for example, including any of (i) enhanced recombinant protein expression and / or yield, and / or (ii) improved protein folding, and / or (iii) enhanced solubility of the recombinant cGAS polypeptide in the cell, results in minimal endotoxin production by the host recombinant cell. In some embodiments, the stabilizing peptide is a SUMO peptide as disclosed herein. In some embodiments, one of ordinary skill in the art can substitute SUMO for a different stabilizing peptide known in the art or disclosedAttorney Docket No: AF23853.P206WO herein. In some embodiments, the compositions and methods disclosed herein has fewer steps and fewer required raw materials and equipment, thereby reducing the production cost and having higher yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present teachings described herein will be more fully understood from the following description of various illustrative embodiments, when read together with the accompanying drawings. It should be understood that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0013] FIGS. 1A-1C4 provide schematics and data of whole cell production platforms for 2’3-cGAMP using E. coli. FIG. 1A is a schematic showing activation of cyclic GMP–AMP synthase (cGAS), where mouse cGAS (mcGAS) is allosterically activated by dsDNA to catalyze the cyclization reaction between GTP and ATP to yield 2’,3’-cGAMP (cGAMP). FIG.1B is a schematic showing the overall process workflow to express and purify 2’3-cGAMP. Recombinant bacterial cells comprising a heterologous nucleic acid sequence encoding cGAS fused to a small ubiquitin modifying (SUMO) protein are cultured in a defined culture media for a sufficient period of time for expression of cGAS, and production and secretion of 2’3-cGAMP into the culture media, where the culture is collected and centrifuged to separate the supernatant from the bacterial cell pellet. The supernatant is processed through anion exchange chromatography (AEX), and the elutant fractions comprising 2’3-cGAMP are collected. The collected fractions are concentrated and filtered through a 3kDa filtration. FIGS.1C1-1C4 shows the results of HPLC chromatograms at 256 nm illustrating the detection of 2’3-cGAMP from the fractions from the AEC, showing 2’3-cGAMP peaks in supernatant before AEC, the load flow through, the eluate and the final product.

[0014] FIGS.2A-2E show validation of the identity of the 2’3’-cGAMP produced from the recombinant cGAS, as assessed using liquid chromatography / double mass spectrometry (LC / MS- MS). FIG.2A shows the peak at 4 mins, and FIG.2B shows the MRM (Da / Da) peaks, showing fragmentation peaks for the unique identification of 2’3-cGAMP in comparison to other cyclic dinucleotides. FIG.2C shows the LC-MS 2’3-cGAMP standard curve. FIG.2D shows the peaks for 100µg / ml 2’3’-cGAMP, and FIG. 2E shows the LC-MS of 2’3’-cGAMP present in the supernatant produced from bacterial cell expressing wt mcGAS.Attorney Docket No: AF23853.P206WO

[0015] FIGS.3A-3I show the impact of different culture conditions on the yield and purity of 2’3-cGAMP produced using E. coli. FIG.3A-3C shows high pressure liquid chromatograph (HPLC) chromatograms at 256 nm illustrating the purity and titers of 2’3-cGAMP produced under the different media conditions, showing 2’3-cGAMP production in the of 2xYT media (FIG.3A), or in the presence of MN2+ supplemented media (FIG. 3B), or in the presence of M9 media (FIG. 3C), showing the effect of different divalent cation combinations. FIG. 3D shows the concentration of 2’3-cGAMP in the culture supernatants as a function of different strains of E. coli, demonstrating E. coli strains BL21 (DE3) and MG1655 were effective at robust 2’3-cGAMP production. (“NI” refers to not induced, i.e., unstimulated cGAS, “I” refers to induced, e.g., shows cGAS concentration after stimulation). FIG.3E shows the concentration of 2’3-cGAMP in the culture supernatants as a function of different media composition and supplementation with divalent cations, demonstrating M9 and 2xYT media were effective for producing high yields of 2’3-cGAMP production, and unexpectedly demonstrated that presence of divalent cations had a negative impact on 2’3-cGAMP yield. The first bar in the set represents unstimulated cGAS, the second bar in the set shows cGAS concentration after stimulation. FIG. 3F shows the concentration of 2’3-cGAMP in the culture supernatants as a function of different inducer concentration. FIG.3G shows the concentration of 2’3-cGAMP in the culture supernatants as a function of different temperatures, demonstrating that high yields of 2’3-cGAMP were still produced at a lower temperature of 20ºC. FIG. 3H shows the concentration of 2’3-cGAMP in the culture supernatants as a function of the concentration of bacteria suspension, demonstrating that OD of 0.8 was more effective than an overgrown culture having an OD of 2.0 or above. FIG. 3I shows 2’3-cGAMP in the culture supernatants produced as a function of the amount of plasmid encoding the cGAS fusion protein.

[0016] FIG. 4A-4J show the impact of genetic engineering on the yields of 2’3-cGAMP secreted by E. coli. expressing wild-type mouse cGAS (wt mcGAS) or modified mouse cGAS (mcGAS) proteins. FIG. 4A shows 2’3-cGAMP yield in E. coli expressing wild-type mouse cGAS (wt mcGAS). FIG.4B shows 2’3-cGAMP yield in E. coli expressing a modified mcGAS polypeptide with the immunostimulatory domain removed (“mcGAS ISD”). Therefore, the modified mcGAS comprises only the catalytic domain. FIG.4C shows 2’3-cGAMP yield in E. coli expressing a modified mcGAS polypeptide comprising the R222E modification (“mcGAS R222E”). FIG.4D shows 2’3-cGAMP yield in E. coli expressing a modified mcGAS polypeptideAttorney Docket No: AF23853.P206WO comprising the R241E modification (“mcGAS R241E”). FIG.4E shows 2’3-cGAMP yield in E. coli expressing a mcGAS polypeptide with a modified puc19 origin (“mcGAS puc19 ori”). FIG. 4F is a schematic representation of the binding site of wt mcGAS for double stranded DNA (dsDNA) showing the amino acid residues R222 and R241 interacting with dsDNA. FIG.4G is a schematic of the catalytic reaction to produce 2’3-cGAMP, where dsDNA serves as a co-factor that binds to and activates mcGAS (mcGAS*), and activated mcGAS* can catalyze ATP and GTP to produce 2’3-cGAMP. FIG.4H shows the effect of mutations at residues R222 and R241 to R222E and R241E respectively, on decreasing the production of 2’3-cGAMP relative to wild- type mcGAS. FIG. 4I shows a western blot of wt mcGAS and mutant mcGAS (mod-mcGAS) proteins expressed by bacterial cells (NI = not induced, IDS = mcGAS with the immunostimulatory domain removed, NC = negative control). FIG.4J is a table of the summary of 2’3-cGAMP yields from FIGS. 4A-4E, demonstrating wt-mcGAS and mcGAS with immunostimulatory domain removed (“mcGAS ISD”) resulted in the greatest 2’3-cGAMP yield.

[0017] FIG.5A-5E show 2’3-cGAMP recovery and purity during purification. FIG.5A is a schematic of the process of harvesting (e.g., collecting) the 2’3-cGAMP from the culture media. The supernatant of the culture media comprising the secreted 2’3-cGAMP is collected and processed through anion exchange chromatography (e.g,, High Q AEX), and 3 elutant fractions having peaks at 256nm are collected, each of which comprise 2’3-cGAMP. FIG.5B shows the yield of 2’3-cGAMP from each anion exchange chromatography (AEX) fraction (Load Flow through (“load FT”), Wash A and Eluate [100nm NaCl]), as quantified by HPLC via comparisons to standards. FIG.5C shows HPLC chromatograms at 256 nm, illustrating the purity and titers of 2’3-cGAMP from each elutant fraction; Load, Load Flow through, and Eluate (100mM NaCl). FIG.5D shows the percentage recovery of the total 2’3-cGAMP, with 38% and 42% 2’3-cGAMP recovered from a 50 nM NaCl Wash (Wash A) and 100 nM NaCl Wash (Wash B) elutant fractions, respectively. FIG. 5E shows the quantification of LPS in the 2’3-cGAMP elutant fractions during purification, showing minimal endotoxin in the wash flow through (Wash FT) and elutant. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0018] Double stranded DNA (dsDNA) in the cytoplasm of mammalian cells acts as potent danger signal indicative of either pathogenic infection, cellular damage or cancer. This mislocalized dsDNA allosterically activates the cyclic GMP–AMP synthase (cGAS), which inAttorney Docket No: AF23853.P206WO turn synthesizes the cyclic dinucleotide, 2’3’ cyclic GMP–AMP (referred to herein as cGAMP or 2’3-cGAMP). 2’3-cGAMP binds to the stimulator of interferon genes (STING), leading to the oligomerization of STING and its translocation to the trans-Golgi network initiating a signaling cascade that culminates in the secretion of type I and type III interferons. 2’3-cGAMP acts as the messenger that ties recognition of dsDNA by cGAS to the subsequent activation of STING. 2’3- cGAMP is easily exported out of the cell and hence functions as a soluble extracellular immunotransmitter. This ensures that the effect of recognition of mislocalized dsDNA is transmitted to all cells within the microenvironment facilitating a broad innate immune response. Therefore, 2’3-cGAMP and STING agonists are being actively investigated as immunotherapeutics for the treatment of cancers and as mucosal adjuvants for the development of vaccines against infectious diseases.

[0019] At present, cyclic dinucleotides, including 2’3’ cyclic GMP–AMP (cGAMP) is mainly prepared by methods such as chemical synthesis, enzyme synthesis and the like. However, the chemical synthesis method uses a plurality of protective groups, is very time consuming, and has a plurality of steps. Furthermore, the chemical synthesis method is not environmentally friendly.

[0020] Enzymatic-based synthetic methods can also be used to produce cGAMP. However, enzymatic synthesis methods need to use a substrate (e.g., GTP) with higher cost, higher efficiency, and higher purity. Such requirements lead to a high price of cyclic dinucleotide (CDN) production.

[0021] As an alternate to chemical synthesis, heterologous cGAS, expressed and purified from E. coli cells, is used for the enzymatic biosynthesis of 2’3-cGAMP13,14. The cGAS enzyme is incubated with dsDNA as an allosteric activator and converts ATP and GTP into 2’3-cGAMP with 85-90% yield in HEPES buffer. The advantage of the enzymatic synthesis is that it does not need organic solvents and results in high-yield conversion15,16. The disadvantage of enzymatic biosynthesis however is that the substrate GTP is expensive and the expression and purification of cGAS has a significant environmental impact.

[0022] To overcome the disadvantages of purification of cGAS, intact E. coli cells transformed with cGAS have been used as bioreactors for the production of 2’3-cGAMP. When E. coli were used as bioreactors for production of 2’3-cGAMP, 2’3-cGAMP was secreted into theAttorney Docket No: AF23853.P206WO culture medium. However, one of the primary disadvantages of using E. coli cells as biocatalysts is the significant need to eliminate endotoxin since it will interfere with all downstream immunological assays. Another significant drawback of such a process is that the downstream process requires cGAS purification and utilizes a protein affinity chromatography step (STING- ligand binding domain protein, STING-LBD). That is, the method necessitates the expression and purification of STING-LBD for purification of cGAS, which increases process economics and environmental impact. As such, purification steps that do not rely on protein-affinity chromatography are desirable. Second, the process workflow does not remove endotoxin.

[0023] Therefore, there exists a need for a method to produce 2’3-cGAMP without the need to remove the endotoxin from the final 2’3-cGAMP product, or the need for a purification process of 2’3-cGAMP or cGAS. Numerous embodiments of the present disclosure aim to address the aforementioned need.

[0024] cGAS nucleic acids

[0025] In some embodiments, the present disclosure pertains to a nucleic acid sequence comprising a promoter operatively linked to a heterologous nucleotide sequence encoding a cyclic GMP–AMP synthase (cGAS) polypeptide. In some embodiments, the nucleic acid sequence comprises a codon optimized cGAS nucleic acid sequence selected from SEQ ID NO: 1 (mcGAS), SEQ ID NO: 2 (hcGAS), SEQ ID NO: 23 (hcGAS), or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 23.

[0026] In some embodiments, the nucleotide sequence also encodes a stabilizing peptide. In some embodiments, the expressed stabilizing peptide forms a fusion protein with the expressed cGAS polypeptide in any order. In some embodiments, the stabilizing peptide is selected from the group consisting of a small ubiquitin modifying protein (SUMO) polypeptide, a maltose- binding protein (MBP), N-utilization substance (NusA), Thioredoxin (Trx), disulfide bond C (DsbC), glutathione S-transferase (GST), ubiquitin (Ub), fragments thereof, or combinations thereof.

[0027] In some embodiments, the nucleic acid sequence further comprises a nucleic acid sequence encoding a linker located between the nucleic sequence encoding the cGAS polypeptide and the stabilizing peptide. In some embodiments, the stabilizing peptide comprises a smallAttorney Docket No: AF23853.P206WO ubiquitin modifying protein (SUMO) polypeptide. In some embodiments, the fusion protein comprises a SUMO polypeptide fused to the N-terminus of the cGAS polypeptide. In some embodiments, the nucleic acid encoding the SUMO polypeptide comprises a sequence selected from SEQ ID NO: 5 or SEQ ID NO: 6, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.

[0028] In some embodiments, the nucleic acid sequence is selected from the group consisting of: a nucleic acid comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 1, and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5 (co-mcGAS-SUMO); a nucleic acid comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 1. (SUMO-mcGAS); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 23 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 23. (SUMO-co1-hcGAS); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 23 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 23, and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5. (co1-hcGAS-SUMO); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 2 (SUMO- co2-hcGAS); or a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 2, and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5 (co2-hcGAS-SUMO).

[0029] In some embodiments, the polynucleotide encoding the hcGAS polypeptide comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the polynucleotide encoding the cGAS polypeptide comprises a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 orAttorney Docket No: AF23853.P206WO SEQ ID NO: 23. In some embodiments, the polynucleotide encoding the cGAS polypeptide comprises a nucleic acid sequence that is 100% identical to the polynucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the polynucleotide encoding the cGAS polypeptide consists of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the polynucleotide encoding the cGAS polypeptide consists of a nucleic acid sequence that is 100% identical to the polynucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 23.

[0030] In some embodiments, the polynucleotide encoding the mouse cGAS (mcGAS) polypeptide comprises a codon-optimized nucleic acid sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the codon-optimized polynucleotide encoding the mcGAS polypeptide comprises a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the polynucleotide encoding the cGAS polypeptide comprises a nucleic acid sequence that is 100% identical to the polynucleotide sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide encoding the mcGAS polypeptide consists of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the codon-optimized polynucleotide encoding the mcGAS polypeptide consists of a codon-optimized nucleic acid sequence that is 100% identical to the polynucleotide sequence of SEQ ID NO: 1.

[0031] In one embodiment, the cGAS gene sequence is a mouse cGAS gene sequence, referred to herein as “mcGAS”. In some embodiments, the cGAS gene sequence is a codon- optimized mouse cGAS gene sequence, referred to herein as co-mcGAS. In one embodiment, the cGAS gene sequence is a human cGAS gene sequence, referred to herein as “hcGAS”. In some embodiments, the cGAS gene sequence is a codon-optimized human cGAS gene sequence, referred to herein as co-hcGAS. Different exemplary co-hGAS sequence as disclosed herein (e.g., SEQ ID NO: 2 or SEQ ID NO: 23) can be referred to as co2-hcGAS and co1-hcGAS, respectively, and can be used interchangeably.

[0032] In one embodiment, the cGAS gene sequence is a human hcGAS sequence. In alternative embodiments, the cGAS sequence is selected from a Verminephrobacter eiseniae cGAS gene sequence, Kingella denitrificans cGAS gene sequence, and a Neisseria bacilliformis cGAS gene sequence.Attorney Docket No: AF23853.P206WO

[0033] In some embodiments, the heterologous nucleic acid sequence comprises a cGAS gene sequence and / or SUMO gene sequence where one, or both sequences are codon-optimized for expression in a specific microorganism host cell. In one embodiment, the genetically engineered bacteria comprise heterologous nucleic acid sequence that is codon-optimized for the bacterial host cell, which encodes the human cGAS (hcGAS) polypeptide or the mouse cGAS (mcGAS) polypeptide, where the hcGAS or mcGAS is fused to a SUMO polypeptide.

[0034] In certain embodiments, the codon-optimized hcGAS nucleic acid sequence has at least about 80% identity with SEQ ID NO: 2 or SEQ ID NO: 23. In certain embodiments, the hcGAS nucleic acid sequence has at least about 90% identity with SEQ ID NO: 2 or SEQ ID NO: 23. In certain embodiments, the hcGAS nucleic acid sequence has at least about 95% identity with SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the hcGAS nucleic acid sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2 or SEQ ID NO: 23. In some specific embodiments, the hcGAS nucleic acid sequence comprises SEQ ID NO: 2 or SEQ ID NO: 23. In other specific embodiments, the hcGAS nucleic acid sequence consists of SEQ ID NO: 2 or SEQ ID NO: 23.

[0035] In certain embodiments, the codon-optimized hcGAS nucleic acid sequence has at least about 80% identity with SEQ ID NO: 2 or SEQ ID NO: 23. In certain embodiments, the codon-optimized hcGAS nucleic acid sequence has at least about 90% identity with SEQ ID NO: 2 or SEQ ID NO: 23. In certain embodiments, the codon-optimized hcGAS nucleic acid sequence has at least about 95% identity with SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the codon-optimized hcGAS nucleic acid sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2 or SEQ ID NO: 23. In some specific embodiments, codon-optimized hcGAS nucleic acid sequence comprises SEQ ID NO: 2 or SEQ ID NO: 23. In other specific embodiments, the codon-optimized hcGAS nucleic acid sequence consists of SEQ ID NO: 2 or SEQ ID NO: 23.

[0036] In other specific embodiments, the mouse cGAS polypeptide consists of SEQ ID NO: 3. In certain embodiments, the codon-optimized mcGAS nucleic acid sequence has at least about 80% identity with SEQ ID NO: 1. In certain embodiments, the mcGAS nucleic acid sequence has at least about 90% identity with SEQ ID NO: 1. In certain embodiments, the mcGAS nucleic acid sequence has at least about 95% identity with SEQ ID NO: 1. In some embodiments, the mcGASAttorney Docket No: AF23853.P206WO nucleic acid sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. In some specific embodiments, the mcGAS nucleic acid sequence comprises SEQ ID NO: 1. In other specific embodiments, the mcGAS nucleic acid sequence consists of SEQ ID NO: 1.

[0037] In some embodiments, the heterologous nucleic acid sequence is operatively linked to a promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a constitutive promoter (e.g., a constitutive or inducible promoter described herein). In some embodiments, the heterologous nucleic acid sequence encoding a cGAS polypeptide as disclosed herein is integrated into the chromosome of the bacterial cell or recombinant cell disclosed herein. In some embodiments, the nucleic acid is present in an expression vector. In some embodiments, the expression vector is pET28A.

[0038] In some embodiments, the present disclosure provides nucleic acids (e.g., DNA, RNA, or analogs thereof), encoding one or more of the polypeptides and / or fusion proteins described herein. An underlying DNA sequence for the polypeptides described herein may be modified in ways that do not affect the sequence of the protein product, and such sequences are included in the invention. In particular embodiments, a DNA sequence may be codon-optimized to improve expression in a host such as a specific bacterial cell line (e.g., E. coli as disclosed herein), an insect cell line, a mammalian cell line (e.g., human or Chinese Hamster Ovary), or other microorganisms (e.g., a modified microorganism, a recombinant microalgae or cyanobacteria, as disclosed herein).

[0039] In some embodiments, the present disclosure provides nucleic acids (e.g., DNA, RNA, or analogs thereof) that are at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identical to a nucleic acid sequence provided in Table 1, Table 2, or a variant or portion thereof. In some embodiments, the nucleic acid is 600-2000, 800-1800, 1000-1600, 1200-1400 nucleotides in length. In some embodiments, the nucleic acid is 600-1600, 800-1800, 1000-2000, 2000-3000, or 3000-4000 nucleotides in length. In some embodiments, a nucleic acid may be used for recombinant production of a polypeptide or fusion protein of Table 1 or Table 2, or fragments thereof.

[0040] Codon-optimized nucleic acid sequences encoding mcGAS and hcGAS (SEQ ID NOs: 3 and 4) are provided as SEQ ID NOs: 1 and 2 and SEQ ID NO: 23 (see Table 1). Nucleic acid sequences encoding different SUMO-cGAS fusion proteins (e.g., exemplary fusion proteinsAttorney Docket No: AF23853.P206WO of SEQ ID NOs: 13-22), are provided as SEQ ID NOs: 26-29 and SEQ ID NO: 75 (See table 2). In some embodiments, due to degeneracy in the genetic code, other DNA sequences (including multiple codon-optimized sequences) could be contemplated by those of ordinary skill to encode such polypeptides and fusion proteins.

[0041] Nucleic acids encoding polypeptides or fusion proteins of Table 1 or Table 2, or fragments thereof, can be cloned into any of a variety of expression vectors, under the control of a variety of regulatory elements, and fusions can be created with other sequences of interest. Methods of cloning nucleic acids are routine and conventional in the art. For general references describing methods of molecular biology which are mentioned in this application (e.g., isolating, cloning, modifying, labeling, manipulating, sequencing and otherwise treating or analyzing nucleic acids and / or proteins), see, e.g., Sambrook et al.,1989; Ausubel et al.,1995; Davis et al., 1986; Hames et al., 1985; Dracopoli et al., 2018; and Coligan et al., 2018.

[0042] Nucleic acids as disclosed herein may be delivered to a microorganism of the invention using any method known in the art. For example, nucleic acids may be delivered as naked nucleic acids or may be formulated with one or more agents, such as liposomes. The nucleic acids may be DNA, RNA, cDNA, or combinations thereof, as is appropriate. Restriction inhibitors may be used in certain embodiments. Additional vectors may include plasmids, viruses, bacteriophages, cosmids, and artificial chromosomes. In a preferred embodiment, nucleic acids are delivered to the microorganism of the invention using a plasmid. By way of example, transformation (including transduction or transfection) may be achieved by electroporation, ultrasonication, polyethylene glycol-mediated transformation, chemical or natural competence, protoplast transformation, prophage induction, or conjugation. In certain embodiments having active restriction enzyme systems, it may be necessary to methylate a nucleic acid before introduction of the nucleic acid into a microorganism.

[0043] Furthermore, nucleic acids may be designed to comprise a regulatory element, such as a promoter, to increase or otherwise control expression of a particular nucleic acid. The promoter may be a constitutive promoter or an inducible promoter. Ideally, the promoter is a Wood-Ljungdahl pathway promoter, a ferredoxin promoter, a pyruvate:ferredoxin oxidoreductase promoter, an Rnf complex operon promoter, an ATP synthase operon promoter, or a phosphotransacetylase / acetate kinase operon promoter.Attorney Docket No: AF23853.P206WO

[0044] Promoters

[0045] In some embodiments, the polynucleotide encoding a cGAS polypeptide (e.g., with or without a SUMO fusion polypeptide) is operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter specific to the recombinant host cell in which it is to be expressed. In some embodiments, the promoter is an inducible promoter that functions effectively in the recombinant host cell in which it is to be expressed. In some embodiments, the promoter is a ubiquitous promoter, a tissue-specific promoter, or a tumor- specific promoter. In some embodiments, the ubiquitous promoter is selected from EFl-a, CMV, SV40, PGK1, Ubc, Beta actin, CAG, Polyhedrin, CaMV35S, Hl, U6, and Ubiquitin B. In some embodiments, the tissue- specific promoter is selected from the tyronsinase (Tyr) promoter, the B29 promoter, the CD 14 promoter, the CD43 promoter, the CD45 promoter, the CD68 promoter, the Desmin promoter, the Elastase-l promoter, the Endoglin promoter, the Fibronectin promoter, the Flt-l promoter, the GFAP promoter, the GPIIb promoter, the ICAM-2 promoter, the mIFN-b promoter, the Mb promoter, the Nphsl promoter, the OG-2 promoter, the SP-B promoter, the SYN1 promoter, the WASP promoter, and the CaMKII promoter. In some embodiments, the tumor-specific promoter is selected from the alpha fetal protein (AFP) promoter, the cholecystokinin type-A receptor (CCKAR) promoter, the carcinoembryonic antigen (CEA) promoter, the c-ErbB2 promoter, the COX-2 promoter, the CXCR4 promoter, the E2F-1 promoter, the human epididymis protein 4 (HE4) promoter, the lipoprotein lipase (LP) promoter, the MUC1 promoter, the prostate- specific antigen (PSA) promoter, the Survivin (Sur) promoter, and the phosphoribosyl-anthranilate isomerase (TRP-l) promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a tetracycline inducible promoter and comprises one or more tetracycline response elements (TRE).

[0046] Recombinant cells expressing cGAS

[0047] In some embodiments, the present disclosure pertains to a recombinant cell. In some embodiments, the recombinant cell expresses a heterologous nucleotide sequence encoding a cyclic GMP–AMP synthase (cGAS) polypeptide. Suitable nucleic acids were described supra and are incorporated herein by reference. For instance, in some embodiments, the nucleic acid sequence comprises a codon optimized cGAS nucleic acid sequence selected from SEQ ID NO: 1 (mcGAS), SEQ ID NO: 2 (hcGAS), SEQ ID NO: 23 (hcGAS), or a nucleic acid sequenceAttorney Docket No: AF23853.P206WO having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 23.

[0048] In some embodiments, the nucleotide sequence also encodes a stabilizing peptide. In some embodiments, the expressed stabilizing peptide forms a fusion protein with the expressed cGAS polypeptide in any order. In some embodiments, the stabilizing peptide is selected from the group consisting of a small ubiquitin modifying protein (SUMO) polypeptide, a maltose- binding protein (MBP), N-utilization substance (NusA), Thioredoxin (Trx), disulfide bond C (DsbC), glutathione S-transferase (GST), ubiquitin (Ub), fragments thereof, or combinations thereof. In some embodiments, the nucleotide sequence further comprises a nucleic acid sequence encoding a linker located between the nucleic sequence encoding the cGAS polypeptide and the stabilizing peptide.

[0049] In some embodiments, the stabilizing peptide comprises a small ubiquitin modifying protein (SUMO) polypeptide. In some embodiments, the fusion protein comprises a SUMO polypeptide fused to the N-terminus of the cGAS polypeptide.

[0050] In some embodiments, the nucleic acid sequence is selected from the group consisting of: a nucleic acid comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 1, and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5 (co-mcGAS-SUMO); a nucleic acid comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 1. (SUMO-mcGAS); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 23 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 23. (SUMO-co1-hcGAS); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 23 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 23, and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5. (co1-hcGAS-SUMO); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acidAttorney Docket No: AF23853.P206WO sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 2 (SUMO- co2-hcGAS); or a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 2, and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5 (co2-hcGAS-SUMO).

[0051] In some embodiments, the recombinant cells of the present disclosure include a bacterial cell. In some embodiments, the bacterial cell is E. coli. In some embodiments, the E. coli is an E. coli strain selected from the group consisting of E. cloni EC10, E. coli MG1655 (DE3), E. coli Bl21(DE3), and E. coli BL21(DE3) RIL.

[0052] In some embodiments, the recombinant cells of the present disclosure are operational for producing 2’3’ cyclic GMP–AMP (2’3’-cGAMP). In some embodiments, the recombinant cells of the present disclosure produce a yield of 2’3’-cGAMP, which is at least 30% higher than the yield of 2’3’-cGAMP produced from a cell expressing a cGAS polypeptide encoded by a nucleic acid that is at least 98% identical to SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25. In some embodiments, the recombinant cells of the present disclosure produce a yield of 2’3’-cGAMP which is at least 30% higher than the yield of 2’3’-cGAMP produced from a cell expressing a cGAS polypeptide having an amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 24. In some embodiments, the recombinant cells of the present disclosure produce a yield of 2’3’-cGAMP which has less than 20 EU / ml endotoxin.

[0053] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a hcGAS polypeptide, wherein the polynucleotide encoding the hcGAS polypeptide comprises a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the polynucleotide encoding the hcGAS polypeptide consists of a codon-optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the codon-optimized polynucleotide encoding the hcGAS polypeptide comprises a nucleotide sequence that is 100% identical to SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the codon-optimized polynucleotide encoding the hcGAS polypeptide consists of SEQ ID NO: 2 or SEQ ID NO: 23 (co-hcGAS).Attorney Docket No: AF23853.P206WO

[0054] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a mcGAS polypeptide, wherein the polynucleotide encoding the mcGAS polypeptide comprises a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the polynucleotide encoding the mcGAS polypeptide consists of a codon-optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the codon- optimized polynucleotide encoding the mcGAS polypeptide comprises a nucleotide sequence that is 100% identical to SEQ ID NO: 1. In some embodiments, the codon-optimized polynucleotide encoding the mcGAS polypeptide consists of SEQ ID NO: 1 (co-mcGAS).

[0055] In some embodiments, the codon-optimized nucleic acid encodes a hcGAS polypeptide that is a truncated modified hcGAS polypeptide (hcGAS-trunc-mod) and comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the codon-optimized nucleic acid encodes a hcGAS-trunc-mod polypeptide that comprises an amino acid sequence that is 100% identical to SEQ ID NO: 4. In some embodiments, the codon-optimized nucleic acid encodes a hcGAS-trunc-mod polypeptide that consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the codon-optimized nucleic acid encodes a hcGAS-trunc- mod polypeptide that consists of SEQ ID NO: 4.

[0056] In some embodiments, the codon-optimized nucleic acid encoding a mcGAS polypeptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. In some embodiments, the codon-optimized nucleic acid encodes a mcGAS polypeptide that comprises an amino acid sequence that is 100% identical to SEQ ID NO: 3. In some embodiments, the codon-optimized nucleic acid encodes a mcGAS polypeptide consisting of an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. In some embodiments, the codon-optimized nucleic acid encodes a mcGAS polypeptide consisting of SEQ ID NO: 3.

[0057] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a fusion protein as disclosed herein, comprising a codon-optimized nucleic acid sequence encoding a SUMO peptide as disclosed herein and at least one of: a mcGAS or hcGAS polypeptide, or both a mcGAS and hcGAS polypeptide, wherein theAttorney Docket No: AF23853.P206WO nucleic acid encoding SUMO is codon-optimized, as is the nucleic acid encoding the one or more cGAS polypeptides. In some embodiments, the codon-optimized nucleic acid encoding the fusion protein comprises a codon-optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the codon-optimized polynucleotide encoding the SUMO peptide consists of a codon-optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the codon- optimized polynucleotide encoding the SUMO peptide comprises a nucleotide sequence that is 100% identical to SEQ ID NO: 7. In some embodiments, the codon-optimized polynucleotide encoding the SUMO peptide consists of SEQ ID NO: 7 (co-mcGAS).

[0058] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a fusion protein as disclosed herein, comprising a codon-optimized nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:26 (co-SUMO-mcGAS). In some embodiments, the codon-optimized polynucleotide encoding a SUMO-mcGAS fusion protein consists of a codon-optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26 (co-SUMO-mcGASn). In some embodiments, the codon-optimized polynucleotide encoding a SUMO-mcGAS fusion protein as disclosed herein comprises a nucleotide sequence that is 100% identical to SEQ ID NO: 26. In some embodiments, the codon-optimized polynucleotide encoding a SUMO-mcGAS fusion protein as disclosed herein consists of SEQ ID NO: 26 (co-SUMO-mcGAS).

[0059] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a fusion protein as disclosed herein, comprising a codon-optimized nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28 (co-mcGAS-SUMO). In some embodiments, the codon-optimized polynucleotide encoding a mcGAS-SUMO fusion protein as disclosed herein consists of a codon- optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28 (co-mcGAS-SUMO). In some embodiments, the codon-optimized polynucleotide encoding a mcGAS-SUMO fusion protein as disclosed herein comprises a nucleotide sequence that is 100% identical to SEQ ID NO: 28. In some embodiments, the codon-optimized polynucleotide encoding a mcGAS-SUMO fusion protein as disclosed herein protein as disclosed herein consists of SEQ ID NO: 28 (co-mcGAS-SUMO)).Attorney Docket No: AF23853.P206WO

[0060] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a fusion protein as disclosed herein, comprising a codon-optimized nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:27 or SEQ ID NO: 74 (co-SUMO-hcGAS). In some embodiments, the codon- optimized polynucleotide encoding a SUMO-mcGAS fusion protein consists of a codon- optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27 or SEQ ID NO: 74 (co-SUMO-hcGASn). In some embodiments, the codon-optimized polynucleotide encoding a SUMO-hcGAS fusion protein as disclosed herein comprises a nucleotide sequence that is 100% identical to SEQ ID NO: 27. In some embodiments, the codon- optimized polynucleotide encoding a SUMO-hcGAS fusion protein as disclosed herein consists of SEQ ID NO: 27 or SEQ ID NO: 74 (co-SUMO-hcGAS).

[0061] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a fusion protein as disclosed herein, comprising a codon-optimized nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29 or SEQ ID NO: 75 (co-hcGAS-SUMO). In some embodiments, the codon- optimized polynucleotide encoding a hcGAS-SUMO fusion protein as disclosed herein consists of a codon-optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29 or SEQ ID NO: 75 (co-hcGAS-SUMO). In some embodiments, the codon- optimized polynucleotide encoding a hcGAS-SUMO fusion protein as disclosed herein comprises a nucleotide sequence that is 100% identical to SEQ ID NO: 29 or SEQ ID NO: 75. In some embodiments, the codon-optimized polynucleotide encoding a hcGAS-SUMO fusion protein as disclosed herein protein as disclosed herein consists of SEQ ID NO: 29 or SEQ ID NO: 75 (co- hcGAS-SUMO).

[0062] In some embodiments, the recombinant cell as described herein comprise a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 26-29. In some embodiments, the recombinant cell as described herein comprise a nucleotide sequence that is 100% identical to any of SEQ ID NO: 26-29. In some embodiments, the recombinant cell as described herein consist of a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 26-29. In some embodiments, the recombinant cell described herein consist of a nucleotide sequence that is 100% identical to any of SEQ ID NO: 26-29 or SEQ ID NO: 74-75.Attorney Docket No: AF23853.P206WO

[0063] In some embodiments, the present disclosure relates, generally, to recombinant bacterial cells comprising a heterologous nucleic acid encoding a fusion polypeptide. In some embodiments, the fusion polypeptide includes, in any order, a cyclic GMP–AMP synthase (cGAS) polypeptide and a small ubiquitin modifying protein (SUMO) polypeptide. In some embodiments, the nucleic acid encoding the cGAS polypeptide is a codon optimized sequence for optimal expression of cGAS in bacterial cells, resulting in a high yield of production of 2-3’- GMP-AMP (cGAMP) without the need for purification of 2’3-cGAMP, and / or the removal of endotoxins.

[0064] In some embodiments, the genetically engineered bacteria, or recombinant bacterial cell comprise human cGAS (hcGAS) codon-optimized gene sequence(s) encoding one or more hcGAS polypeptide(s) comprising SEQ ID NO: 4 or functional fragments thereof. In some embodiments, genetically engineered bacteria or recombinant bacterial host cell comprise a codon-optimized hcGAS nucleic acid encoding a hcGAS polypeptide that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to SEQ ID NO: 4 or a functional fragment thereof. In some embodiments, the hcGAS polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. In some specific embodiments, the hcGAS polypeptide comprises SEQ ID NO: 4. In other specific embodiments, the hcGAS polypeptide consists of SEQ ID NO: 4.

[0065] In some embodiments, the genetically engineered bacteria, or recombinant bacterial cell comprise human cGAS (hcGAS) codon-optimized nucleic acid sequence comprising SEQ ID NO: 2 or SEQ ID NO: 23 or a functional fragments thereof of at least 850 consecutive nucleotides. In some embodiments, genetically engineered bacteria or recombinant bacterial host cell comprises a codon-optimized hcGAS nucleic acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the codon-optimized hcGAS nucleic acid sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2 or SEQ ID NO: 23. In other specific embodiments, the codon-optimized hcGAS nucleic acid sequence consists of SEQ ID NO: 2 or SEQ ID NO: 23.Attorney Docket No: AF23853.P206WO

[0066] In some embodiments, the present disclosure provides a recombinant bacterial cell comprising a polynucleotide encoding a cyclic GMP-AMP synthase (cGAS) polypeptide. In humans, the wild-type cGAS protein of SEQ ID NO: 24 is encoded by the human CGAS gene (NM_138441.3), also referred to as the MB21D1 or C6orfl50 gene, which is SEQ ID NO: 25. The amino acid sequence of full-length human cGAS protein is provided in SEQ ID NO: 24 (encoded by codon SEQ ID NO: 25). In some embodiments, the cGAS protein comprises a truncated hcGAS polypeptide, which corresponds to residues 157-552 of the full-length wt hcGAS of SEQ ID NO: 24 (which corresponds to SEQ ID NO: 12 herein), and is encoded by SEQ ID NO: 10. However, in particular embodiments, the fusion protein as disclosed herein comprises a modified hcGAS polypeptide, comprising K187 and L19R modifications (nomenclature based on the full-length protein of SEQ ID NO: 24).

[0067] In some embodiments, the genetically engineered bacteria, or recombinant bacterial cell comprise mouse cGAS (mcGAS) codon-optimized gene sequence(s) encoding one or more mouse cGAS polypeptide(s) comprising SEQ ID NO: 3 or functional fragments thereof. In some embodiments, genetically engineered bacteria or recombinant bacterial host cell comprise a codon-optimized mcGAS nucleic acid encoding a mouse cGAS polypeptide that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to SEQ ID NO: 3 or a functional fragment thereof. In some embodiments, the mouse cGAS polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. In some specific embodiments, the mouse cGAS polypeptide comprises SEQ ID NO: 3.

[0068] In some embodiments, the genetically engineered bacteria, or recombinant bacterial cell comprise mouse cGAS (mcGAS) codon-optimized gene sequence(s) encoding one or more mcGAS polypeptide(s) comprising SEQ ID NO: 3 or functional fragments thereof. In some embodiments, genetically engineered bacteria or recombinant bacterial host cell comprise a mcGAS gene sequence encoding a polypeptide that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to SEQ ID NO: 1 or a functional fragment thereof. In some embodiments, the polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. In some specific embodiments, the polypeptide comprises SEQ ID NO: 1. In other specific embodiments, the polypeptide consists of SEQ ID NO: 1. In certain embodiments, the codon-Attorney Docket No: AF23853.P206WO optimized hcGAS nucleic acid sequence has at least about 80% identity with SEQ ID NO: 1. In certain embodiments, the gene sequence has at least about 90% identity with SEQ ID NO: 1. In certain embodiments, the gene sequence has at least about 95% identity with SEQ ID NO: 1. In some embodiments, the gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. In some specific embodiments, the gene sequence comprises SEQ ID NO: 1. In other specific embodiments, the gene sequence consists of SEQ ID NO: 1.

[0069] In some embodiments, the present disclosure provides a method of expressing a cGAS polypeptide in a cell (e.g., a recombinant cell as disclosed herein) by stably expressing a codon-optimized nucleic acid encoding a cGAS (e.g., an mcGAS or hcGAS polypeptide as described herein) or a composition thereof. In some embodiments, expression of the cGAS polypeptide induces production of 2’3-cGAMP in the cell. In some embodiments, the method further comprises administering a cGAS activator to the cell. In some embodiments, administration of the cGAS activator induces production of 2’3-cGAMP in the cell.

[0070] In some embodiments, administration of an inducer results in expression of cGAS and activation of the STING pathway in the cell. In some embodiments, the inducer is an inducer of an inducible promoter, as disclosed herein. In some embodiments, the method further comprises administering a cGAS activator to the cell, wherein the activator is dsDNA. In some embodiments, administration of a nucleotide expressing cGAS (e.g., a recombinant virus with nucleotides expressing cGAS) results in expression of cGAS in the cell and administration of the cGAS activator results in the activation of the STING pathway.

[0071] In some embodiments, the cGAS activator is selected from a double stranded DNA (dsDNA) molecule. In some embodiments, the cell is in vitro.

[0072] In some embodiments, the recombinant cell is a modified microorganism (e.g., a recombinant microalgae or cyanobacteria). In some embodiments, the modified microorganism is a bacterial cell as disclosed herein. Examples of microorganisms include bacteria, viruses, parasites, fungi, certain algae, protozoa, and yeast. In some embodiments, the microorganism is modified (“modified microorganism”) from its native state to express one or more cGAS fusion proteins as disclosed herein. In certain embodiments, the modified microorganism is a modified bacterium. In some embodiments, the modified microorganism is a genetically engineeredAttorney Docket No: AF23853.P206WO bacterium. In certain embodiments, the modified microorganism is a modified yeast. In other embodiments, the modified microorganism is a genetically engineered yeast.

[0073] Further embodiments of the present disclosure relate to a modified microorganism comprising at least one heterologous nucleic acid sequence that encodes a cGAS polypeptide, such that when the cGAS polypeptide is expressed, and in the presence of an activator of the expressed cGAS polypeptide, 2’3-cGAMP is produced. In some embodiments, the modified microorganism comprises at least one heterologous nucleic acid sequence, where the heterologous sequence is a codon-optimized nucleic acid sequence that encodes a cGAS polypeptide for optimal expression of the cGAS polypeptide in the modified microorganism. In some embodiments, the heterologous nucleic acid sequence comprises a codon-optimized nucleic acid sequence encoding a cGAS polypeptide and a nucleic acid sequence encoding a SUMO polypeptide, where nucleic acid sequence encoding a SUMO polypeptide can be codon-optimized for expression in the particular microorganism host cell. That is, at least one, or both of: the nucleic acid sequences encoding cGAS and the nucleic acid sequence encoding SUMO are codon- optimized for the optimal expression of the polypeptides in the selected microorganism. In some embodiments, the heterologous nucleic acid sequence comprises a codon-optimized nucleic acid sequence encoding a cGAS polypeptide and a nucleic acid sequence encoding a SUMO polypeptide, and expression of the heterologous nucleic acid sequence produces a fusion protein comprising a cGAS polypeptide and a SUMO polypeptide, where the cGAS polypeptide and SUMO polypeptide can be in any order.

[0074] In some embodiments, the recombinant cell comprising a codon-optimized nucleic acid encoding a SUMO-cGAS fusion protein as disclosed herein is a bacterial cell. In some embodiments, the bacterial cell includes any of: E. cloni EC10, E. coli MG1655 (DE3), E. coli Bl21(DE3), and E. coli BL21(DE3) RIL (Agilent Inc.).

[0075] Other organisms are encompassed for use as host cells. For instance, in some embodiments, the host cells are selected from a bacterium (e.g., selected from any of: Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Escherichia coli, Saccharomyces cerevisiae, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharbutyricum, Clostridium saccharoperbutylacetonicum, Clostridium butyricum, Clostridium diolis, Clostridium kluyveri, Clostridium pasterianium, Clostridium novyi,Attorney Docket No: AF23853.P206WO Clostridium difficile, Clostridium thermocellum, Clostridium cellulolyticum, Clostridium cellulovorans, Clostridium phytofermentans, Lactococcus lactis, Bacillus subtilis, Bacillus licheniformis, Zymomonas mobilis, Klebsiella oxytoca, Klebsiella pneumonia, Corynebacterium glutamicum, Trichoderma reesei, Cupriavidus necator, Pseudomonas putida, Lactobacillus plantarum, and Methylobacterium extorquens).

[0076] The microorganism of the present disclosure may be further classified based on functional characteristics. For example, the microorganisms of the present disclosure may be or may be derived from a C1-fixing microorganism, an anaerobe, an acetogen, an ethanologen, a carboxydotroph, and / or a methanotroph. Exemplary microorganisms can be selected from any disclosed in Table 1 as disclosed in U.S. Patent 9,738,875, which is incorporated herein in its entirety by reference, and provides a representative list of microorganisms and identifies their functional characteristics useful for expressing cGAS fusion proteins as disclosed herein for the production of 2’3-cGAMP.

[0077] A “microorganism” is a microscopic organism, especially a bacterium, archea, virus, or fungus. The microorganism of the invention is typically a bacterium. As used herein, recitation of “microorganism” should be taken to encompass “bacterium.”

[0078] A “parental microorganism” is a microorganism used to generate a microorganism of the invention. The parental microorganism may be a naturally-occurring microorganism (i.e., a wild-type microorganism) or a microorganism that has been previously modified (i.e., a mutant or recombinant microorganism). The microorganism of the present disclosure may be modified to express or overexpress one or more enzymes that were not expressed or overexpressed in the parental microorganism. Similarly, the microorganism of the present disclosure may be modified to contain one or more genes that were not contained by the parental microorganism. The microorganism of the present disclosure may also be modified to not express or to express lower amounts of one or more enzymes that were expressed in the parental microorganism. In one embodiment, the parental microorganism is Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred embodiment, the parental microorganism is Clostridium autoethanogenum LZ1561, which is deposited under DSMZ accession number DSM23693.Attorney Docket No: AF23853.P206WO

[0079] The term “derived from” indicates that a nucleic acid, protein, or microorganism is modified or adapted from a different (e.g., a parental or wild-type) nucleic acid, protein, or microorganism, so as to produce a new nucleic acid, protein, or microorganism. Such modifications or adaptations typically include insertion, deletion, mutation, or substitution of nucleic acids or genes. Generally, the microorganism of the present disclosure is derived from a parental microorganism. In one embodiment, the microorganism of the present disclosure is derived from Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred embodiment, the microorganism of the present disclosure is derived from Clostridium autoethanogenum LZ1561, which is deposited under DSMZ accession number DSM23693.

[0080] In some embodiments, the recombinant cells include, without limitation, genetically engineered recombinant microorganism or bacterial cell that increases 2’3-cGAMP production by at least about 80% to 100% relative to unmodified bacteria of the same bacterial subtype under the same conditions, or bacteria without codon-optimized nucleic acid encoding the cGAS fusion protein, after 4 hours. In one embodiment, the recombinant cells (e.g., without limitation, a genetically engineered recombinant microorganism or bacterial cell) increase 2’3- cGAMP production by at least about 80% to 100% relative to a bacteria of the same bacterial subtype that comprises a cGAS polypeptide encoded by a cGAS nucleic acid sequence that is not codon-optimized, or comprises a fusion polypeptide comprising cGAS and SUMO, as disclosed herein, under the same conditions, after 4 hours.

[0081] In one embodiment, the recombinant cells (e.g., without limitation, genetically engineered recombinant microorganism or bacterial cell) increases 2’3-cGAMP production by at least about 90% to 100% relative to unmodified bacteria of the same bacterial subtype that comprises a cGAS polypeptide encoded by a cGAS nucleic acid sequence that is not codon- optimized, or comprises a fusion polypeptide comprising cGAS and SUMO, as disclosed herein, under the same conditions, after 4 hours.

[0082] In one specific embodiment, the recombinant cells (e.g., without limitation, genetically engineered recombinant microorganism or bacterial cell) increases 2’3-cGAMP production by at least about 95% to 100% relative to unmodified bacteria of the same bacterial subtype that comprises a cGAS polypeptide encoded by a cGAS nucleic acid sequence that is notAttorney Docket No: AF23853.P206WO codon-optimized, or comprises a fusion polypeptide comprising cGAS and SUMO, as disclosed herein, under the same conditions, after 4 hours.

[0083] In one specific embodiment, the recombinant cells (e.g., without limitation, genetically engineered recombinant microorganism or bacterial cell) increases 2’3-cGAMP production by at least about 99% to 100% relative to unmodified bacteria of the same bacterial subtype that comprises a cGAS polypeptide encoded by a cGAS nucleic acid sequence that is not codon-optimized, or comprises a fusion polypeptide comprising cGAS and SUMO, as disclosed herein, under the same conditions, after 4 hours.

[0084] In yet another embodiment, the recombinant cells (e.g., without limitation, genetically engineered recombinant microorganism or bacterial cell) increases 2’3-cGAMP production by at least about 10-50 fold after 4 hours. In yet another embodiment, the recombinant cells (e.g., without limitation, a genetically engineered recombinant microorganism or bacterial cell), increase 2’3-cGAMP production by at least about 50-100 fold relative to unmodified bacteria of the same bacterial subtype that comprises a cGAS polypeptide encoded by a cGAS nucleic acid sequence that is not codon-optimized, or comprises a fusion polypeptide comprising cGAS and SUMO, as disclosed herein, under the same conditions, after 4 hours. In yet another embodiment, the recombinant cells (e.g., without limitation, a genetically engineered recombinant microorganism or bacterial cell) increase 2’3-cGAMP production by at least about 100-500 fold relative to unmodified bacteria of the same bacterial subtype that comprises a cGAS polypeptide encoded by a cGAS nucleic acid sequence that is not codon-optimized, or comprises a fusion polypeptide comprising cGAS and SUMO, as disclosed herein, under the same conditions, after 4 hours. In yet another embodiment, the recombinant cells (e.g., without limitation, a genetically engineered recombinant microorganism or bacterial cell) increase 2’3-cGAMP production by at least about 500-1000 fold after 4 hours. In yet another embodiment, the recombinant cells (e.g., without limitation, a genetically engineered recombinant microorganism or bacterial cell), increases 2’3-cGAMP production by at least about 1000-5000 fold relative to unmodified bacteria of the same bacterial subtype that comprises a cGAS polypeptide encoded by a cGAS nucleic acid sequence that is not codon-optimized, or comprises a fusion polypeptide comprising cGAS and SUMO, as disclosed herein, under the same conditions, after 4 hours.

[0085] In yet another embodiment, the recombinant cells (e.g., without limitation, genetically engineered recombinant microorganism or bacterial cell), increases 2’3-cGAMPAttorney Docket No: AF23853.P206WO production by at least about 5000-10000 fold after 4 hours, or relative to unmodified bacteria of the same bacterial subtype that comprises a cGAS polypeptide encoded by a cGAS nucleic acid sequence that is not codon-optimized, or comprises a fusion polypeptide comprising cGAS and SUMO, as disclosed herein, under the same conditions, after 4 hours.

[0086] In yet another embodiment, the recombinant cells (e.g., without limitation, genetically engineered recombinant microorganism or bacterial cell) increase 2’3-cGAMP production by at least about 10000-1000 fold after 4 hours, or relative to unmodified bacteria of the same bacterial subtype that comprises a cGAS polypeptide encoded by a cGAS nucleic acid sequence that is not codon-optimized, or comprises a fusion polypeptide comprising cGAS and SUMO, as disclosed herein, under the same conditions, after 4 hours.

[0087] As used herein, the term “recombinant microorganism” refers to a microorganism (e.g., a bacteria, yeast, or virus) that has been genetically modified from its native state. Thus, a “recombinant bacterial cell” or “recombinant bacteria” refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, a recombinant bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Recombinant bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, recombinant bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.

[0088] A “programmed or engineered microorganism” refers to a microorganism (e.g., a bacteria, yeast, or virus) that has been genetically modified from its native state to perform a specific function. Thus, a “programmed or engineered bacterial cell” or “programmed or engineered bacteria” refers to a bacterial cell or bacteria that has been genetically modified from its native state to perform a specific function. In certain embodiments, the programmed or engineered bacterial cell has been modified to express one or more proteins, for example, one or more proteins that have a therapeutic activity or serve a therapeutic purpose. The programmed or engineered bacterial cell may additionally have the ability to stop growing or to destroy itself once the protein(s) of interest have been expressed. In alternative embodiments, a cGAS polypeptide as disclosed herein can be present in a cell-free system.Attorney Docket No: AF23853.P206WO

[0089] In some embodiments, the present disclosure provides a recombinant cell comprising a polynucleotide encoding a cGAS-SUMO fusion polypeptide as disclosed herein and further comprising a polynucleotide encoding a detectable label. In some embodiments, the detectable label is selected from a fluorescent protein.

[0090] Uses of cGAS polypeptides in 2’3-cGAMP production

[0091] In some embodiments, the cGAS polypeptides of the present disclosure (e.g., cGAS- SUMO fusion polypeptides as described herein) have utility in the production of 2’3-cGAMP for various purposes (e.g., therapeutic purposes).

[0092] In some embodiments, the present disclosure pertains to methods of producing 2’3’- cGAMP through the following methods: (a) culturing a recombinant cell of the present disclosure in a culture media, where the culturing induces the expression and activation of the cGAS polypeptide; (b) collecting the culture media; (c) performing anion exchange chromatography (AEC) of the collected culture media; and (d) collecting the elutant from an AEC column, where the elutant comprises the 2’3’-cGAMP. In some embodiments, the methods of the present disclosure also include a step of diluting the culture media collected in step (b) with a buffer. In some embodiments, the buffer comprises 20mM Tris at pH7.5 (Buffer A) at a 1:5 ratio of culture media to buffer. In some embodiments, the collecting step (d) comprises eluting the 2’3’-cGAMP from the AEC column. In some embodiments, the eluting occurs with incremental concentrations of NaCl at 50mM and 100mM.

[0093] In some embodiments, the methods of the present disclosure also includes a step of filtering the elutant. In some embodiments, the elutant is filtered through an appropriate filter, such as a filtration column (e.g., a 3kDa MWCO column). In some embodiments, the filtration occurs through a 0.2 micrometer filter. In some embodiments, a bacterial culture may be spun (e.g., at 4000 × g for 45 min at 4°C). Thereafter, the supernatant may filtered (e.g., by using a 0.2 μm filter). Thereafter, the cell pellet and supernatant may be stored separately at −80°C.

[0094] In some embodiments, the methods of the present disclosure do not require protein purification of the cGAS polypeptide using protein affinity chromatography. In some embodiments, the methods of the present disclosure do not require endotoxin removal from the elutant. In some embodiments, the elutant comprises less than 20EU / ml endotoxin. In some embodiments, the endotoxin is lipopolysaccharide (LPS).Attorney Docket No: AF23853.P206WO

[0095] In some embodiments, activation of the cGAS polypeptide comprises activation by double stranded DNA (dsDNA). In some embodiments, the culture media is selected from the group consisting of M9, 2xTT, or combinations thereof. In some embodiments, the culture media is supplemented with at least one divalent cation. In some embodiments, the at least one divalent cation is selected from the group consisting of Zn, Mn, and Mg.

[0096] In some embodiments, the recombinant cell produces a yield of 2’3’-cGAMP which is at least 30% higher than the yield of 2’3’-cGAMP produced from a cell expressing a cGAS polypeptide encoded by a nucleic acid that is at least 98% identical to SEQ ID NO: 9 or SEQ ID NO: 10. In some embodiments, the recombinant cell produces a yield of 2’3’-cGAMP which is at least 50% higher than the yield of 2’3’-cGAMP produced from a cell expressing a cGAS polypeptide encoded by a nucleic acid that is at least 98% identical to SEQ ID NO: 9 or SEQ ID NO: 10.

[0097] In some embodiments, the present disclosure pertains to bacterial production of 2’3- cGAMP using bacterial cells comprising the heterologous nucleic acid that encodes a cGAS- SUMO fusion polypeptide. In some embodiments, such methods represent a substantial advance over the currently available options for bacterial 2’3-cGAMP production, including but not limited to, (i) a higher yield of 2’3-cGAMP than the yield of 2’3-cGAMP from other systems or bacterial cells expressing a cGAS polypeptide that is expressed from non-codon optimized nucleic acid sequence, and (ii) minimal production of endotoxins, thereby alleviating a need to purify the cGAS or remove endotoxins from the 2’3-cGAMP. Thus, the methods, systems and compositions disclosed herein provide significant advantages in producing higher-yield and higher-purity 2’3-cGAMP as compared to system and recombinant cells comprising non-codon optimized nucleic acid sequences encoding cGAS.

[0098] In some embodiments, the recombinant microorganisms, as disclosed herein, are useful for the bacterial production of 2’-3’-GMP-AMP (cGAMP) at a high yield and / or without the need for a process for endotoxin removal.

[0099] Endotoxin (ET) describes a family of lipopolysaccharides which together with proteins and phospholipids form the outer cell wall of Gram-negative bacteria. Endotoxins occur exclusively in this bacterial group and play an important role in the organization, stability and barrier function of the outer membrane. Numerous bacteriophages use endotoxin or general lipopolysaccharide for specific detection of their host bacteria. Endotoxins (ET) are ubiquitousAttorney Docket No: AF23853.P206WO contaminants of biological preparations, which poses a serious problem because they are broad- spectrum cytotoxins that can confound research results, give false results in diagnostic assays, and render intended therapeutic products unsafe for use. This makes it important to have effective materials and methods for their removal from the biological preparations in which they reside. [000100] Endotoxins are biomolecules which can be found in practically all aqueous solutions without corresponding precautionary measures. Endotoxins in humans and animals can lead to sepsis, to a strong incorrect response of the immune system. Hence, for example when producing pharmaproteins, contamination with endotoxin should be detected precisely and should be removed completely subsequently. Endotoxin represents a problem with genetically engineered pharmaceuticals, gene therapeutics or substances, which are injected into humans or animals (e.g., veterinary treatment or in animal tests). However, not only in medicinal but also in research applications, such as transfection experiments of mammal cells, inhibition or lowering of the transfection efficiency by means of endotoxin can be observed. [000101] In order to be able to use proteins or products produced by bacteria (e.g., bioproduced) within the framework of clinical studies, the European and American pharmacopoeia demand that the proteins fall below specific boundary values for endotoxin level (e.g., immune serum globulin 0.91 EU / ml, this corresponds to 5 EU / kg bodyweight and hour (dosage=EU / kg*h); EU=endotoxin unit; FDA (Food and Drug Administration): Guideline on Validation of LAL as End Product). If a medicine or proteins contained therein have too high an endotoxin level, this can lead to the death of the subject. The misdirected immune defense damages the patient due to overreaction. This can lead to tissue inflammation, drop in blood pressure, heart racing, thrombosis, and / or shock. Even a longer enduring endotoxin exposition in picogram quantities can lead to chronic side effects, such as immune deficiencies and / or septic symptoms. Within the framework of substance production, in particular in processes with “good manufacturing practice” (GMP) conditions, it is therefore attempted to deplete endotoxin as far as possible. [000102] In some embodiments, the present disclosure relates to a method for enzymatically producing 2’3-cGAMP in microorganisms that has minimal endotoxin contamination in a high yield 2’3-cGAMP sample.Attorney Docket No: AF23853.P206WO [000103] In particular, the methods and compositions as disclosed herein can be used in a simplified purification protocol to yield a high final yield of purified 2’3-cGAMP with < 20 EU / ml endotoxin. Herein, the present disclosure pertains to the production of 2’3-cGAMP at a final yield of 58.8 ± 2.1 mg / L with < 20 EU / mL of endotoxin. [000104] Therefore, the methods and compositions disclosed herein relate to 2’3-cGAMP production in an efficient and cost-effective manner that have endotoxin depletion. The term “endotoxin depletion” as used here means complete or partial removal of endotoxin from a sample material. The term “endotoxin” as used here describes bacterial lipopolysaccharide which is a component of the outer membrane of Gram-negative bacteria. [000105] In some embodiments, the methods and compositions as disclosed herein enable the reduction of endotoxin in a 2’3-cGAMP preparation produced by recombinant bacterial cell comprising a nucleic acid encoding a SUMO-cGAS fusion protein, where the reduction in endotoxin is by more than about 50%, or about 60%, or about 70%, or about 80%,or about 90%, or 95% as compared to the endotoxin levels in a 2’3-cGAMP preparation produced by a recombinant bacterial cell comprising a nucleic acid encoding a cGAS gene alone (e.g., not fused to SUMO or a tag protein), or a recombinant bacterial cell comprising a nucleic acid encoding a SUMO-cGAS fusion protein where the nucleic acid is not codon-optimized. [000106] In some embodiments, the methods and compositions as disclosed herein enable the production of a 2’3-cGAMP preparation produced by recombinant bacterial cell comprising a nucleic acid encoding a SUMO-cGAS fusion protein, where the 2’3-cGAMP preparation has more than about 70%, or about 80%,or about 81%, or about 82%,or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%,or about 96%,or about 97%, or 98% or 99% or more of the endotoxin removed as compared to the endotoxin levels in a 2’3- cGAMP preparation produced by a recombinant bacterial cell comprising a nucleic acid encoding a cGAS gene alone (e.g., not fused to SUMO or a tag protein), or a recombinant bacterial cell comprising a nucleic acid encoding a SUMO-cGAS fusion protein where the nucleic acid is not codon-optimized. [000107] In some embodiments, the methods and compositions as disclosed herein enable the production of a 2’3-cGAMP preparation produced by recombinant bacterial cell comprisingAttorney Docket No: AF23853.P206WO a nucleic acid encoding a SUMO-cGAS fusion protein, where the 2’3-cGAMP preparation has less than 1%, or about 2%, or about 3%, or about 4%,or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 11%, or about 12%, or about 13%, or about 14%, or about 15%, or about 16%, or about 17%,or about 18%,or about 19%, or about 20% or less than 20% endotoxin contamination. [000108] In some embodiments, the methods and compositions as disclosed herein enable the production of a 2’3-cGAMP preparation produced by recombinant bacterial cell comprising a nucleic acid encoding a SUMO-cGAS fusion protein, where the 2’3-cGAMP preparation has less than about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19 or about 20 EU / ml of endotoxin. [000109] In some embodiments, the methods and compositions as disclosed herein enable the production of a 2’3-cGAMP preparation produced by recombinant bacterial cell comprising a nucleic acid encoding a SUMO-cGAS fusion protein, where the 2’3-cGAMP preparation has 0.0001 EU / ml endotoxin, or between 0.0001-0.001 EU / ml endotoxin, or between 0.001-0.01 EU / ml endotoxin, or between 0.01-0.1 EU / ml endotoxin, or between 0.1-1.0 EU / ml endotoxin, or between 1.0-2.0 EU / ml endotoxin, or between 2.0-3.0 EU / ml endotoxin, or between 3.0-4.0 EU / ml endotoxin, or between 4.0-5.0 EU / ml endotoxin, or between 5.0-6.0 EU / ml endotoxin, or between 6.0-7.0 EU / ml endotoxin, or between 7.0-8.0 EU / ml endotoxin, or between 8.0-9.0 EU / ml endotoxin, or between 9.0-10.0 EU / ml endotoxin, or between 10.0-11.0 EU / ml endotoxin, or between 11.0-12.0 EU / ml endotoxin, or between 12.0-14.0 EU / ml endotoxin, or between 14.0- 16.0 EU / ml endotoxin, or between 16.0-18.0 EU / ml endotoxin, or between 18.0-20.0 EU / ml endotoxin, or between 1.0-5.0 EU / ml endotoxin, or between 5.0-10.0 EU / ml endotoxin, or between 10.0-15.0 EU / ml endotoxin, or between 15.0-20.0 EU / ml endotoxin, or less than 20.0 EU / ml endotoxin contamination. [000110] Fusion Proteins [000111] In some embodiments, the present disclosure pertains to a fusion protein that includes a cyclic GMP–AMP synthase (cGAS) polypeptide and a stabilizing peptide. In some embodiments, the fusion protein also includes a linker located between the nucleic sequence encoding the cGAS polypeptide and the stabilizing peptide.Attorney Docket No: AF23853.P206WO [000112] In some embodiments, the cGAS polypeptide is produced by the expression of a nucleic acid sequence comprising a codon optimized cGAS nucleic acid sequence selected from SEQ ID NO: 1 (mcGAS), SEQ ID NO: 2 (hcGAS), SEQ ID NO: 23 (hcGAS), or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 23. In some embodiments, the cGAS polypeptide comprises a sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 3, or SEQ ID NO: 4. [000113] In some embodiments, the stabilizing peptide is selected from the group consisting of a small ubiquitin modifying protein (SUMO) polypeptide, a maltose-binding protein (MBP), N- utilization substance (NusA), Thioredoxin (Trx), disulfide bond C (DsbC), glutathione S- transferase (GST), ubiquitin (Ub), fragments thereof, or combinations thereof. [000114] In some embodiments, the stabilizing peptide comprises a small ubiquitin modifying protein (SUMO) polypeptide. In some embodiments, the fusion protein comprises a SUMO polypeptide fused to the N-terminus of the cGAS polypeptide. In some embodiments, the SUMO polypeptide is produced by the expression of a nucleic acid sequence selected from SEQ ID NO: 5 or SEQ ID NO: 6, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the SUMO polypeptide comprises a sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 7, or SEQ ID NO: 8. [000115] In some embodiments, the fusion protein comprises one or more of the polypeptides listed in Table 1. In some embodiments, the fusion protein comprises two of the polypeptides listed in Table 1. In some embodiments, the fusion protein comprises three of the polypeptides listed in Table 1. In some embodiments, the fusion protein comprises one or more of the polypeptides encoded by one or more of the genes listed in Table 1. In some embodiments, the fusion protein comprises two of polypeptides encoded by two or more of the genes listed in Table 1. In some embodiments, the fusion protein comprises three polypeptides encoded by three of the genes listed in Table 1.Attorney Docket No: AF23853.P206WO Table 1. Exemplary Polypeptide Components of Fusion Proteins Protei Description n DNA NCBI Accession SEQ SEQ ID No. No. ID No. co- Mouse cGAS [Codon- mcGASoptimized] (507aa)3 1 --co- hcGAS (trunc- Truncated-Modified SEQ ID NO: 2 mod) human cGAS [residues (co2-hcGAS); 157-5 4 - (“hcGAS 22, K187N / L195R] – SEQ ID NO: 23 -trunc- codon-optimized (366aa) (co1-hcGAS) mod”) SUMO Native SUMO (110aaNM_001180818.1 / (wt)) 8 6NP_010798.1 Co- modified- SUMO Modified SUMO [residues 75(co-mod-1-97] (97aa)SUMO) mcGAS Protein: (wt) Nati NP_775562.2 / (“mcGA ve mcGAS 11 9 nucleic acid: S-wt”) NM_173386.5 hcGAS (trunc.- hcGAS truncated (residues NP_612450.2:157- wt) 157-522 with wt residues 522 cyclic GMP- (“hcGAS- K187 and L195) (not 12 10 AMP synthase trunc- codon-optimized) isoform 1 [Homo sapiens] wt”) hcGAS (full- Protein: length- full-length wild-type NP_612450.2; wt) hcGAS protein 24 25 mRNA (“hcGAS- (NP_612450.2; 522aa) transcript:NM_138 fl-wt”) 441.3 [000116] In some embodiments, a cGAS fusion protein comprises one or more cGAS polypeptides having an amino acid sequence comprising any of SEQ ID NOs: 3 or 4. In some embodiments, a fusion protein comprises two cGAS antigenic polypeptides having an amino acid sequence comprising any of SEQ ID NOs: 3-4, or a functional fragment thereof. In some embodiments, a fusion protein comprises (i) two cGAS polypeptides having an amino acidAttorney Docket No: AF23853.P206WO sequence comprising any of SEQ ID NOs: 3-4, or a functional fragment thereof, and (i) a SUMO protein comprising SEQ ID NO:7 or 8, or fragment thereof that bind to a SUMO activating enzyme (SAE). In some such embodiments, the fusion protein comprises at least one mcGAS polypeptide that is or comprises an mouse cGAS polypeptide (e.g., SEQ ID NO: 3). In some such embodiments, the fusion protein comprises at least one hcGAS polypeptide that is, or comprises, an human cGAS polypeptide (e.g., SEQ ID NO: 4). In some such embodiments, the fusion protein comprises at least one SUMO polypeptide that is or comprises a SUMO attachment or polypeptide (e.g., SEQ ID NOs: 7 or 9). [000117] In some embodiments, a fusion protein comprises one or more polypeptides homologous to the cGAS polypeptides listed in Table 1 (e.g., a mcGAS polypeptide or a human cGAS polypeptide). One of skill in the art may readily substitute an amino acid sequence, or a portion thereof the cGAS sequences of SEQ ID NO: 3 or 4, with the homologous amino acid sequence from a cGAS enzymes. In some embodiments, cGAS polypeptides have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to the polypeptides of SEQ ID NO: 3 or SEQ ID NO: 4 listed in Table 1, or functional fragments thereof. Serotypic variation may be used to design such variants of the polypeptides listed in Table 1. [000118] In some embodiments, the fusion protein comprises a human cGAS polypeptide that comprises K187N mutation. In some embodiments, the fusion protein comprises a human cGAS polypeptide that comprises L195R mutation. In some embodiments, the fusion protein comprises a human cGAS polypeptide that comprises both a K187N and a L195R mutation. In some embodiments, the fusion protein comprises a human cGAS polypeptide that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to the polypeptide of SEQ ID NO: 4, where amino acid residue 31 of SEQ ID NO: 4 is N, or a conservative amino acid of N. In some embodiments, the fusion protein comprises a human cGAS polypeptide that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to the polypeptide of SEQ ID NO: 4, where amino acid residue 39 of SEQ ID NO: 4 is R, or a conservative amino acid of R. In some embodiments, the fusion protein comprises a human cGAS polypeptide that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to the polypeptide of SEQ ID NO: 4, where amino acid residue 31 of SEQ ID NO: 4 is N or a conservative amino acid of N, and where amino acid residue 39 of SEQ ID NO: 4 is R, or a conservative amino acid of R.Attorney Docket No: AF23853.P206WO [000119] In some embodiments, fusion proteins described herein comprise one or more fragments of polypeptides listed in Table 1 (e.g., SUMO activating enzyme (SAE)-binding fragments of SUMO, and a cGAS polypeptide selected from SEQ ID NO: 3 or SEQ ID NO: 4 with, or without a signal sequence). [000120] In some embodiments, fusion proteins described herein comprise cGAS truncation mutants that are close in size to the cGAS polypeptides of SEQ ID NO: 3 or 4 as listed in Table 1. For example, they may lack at most one, two, three, four, five, ten, or twenty amino acids from one or both termini (referring to component polypeptides in a fusion protein). In some embodiments, a fragment is a truncated fragment of any of SEQ ID NOs: 3-4 lacking 1-5, 1-10, or 1-20 amino acid residues from the N-terminus, C-terminus, or both, of any one of SEQ ID NOs: 3-4. In some embodiments, a fragment is a truncated fragment of any of SEQ ID NOs:3-4 lacking 1-10 amino acid residues from the N-terminus, C-terminus, or both, of any one of SEQ ID Nos: 3-4. For instance, in some embodiments, a fragment may lack 10 amino acid residues at both the N-terminus and C-terminus of any one of SEQ ID NOs: 3-4, resulting in a protein lacking 20 amino acid residues. Internal deletions, e.g., of 1-10, 11-20, 21-30, or 31-40 amino acids, are also contemplated. [000121] In some embodiments, a SUMO-cGAS fusion protein comprises an N-terminal SUMO polypeptide or a C-terminal SUMO polypeptide fused to the cGAS polypeptide of SEQ ID NO: 3 or 4, or a functional variant thereof as described herein. [000122] In some embodiments, the SUMO polypeptide and the cGAS polypeptide are directly bound to each other. In some embodiments, the N-terminal polypeptide and the C-terminal polypeptide are linked via a linker peptide. In some embodiments, the length and / or amino acids of a linker, when present, can be adjusted to obtain a more flexible, semi-rigid, or rigid linker. Exemplary flexible peptide linkers are shown as SEQ ID NOs: 37A-40. A linker can generally be from 1-40, such as 3-10 or 10-30 and specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In some embodiments, the fusion protein comprises one linker. In some embodiments, the fusion protein comprises two linkers. In some embodiments, the one or two linkers are selected from SEQ ID NO:37A (GGGGSSS) and AAA. In some embodiments, the fusion protein comprises SEQ ID NO:37A (GGGGSSS) and AAA. In some embodiments, the fusion protein comprises an amino acid sequence AAA residual from a Not IAttorney Docket No: AF23853.P206WO restriction site. In some embodiments, the fusion protein comprises a linker of SEQ ID NO:37A (GGGGSSS) and an amino acid sequence AAA residual from a Not I restriction site. [000123] Exemplary fusion proteins are shown in Table 2. Table 2. Exemplary Fusion Proteins Protein DNA Locus tag name and description SEQ ID No. SEQ ID No. SUMO-mcGAS 13 26 SUMO-hcGAS 14 27 or 74 mcGAS-SUMO 15 28 hcGAS-SUMO 16 29 or 75 SUMO-(linker)-mcGAS 17 SUMO-(linker)-hcGAS 18 mcGAS-(linker)-SUMO 19 hcGAS-(linker)-SUMO 20 SUMO-mcGAS-(linker)-mcGAS 21 SUMO-hcGAS-(linker)-hcGAS 22 [000124] In some embodiments, the present disclosure provides SUMO-cGAS fusion proteins with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to a fusion protein listed in Table 2. In some embodiments, a fusion protein is or includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOs:13-21. In some embodiments, a SUMO-cGAS fusion protein is or includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 13. In some embodiments, a fusion protein is or includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identity to SUMO-hcGAS of SEQ ID NO: 14. [000125] In some embodiments, a fusion protein described herein comprises a SUMO polypeptide or a functional fragment thereof which binds to a SUMO activating enzyme (SAE). In some embodiments, the fusion protein comprises a SUMO polypeptide, and one or more cGAS polypeptides. In some embodiments, the fusion protein comprises a SUMO polypeptide and two or more cGAS polypeptides.Attorney Docket No: AF23853.P206WO [000126] In some embodiments, a SUMO-cGAS fusion protein described herein comprises a SUMO attachment, or SUMO activating enzyme (SAE)-binding moiety that is, or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 7, or a fragment thereof that binds to a SUMO activating enzyme (SAE). In some embodiments, the fusion protein comprises a SUMO attachment, or SUMO activating enzyme (SAE)-binding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:7 (amino acids 1- 97 of native SUMO), or an SAE-binding fragment thereof. [000127] In some embodiments, the SUMO-cGAS fusion protein comprises a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:13. In some embodiments, the SUMO-cGAS fusion protein comprises a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 14. [000128] In some embodiments, a fusion protein described herein comprises each of: (a) a SUMO polypeptide, or SAE-binding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:7 (SUMO), or a SAE-binding fragment thereof; (b) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:3 (mcGAS), or a functional fragment thereof; and (c) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:3, where (a),(b) and (c) can be in any order. [000129] In some embodiments, a fusion protein described herein comprises each of: (a) a SUMO polypeptide, or SAE-binding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:7 (SUMO), or a SAE-binding fragment thereof; (b) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:4 (hcGAS), or a functional fragment thereof; and (c) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:4, where (a),(b) and (c) can be in any order. [000130] In some embodiments, a fusion protein described herein comprises each of: (a) a SUMO polypeptide, or SAE-binding moiety that is or comprises a polypeptide having at leastAttorney Docket No: AF23853.P206WO 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:7 (SUMO), or a SAE-binding fragment thereof; (b) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 3 (mcGAS), or a functional fragment thereof; and (c) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:4 (hcGAS), where (a),(b) and (c) can be in any order. [000131] In some embodiments, the SUMO-cGAS fusion protein further comprises one or more linkers. In some embodiments, the one or more linkers are selected from SEQ ID NO:37A (GGGGSSS) and AAA. In some embodiments, the fusion protein comprises an amino acid sequence AAA residual from a Not I restriction site. In some embodiments, the fusion protein comprises a linker of SEQ ID NO:37A (GGGGSSS) and an amino acid sequence AAA. [000132] In some embodiments, a SUMO-cGAS fusion protein described herein comprises each of: (a) a SUMO polypeptide or SAE-binding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 7, or SAE-binding fragment thereof; (b) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 3 (amino mcGAS polypeptide) or a functional fragment thereof. In some embodiments, the SUMO-cGAS fusion protein can further optionally comprise, (c) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 4 (hcGAS polypeptide) or a functional fragment thereof. [000133] In some embodiments, a SUMO-cGAS fusion protein described herein comprises each of: (a) a SUMO polypeptide or SAE-binding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 7, or SAE-binding fragment thereof; (b) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 4 (amino mcGAS polypeptide) or a functional fragment thereof. In some embodiments, the SUMO-cGAS fusion protein can further optionally comprise, (c) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%,Attorney Docket No: AF23853.P206WO 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 3 (mcGAS polypeptide) or a functional fragment thereof. [000134] In some embodiments, the fusion protein further comprises one or more linkers. In some embodiments, the one or more linkers are selected from SEQ ID NO:37A (GGGGSSS) and AAA. In some embodiments, the fusion protein comprises an amino acid sequence AAA residual from a Not I restriction site. In some embodiments, the fusion protein comprises a linker of SEQ ID NO:37A (GGGGSSS) and an amino acid sequence AAA residual from a Not I restriction site. [000135] In some embodiments, a fusion protein described herein comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence SEQ ID NO: 13. In some embodiments, the fusion protein comprises the amino acid sequence SEQ ID NO: 13. In some embodiments, the fusion protein consists of the amino acid sequence SEQ ID NO:13 (SUMO-mcGAS). [000136] In some embodiments, a fusion protein described herein comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence SEQ ID NO: 14. In some embodiments, the fusion protein comprises the amino acid sequence SEQ ID NO: 14. In some embodiments, the fusion protein consists of the amino acid sequence SEQ ID NO:14 (SUMO-hcGAS). [000137] In some embodiments, the fusion protein as disclosed herein comprises a truncated and modified hcGAS polypeptide comprising the polypeptides of SEQ ID NO: 4 (comprising K187 and L19R modification), which is encoded by a nucleic acid sequence that is codon optimized, for example, SEQ ID NO: 2 or SEQ ID NO: 23. [000138] The amino acid sequence of mouse cGAS is provided as SEQ ID NO: 11, which is encoded by SEQ ID NO: 9. However, in particular embodiments, the fusion protein as disclosed herein comprises a modified mcGAS polypeptide of SEQ ID NO: 3, which is encoded by a nucleic acid sequence that is codon optimized, for example, SEQ ID NO: 1. [000139] In some embodiments, a fusion protein described herein includes a variant or fragment of a polypeptide listed in Table 1. In some embodiments, a fusion protein described herein includes a polypeptide encoded by a variant or fragment of a gene listed in Table 1. In some embodiments, a fragment included in a fusion protein described herein is close in size to a full-length polypeptide or a polypeptide listed in Table 1. For example, they may lack at most one, two, three, four, five, ten, twenty, or thirty amino acids from one or both termini. In someAttorney Docket No: AF23853.P206WO embodiments, the fragment is 25-50 amino acids in length, or 50-100, or 100-150, or 150-200, or 200-250, or 250-300, or 300-350 amino acids in length. In some embodiments, the fragments result from processing, or partial processing, of signal sequences by an expression host, e.g. E. coli, an insect cell line (e.g., the baculovirus expression system), or a mammalian (e.g., human or Chinese Hamster Ovary) cell line, or other recombinant microorganism, e.g., microalgae or the like. The fragments described above or sub-fragments thereof (e.g., fragments of 8-50, 8-30, or 8-20 amino acid residues) preferably have one of the biological activities described below (e.g., ability to produce 2’3-cGAMP in the recombinant cell, and / or where the recombinant cell secretes the 2’3-cGAMP into the surrounding culture media). [000140] The DNA and protein sequence of each gene and polypeptide may be identified by searching for the Locus Tag in a publicly available database (e.g., Entrez Gene (on the NCBI NIH web site on the World Wide Web, at www.ncbi.nlm.nih. gov / sites / entrez?db=gene)), in the Streptococcus pneumoniae TIGR4 genome, and the indicated sequences are also included within the scope of the present disclosure. [000141] Certain polypeptides of Table 1, variants thereof, and additional exemplary polypeptides and linkers which constitute components of various embodiments of the fusion proteins are described in greater detail below. [000142] Human cGAS Polypeptides (e.g., SEQ ID NOs: 4) and Variants Thereof [000143] The native human cyclic GMP-AMP synthase isoform 1(hcGAS) comprises the amino acid sequence of Accession No. NP_612450.2, which is 522 amino acid sequence and corresponds to SEQ ID NO: 24 herein (referred to herein as “hcGAS-fl-wt”). The full-length wild-type hcGAS-fl-wt polypeptide is 522 amino acids in length and comprises K amino acid at residue 187 (K187) and L amino acid at residue 195 (L195) amino acid, and is encoded by Accession No: NM_138441.3, corresponding to SEQ ID NO: 25. In some embodiments, a hcGAS polypeptide is, or comprises a full-length wild-type cGAS polypeptide of SEQ ID NO: 24. For example, in some embodiments, a full-length, wild type hcGAS polypeptide has is represented by the amino acid sequence as set forth in SEQ ID NO: 24. [000144] In some embodiments, the hcGAS polypeptide is, or comprises a truncated wild-type cGAS polypeptide (referred to as “hcGAS-trunc-wt”), which lacks residues 1-156 of SEQ ID NO: 24 and where the hcGAS-trunc-wt polypeptide corresponds to the amino acids of SEQ ID NO: 12. For example, in some embodiments, a truncated, wild type hcGAS polypeptide is representedAttorney Docket No: AF23853.P206WO by the amino acid sequence as set forth in SEQ ID NO: 12, and is encoded by SEQ ID NO: 10 herein. [000145] In some embodiments, a fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, or 399 consecutive amino acids of a truncated hcGAS polypeptide as disclosed herein of SEQ ID NO: 4 or SEQ ID NO: 12. [000146] In some embodiments, a hcGAS polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, or 399 consecutive amino acids of the sequence shown in SEQ ID NO: 24 [hcGAS-fl-wt]. In some embodiments, a hcGAS polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, 380, 400, 450, 460, 470, 480, 490, 500, 510, 515 or 522 consecutive amino acids of the sequence shown in SEQ ID NO: 24 [hcGAS-fl-wt] . [000147] In some embodiments, a hcGAS polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, or 399 consecutive amino acids of the sequence shown in SEQ ID NO: 12 [hcGAS-trunc-wt]. In some embodiments, a hcGAS polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, or 399 consecutive amino acids of the sequence shown in SEQ ID NO: 12 [hcGAS-trunc-wt] . [000148] In some embodiments, a hcGAS polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, or 399 consecutive amino acids of the sequence shown in SEQ ID NO: 4 [hcGAS-trunc-mod]. In some embodiments, a hcGAS polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200,Attorney Docket No: AF23853.P206WO 250, 300, 350, 367, or 399 consecutive amino acids of the sequence shown in SEQ ID NO: 4 [hcGAS-trunc-mod]. [000149] In particular embodiments, where a hcGAS polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, or 399 consecutive amino acids of the sequence shown in SEQ ID NO: 4 [hcGAS-trunc-mod], the hcGAS polypeptide is a modified polypeptide that has at least one modification selected from K187N or L195R, or where both modifications K187N and L195R are present. The K187N modification numbering refers to the full-length hcGAS polypeptide, and in the truncated hcGAS (hcGAS-trunc.) polypeptide, corresponds to the presence of an Asn (i.e., N or Asparagine) amino acid at residue 31 of SEQ ID NO: 4. Similarly, reference to the L195R modification is based on the numbering of the full-length hcGAS polypeptide, and corresponds to the presence of an Arg (R or Arginine) amino acid at residue 39 of SEQ ID NO: 4. The modifications K187N or L195R assist to stabilize make direct contact of the human cGAS polypeptide with the DNA molecule that it binds to, increasing 2’3’-cGAMP synthesis and yield as compared to native hcGAS. [000150] In some embodiments, a hcGAS polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a polypeptide comprising the sequence of at least amino acid residues 29-366 of SEQ ID NO: 4, where amino acid residue 31 of SEQ ID NO: 4 is Asn (N). [000151] In some embodiments, a hcGAS polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a polypeptide comprising the sequence of at least amino acid residues 29-366 of SEQ ID NO: 4, where amino acid residue 39 of SEQ ID NO: 4 is Arg (R). [000152] In some embodiments, a hcGAS polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a polypeptide comprising theAttorney Docket No: AF23853.P206WO sequence of at least amino acid residues 29-366 of SEQ ID NO: 4, where amino acid residue 31 of SEQ ID NO: 4 is Asn (N) and amino acid residue 39 of SEQ ID NO: 4 is Arg (R). [000153] An exemplary nucleotide sequence encoding a hcGAS polypeptide of SEQ ID NO: 4 (“hcGAS-trunc-mod”) is provided herein as the codon-optimized sequence of SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, a fusion protein as disclosed herein is encoded by a nucleic acid comprising SEQ ID NO: 2 or a nucleic acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least SEQ ID NO: 2 or SEQ ID NO: 23). [000154] Mouse cGAS Polypeptides (e.g., SEQ ID NO:4) and Variants Thereof [000155] The mouse cGAS (mcGAS) has the amino acid sequence of Gene Accession No: NP_775562.2, which corresponds to SEQ ID NO: 11 herein. The mcGAS polypeptide is encoded by SEQ ID NO: 9 herein, which is the native mRNA transcript corresponding to Accession number NM_173386.5. Herein, in some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 3, or a functional fragment thereof, which is encoded by a codon- optimized nucleic acid sequence of SEQ ID NO: 1 as disclosed herein. [000156] In some embodiments, the mcGAS polypeptide is, or comprises a wild-type mouse cGAS polypeptide (referred to as “mcGAS-wt”), where the mcGAS-wt polypeptide corresponds to the amino acids of SEQ ID NO: 11. For example, in some embodiments, a wild type mcGAS polypeptide is represented by the amino acid sequence as set forth in SEQ ID NO: 11, and is encoded by SEQ ID NO: 9 herein. [000157] In some embodiments, a fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, or 399 consecutive amino acids of a mcGAS polypeptide as disclosed herein of SEQ ID NO: 12 or SEQ ID NO: 3. [000158] In some embodiments, a mcGAS polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, or 399 consecutive amino acids of the sequence shown in SEQ ID NO: 3 [co-mGAS]. In some embodiments, a mcGAS polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200,Attorney Docket No: AF23853.P206WO 250, 300, 350, 367, 380, 400, 450, 460, 470, 480, 490, 500, 505 or 507 consecutive amino acids of the sequence shown in SEQ ID NO: 3 [co-mGAS]. [000159] In some embodiments, a mcGAS polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, or 399 consecutive amino acids of the sequence shown in SEQ ID NO: 11 [mGAS-wt]. In some embodiments, a mcGAS polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, 380, 400, 450, 460, 470, 480, 490, 500, 505 or 507 consecutive amino acids of the sequence shown in SEQ ID NO: 11 [mGAS-wt]. [000160] An exemplary nucleotide sequence encoding a mcGAS polypeptide of SEQ ID NO: 3 (“mcGAS”) is provided herein as the codon-optimized sequence of SEQ ID NO: 1 (co-mcGAS). In some embodiments, a fusion protein as disclosed herein is encoded by a nucleic acid, where the nucleic acid comprises in part, SEQ ID NO: 1 or a nucleic acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least SEQ ID NO: 1 (co-mcGAS). [000161] In some embodiments, a nucleotide sequence encoding a mcGAS polypeptide of SEQ ID NO: 11 (“mcGAS-wt”) is provided herein as the sequence of SEQ ID NO: 9 (mcGAS-wt). In some embodiments, a fusion protein as disclosed herein is encoded by a nucleic acid, where the nucleic acid comprises in part, SEQ ID NO: 9 or a nucleic acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least SEQ ID NO: 9 (co-mcGAS-wt). [000162] SUMO and other Stabilizing sequences [000163] In some embodiments, a fusion protein described herein comprises a stabilizing protein, also referred to herein as a Tag. In some embodiments, the tag is a SUMO peptide. In some embodiments, the tag is a Ub tag. SUMO is a small ubiquitin modifying protein. In some embodiments, the SUMO peptide is encoded by a nucleic acid sequence comprising a start codon, Met. [000164] SUMO has an external hydrophilic surface and inner hydrophobic core, which may exert a detergent-like effect on otherwise insoluble protein. A fusion protein comprising a SUMOAttorney Docket No: AF23853.P206WO peptide sequence can, in some embodiments, enhance expression and facilitate purification with Ni–NTA (nickel–nitriloacetic acid) chromatography. SUMO can be used to increase the expression of a soluble heterologous protein in E. coli. Using Ub or SUMO in a fusion protein requires the use of proteases for removal of the tag, however, the SUMO proteases and the deubiquitylating enzymes (DUBs) are distinct from other proteases that recognize a peptide sequence, as they recognize the tertiary structure of the SUMO or Ub tag. When the target protein (e.g., cGAS as disclosed herein) is fused directly to the C-terminus of SUMO or Ub, cleavage will not result in extraneous residues at the N-terminus of the target protein and therefore will yield native-like cGAS protein. While DUBs for complete cleavage of the Ub tag requires a large amount of enzyme (1:10 molar ratio of DUB to target), SUMO proteases are much more robust, requiring only a 1:5000 molar ratio of protease to target. The new generation of SUMO proteases has been shown to be even more catalytically active, requiring only 1:100,000 molar ratio of protease to target. [000165] In some embodiments, the fusion protein comprises a SUMO peptide at the N- terminus (e.g., a SUMO peptide attached to a cGAS polypeptide as disclosed herein). In some embodiments, the fusion protein comprises a SUMO peptide at the C-terminus (e.g., a SUMO sequence attached to the C-terminus of a cGAS polypeptide as disclosed herein). In some embodiments, the SUMO peptide can be readily replaced with a different stabilization sequence readily known by a skilled artisan, for example but not limited to, selected from any of: MBP (Maltose-Binding Protein), NusA (N-utilization substance), Trx (Thioredoxin), DsbC (Disulfide bond C, which is a prokaryotic disulfide bond isomerase). [000166] In some embodiments, the nucleotide sequence of SUMO is set forth in SEQ ID NO: 5. In some embodiments, the amino acid sequence of SUMO is set forth in SEQ ID NO: 7. In some embodiments, a fusion protein comprises SUMO. In some embodiments, a fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90 or 95, consecutive amino acids of a SUMO polypeptide. [000167] In some embodiments, a SUMO polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 100, or 110 consecutive amino acids of the sequence shown in SEQ ID NO:8 [SUMO- wt]. In some embodiments, a SUMO polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%,Attorney Docket No: AF23853.P206WO 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100 or 110 consecutive amino acids of the sequence shown in SEQ ID NO:8 [SUMO-wt (110aa)]. [000168] In some embodiments, a SUMO polypeptide of the fusion protein is a truncated or modified SUMO polypeptide (mod-SUMO) and comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 96 or 97 consecutive amino acids of the sequence shown in SEQ ID NO:7 [mod-SUMO]. In some embodiments, a SUMO polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g.,, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 96, or 97 consecutive amino acids of the sequence shown in SEQ ID NO: 7 [mod-SUMO]. [000169] In some embodiments, a SUMO polypeptide of the fusion protein is or comprises amino acids 1-97 of SEQ ID NO: 8. In some embodiments, a SUMO polypeptide of the fusion protein is or comprises amino acids 1-79 of SEQ ID NO:8. In some embodiments, a SUMO polypeptide of the fusion protein is or comprises amino acids of SEQ ID NO: 7. In some embodiments, a SUMO polypeptide of the fusion protein is or comprises amino acids of SEQ ID NO:7, or a polypeptide that is at least 60% or more (including, e.g.,, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 7. [000170] In some embodiments, a nucleotide sequence encoding a SUMO polypeptide of SEQ ID NO: 7 (mod-SUMO) is codon-optimized, and provided herein as the sequence of SEQ ID NO: 5 (co-mod-SUMO). In some embodiments, a fusion protein as disclosed herein is encoded by a nucleic acid, where the nucleic acid comprises in part, SEQ ID NO: 5 or a nucleic acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least SEQ ID NO: 5 (co-mod-SUMO). [000171] In some embodiments, a SUMO protein for use in the fusion protein as disclosed herein can be a SUMO protein as disclosed in US Patents 6,872, 255; 7,498,165; 8,034,910; 8,119369; each of which are incorporated herein in their entirety by reference. [000172] In some embodiments, the recombinant cell comprises a codon-optimized nucleic acid encoding a fusion protein, wherein the fusion protein comprises a SUMO selected from any of: human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-Attorney Docket No: AF23853.P206WO 1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, or a combination thereof. The nucleic acid sequences of such SUMO proteins are disclosed in US 8,119,369, which is incorporated herein in its entirety by reference. [000173] In some embodiments, when the fusion protein comprising the SUMO and cGAS polypeptide is expressed by the recombinant cell, e.g., engineered microorganism as disclosed herein, such as, but not limited to a bacterial cell, the SUMO is not cleaved from the cGAS polypeptide, and remains attached or fused to the cGAS polypeptide to stabilize the fusion protein. In alternative embodiments, the SUMO is cleaved from the cGAS polypeptide after the fusion protein is expressed by the recombinant cell. [000174] In some embodiments, SUMO can be replaced by any of: CTHS (C-terminal half of SUMO); NTHS (N-terminal half of SUMO); GST (glutathione S-transferase); MBP (maltose binding protein); Trx (thioredoxin), and Ub (ubiquitin). [000175] In some embodiments, the SUMO peptide can be readily replaced with a different stabilization sequence readily known by a skilled artisan, for example but not limited to, selected from any of: MBP (Maltose-Binding Protein), NusA (N-utilization substance), Trx (Thioredoxin), and DsbC. Exemplary tags are shown in Table 3. Table 3. Exemplary tags. Tag Description Protein DNA SEQ Uniprot Exemplary fusion SEQ ID ID No: number protein No: MBP Maltose- 30 31 Uniprot: 32 (MBP-mcGAS Binding P0AEX9 plasmid) Protein NusA N-utilization 32A 33 Uniprot: 34 (NusA-mcGAS substance P0AFF8 plasmid) DsbC 35 36 Uniprot: 70 (DsbC-mcGAS P0AEG6 plasmid) Trx Thioredoxin 71 72 Uniprot: 73 (TrX-mcGAS P0AA25 plasmid)Attorney Docket No: AF23853.P206WO [000176] MBP-cGAS fusion protein [000177] In some embodiments, the fusion protein expressed by the recombinant cell is a MBP- cGAS fusion protein comprising a MBP tag. In some embodiments, a fusion protein comprises MBP. In some embodiments, a fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90 or 95 consecutive amino acids of a MBP polypeptide. [000178] In some embodiments, a MBP polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 100 or 110 consecutive amino acids of the sequence shown in SEQ ID NO: 30 [MBP peptide]. In some embodiments, a MBP polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100 or 110 consecutive amino acids of the sequence shown in SEQ ID NO: 30 [MBP peptide (396aa)]. [000179] In some embodiments, a nucleotide sequence encoding a MBP polypeptide of SEQ ID NO: 30 is codon-optimized. In some embodiments, a nucleotide sequence encoding a MBP peptide is provided herein as the sequence of SEQ ID NO: 31. In some embodiments, a fusion protein as disclosed herein is encoded by a nucleic acid, where the nucleic acid comprises in part, SEQ ID NO: 31 or a nucleic acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least SEQ ID NO: 31, thereby encoding a MBP-cGAS fusion protein. [000180] In some embodiments, a codon-optimized cGAS nucleic acid as disclosed herein (e.g., SEQ ID NO: 1 or 2) is attached to a nucleic acid sequence, that comprises SEQ ID NO: 31 or a nucleic acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least SEQ ID NO: 31. [000181] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a fusion protein as disclosed herein, where the codon- optimized nucleic acid sequence is expressed by a plasmid of SEQ ID NO: 32 encoding a MBP- mcGAS fusion protein, where the plasmid comprises a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 31 andAttorney Docket No: AF23853.P206WO SEQ ID NO: 1 (MBP-mcGAS). In some embodiments, the codon-optimized polynucleotide encoding a MBP-mcGAS fusion protein consists of a codon-optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 31 and SEQ ID NO: 1 (MBP-mcGAS). In some embodiments, the codon-optimized polynucleotide encoding a MTB-mcGAS fusion protein as disclosed herein comprises a nucleotide sequence that is 100% identical to a nucleic acid sequence comprising SEQ ID NO: 31 and SEQ ID NO: 1. In some embodiments, the codon-optimized polynucleotide encoding a MBT-mcGAS fusion protein as disclosed herein consists of a nucleic acid sequence comprising SEQ ID NO: 31 and SEQ ID NO: 1 (MBP-mcGAS). [000182] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a fusion protein as disclosed herein, where the codon- optimized nucleic acid sequence encodes a MBP-hcGAS fusion protein, where the nucleic acid sequence is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 31 and SEQ ID NO: 2 or SEQ ID NO: 23 (MBP-hcGAS). In some embodiments, the codon-optimized polynucleotide encoding a MBP-hcGAS fusion protein consists of a codon- optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 31 and SEQ ID NO: 2 or SEQ ID NO: 23 (MBP-hcGAS). In some embodiments, the codon-optimized polynucleotide encoding a MTB-hcGAS fusion protein as disclosed herein comprises a nucleotide sequence is 100% identical to a nucleic acid sequence comprising SEQ ID NO: 31 and SEQ ID NO: 2. In some embodiments, the codon- optimized polynucleotide encoding a MBT-hcGAS fusion protein as disclosed herein consists of a nucleic acid sequence comprising SEQ ID NO: 31 and SEQ ID NO: 2 or SEQ ID NO: 23 (MBP- hcGAS). [000183] NusA-cGAS fusion protein [000184] In some embodiments, the fusion protein expressed by the recombinant cell is a NusA-cGAS fusion protein comprising a NusA tag. In some embodiments, a fusion protein comprises NusA. In some embodiments, a fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90 or 95 consecutive amino acids of a NusA polypeptide.Attorney Docket No: AF23853.P206WO [000185] In some embodiments, a NusA polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 100 or 110 consecutive amino acids of the sequence shown in SEQ ID NO: 32A [NusA peptide]. In some embodiments, a NusA polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100 or 110 consecutive amino acids of the sequence shown in SEQ ID NO: 32A [NusA peptide (495aa)]. [000186] In some embodiments, a nucleotide sequence encoding a NusA polypeptide of SEQ ID NO: 32A is codon-optimized. In some embodiments, a nucleotide sequence encoding a NusA peptide is provided herein as the sequence of SEQ ID NO: 33. In some embodiments, a fusion protein as disclosed herein is encoded by a nucleic acid, where the nucleic acid comprises in part, SEQ ID NO: 33 or a nucleic acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least SEQ ID NO: 33, thereby encoding a NusA-cGAS fusion protein. [000187] In some embodiments, a codon-optimized cGAS nucleic acid as disclosed herein (e.g., SEQ ID NO: 1 or 2) is attached to a nucleic acid sequence, that comprises SEQ ID NO: 33 or a nucleic acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least SEQ ID NO: 33. [000188] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a fusion protein as disclosed herein, where the codon- optimized nucleic acid sequence is expressed by a plasmid of SEQ ID NO: 34 encoding a NusA- mcGAS fusion protein, where the plasmid comprises a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 33 and SEQ ID NO: 1 (NusA-mcGAS). In some embodiments, the codon-optimized polynucleotide encoding a NusA-mcGAS fusion protein consists of a codon-optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 33 and SEQ ID NO: 1 (NusA-mcGAS). In some embodiments, the codon-optimized polynucleotide encoding a NusA-mcGAS fusion protein as disclosed herein comprises a nucleotide sequence that is 100% identical to a nucleic acid sequence comprising SEQ ID NO: 33 and SEQ ID NO: 1. In some embodiments, the codon-optimized polynucleotide encoding aAttorney Docket No: AF23853.P206WO NusA-mcGAS fusion protein as disclosed herein consists of a nucleic acid sequence comprising SEQ ID NO: 33 and SEQ ID NO: 1 (NusA-mcGAS). [000189] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a fusion protein as disclosed herein, where the codon- optimized nucleic acid sequence encodes a NusA-hcGAS fusion protein, where the nucleic acid sequence is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 33 and SEQ ID NO: 2 or SEQ ID NO: 23 (NusA-hcGAS). In some embodiments, the codon-optimized polynucleotide encoding a NusA-hcGAS fusion protein consists of a codon- optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 33 and SEQ ID NO: 2 or SEQ ID NO: 23 (NusA-hcGAS). In some embodiments, the codon-optimized polynucleotide encoding a NusA-hcGAS fusion protein as disclosed herein comprises a nucleotide sequence that is 100% identical to a nucleic acid sequence comprising SEQ ID NO: 33 and SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the codon-optimized polynucleotide encoding a NusA-hcGAS fusion protein as disclosed herein consists of a nucleic acid sequence comprising SEQ ID NO: 33 and SEQ ID NO: 2 or SEQ ID NO: 23 (NusA-hcGAS). [000190] DsbC-cGAS fusion protein [000191] In some embodiments, the fusion protein expressed by the recombinant cell is a DsbC-cGAS fusion protein comprising a DsbC tag. In some embodiments, a fusion protein comprises DsbC. In some embodiments, a fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90 or 95 consecutive amino acids of a DsbC polypeptide. [000192] In some embodiments, a DsbC polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 100 or 110 consecutive amino acids of the sequence shown in SEQ ID NO: 35 [DsbC peptide]. In some embodiments, a DsbC polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100 or 110 consecutive amino acids of the sequence shown in SEQ ID NO: 35 [DsbC peptide (236aa)].Attorney Docket No: AF23853.P206WO [000193] In some embodiments, a nucleotide sequence encoding a DsbC polypeptide of SEQ ID NO: 35 is codon-optimized. In some embodiments, a nucleotide sequence encoding a DsbC peptide is provided herein as the sequence of SEQ ID NO: 36. In some embodiments, a fusion protein as disclosed herein is encoded by a nucleic acid, where the nucleic acid comprises in part, SEQ ID NO: 36 or a nucleic acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least SEQ ID NO: 35, thereby encoding a DsbC-cGAS fusion protein. [000194] In some embodiments, a codon-optimized cGAS nucleic acid as disclosed herein (e.g., SEQ ID NO: 1 or 2) is attached to a nucleic acid sequence, that comprises SEQ ID NO: 36 or a nucleic acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least SEQ ID NO: 36. [000195] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a fusion protein as disclosed herein, where the codon- optimized nucleic acid sequence is expressed by a plasmid of SEQ ID NO: 70 encoding a DsbC- mcGAS fusion protein, where the plasmid comprises a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 36 and SEQ ID NO: 1 (DsbC-mcGAS). In some embodiments, the codon-optimized polynucleotide encoding a DsbC-mcGAS fusion protein consists of a codon-optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 36 and SEQ ID NO: 1 (DsbC-mcGAS). In some embodiments, the codon-optimized polynucleotide encoding a DsbC-mcGAS fusion protein as disclosed herein comprises a nucleotide sequence that is 100% identical to a nucleic acid sequence comprising SEQ ID NO: 36 and SEQ ID NO: 1. In some embodiments, the codon-optimized polynucleotide encoding a DsbC-mcGAS fusion protein as disclosed herein consists of a nucleic acid sequence comprising SEQ ID NO: 36 and SEQ ID NO: 1 (DsbC-mcGAS). [000196] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a fusion protein as disclosed herein, where the codon- optimized nucleic acid sequence encodes a DsbC-hcGAS fusion protein, where the nucleic acid sequence is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 36 and SEQ ID NO: 2 or SEQ ID NO: 23 (DsbC-hcGAS). In some embodiments, the codon-optimized polynucleotide encoding a DsbC-hcGAS fusion protein consists of a codon-Attorney Docket No: AF23853.P206WO optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 36 and SEQ ID NO: 2 or SEQ ID NO: 23 (DsbC-hcGAS). In some embodiments, the codon-optimized polynucleotide encoding a DsbC-hcGAS fusion protein as disclosed herein comprises a nucleotide sequence is 100% identical to a nucleic acid sequence comprising SEQ ID NO: 36 and SEQ ID NO: 2. In some embodiments, the codon- optimized polynucleotide encoding a DsbC-hcGAS fusion protein as disclosed herein consists of a nucleic acid sequence comprising SEQ ID NO: 36 and SEQ ID NO: 2 or SEQ ID NO: 23 (DsbC- hcGAS). [000197] TrX-cGAS fusion protein [000198] In some embodiments, the fusion protein expressed by the recombinant cell is a TrX- cGAS fusion protein comprising a TrX tag. In some embodiments, a fusion protein comprises TrX. In some embodiments, a fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90 or 95 consecutive amino acids of a TrX polypeptide. [000199] In some embodiments, a TrX polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 100 or 105 consecutive amino acids of the sequence shown in SEQ ID NO: 71 [TrX peptide]. In some embodiments, a TrX polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100 or 105 consecutive amino acids of the sequence shown in SEQ ID NO: 71 [TrX peptide (109 aa)]. [000200] In some embodiments, a nucleotide sequence encoding a TrX polypeptide of SEQ ID NO: 71 is codon-optimized. In some embodiments, a nucleotide sequence encoding a TrX peptide is provided herein as the sequence of SEQ ID NO: 72. In some embodiments, a fusion protein as disclosed herein is encoded by a nucleic acid, where the nucleic acid comprises in part, SEQ ID NO: 72 or a nucleic acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least SEQ ID NO: 72, thereby encoding a TrX-cGAS fusion protein. [000201] In some embodiments, a codon-optimized cGAS nucleic acid as disclosed herein (e.g., SEQ ID NO: 1 or 2) is attached to a nucleic acid sequence, that comprises SEQ ID NO: 72Attorney Docket No: AF23853.P206WO or a nucleic acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least SEQ ID NO: 72. [000202] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a fusion protein as disclosed herein, where the codon- optimized nucleic acid sequence is expressed by a plasmid of SEQ ID NO: 73 encoding a TrX- mcGAS fusion protein, where the plasmid comprises a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 72 and SEQ ID NO: 1 (TrX-mcGAS). In some embodiments, the codon-optimized polynucleotide encoding a TrX-mcGAS fusion protein consists of a codon-optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 72 and SEQ ID NO: 1 (TrX-mcGAS). In some embodiments, the codon-optimized polynucleotide encoding a TrX-mcGAS fusion protein as disclosed herein comprises a nucleotide sequence that is 100% identical to a nucleic acid sequence comprising SEQ ID NO: 72 and SEQ ID NO: 1. In some embodiments, the codon-optimized polynucleotide encoding a TrX-mcGAS fusion protein as disclosed herein consists of a nucleic acid sequence comprising SEQ ID NO: 72 and SEQ ID NO: 1 (TrX-mcGAS). [000203] In some embodiments, the present disclosure provides a recombinant cell comprising a codon-optimized polynucleotide encoding a fusion protein as disclosed herein, where the codon- optimized nucleic acid sequence encodes a TrX-hcGAS fusion protein, where the nucleic acid sequence is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 72 and SEQ ID NO: 2 or SEQ ID NO: 23 (TrX-hcGAS). In some embodiments, the codon-optimized polynucleotide encoding a TrX-hcGAS fusion protein consists of a codon- optimized nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence comprising SEQ ID NO: 72 and SEQ ID NO: 2 or SEQ ID NO: 23 (TrX-hcGAS). In some embodiments, the codon-optimized polynucleotide encoding a TrX-hcGAS fusion protein as disclosed herein comprises a nucleotide sequence that is 100% identical to a nucleic acid sequence comprising SEQ ID NO: 72 and SEQ ID NO: 2 or SEQ ID NO: 23 . In some embodiments, the codon-optimized polynucleotide encoding a TrX-hcGAS fusion protein as disclosed herein consists of a nucleic acid sequence comprising SEQ ID NO: 72 and SEQ ID NO: 2 or SEQ ID NO: 23 (TrX-hcGAS).Attorney Docket No: AF23853.P206WO [000204] Additional Tags on the Fusion Proteins [000205] In some embodiments, a fusion protein described herein may comprise an additional tag in addition to the stabilizing SUMO peptide. The N-terminus of SUMO is very flexible and can accommodate a variety of affinity tags (e.g., GST), and therefore the fusion protein comprising a SUMO peptide can therefore be tailored with an additional desired affinity tag. A tag may be N-terminal or C-terminal. For instance, tags may be added to a polypeptide (via additions or modifications on the encoding DNA sequence) to facilitate purification, detection, solubility, or confer other desirable characteristics on the protein. In some embodiments, a tag may be a peptide, oligopeptide, or polypeptide that may be used in affinity purification. In some embodiments, a tag is, comprises, or is derived from one or more of polyhistidine (His), Glutathione S-transferase (GST), tandem affinity purification (TAP), FLAG, myc, human influenza hemagglutinin (HA), maltose binding protein (MBP), vesicular Stomatitis viral glycoprotein (VSV-G), thioredoxin, V5, avidin, streptavidin, biotin carboxyl carrier protein (BCCP), Calmodulin, Nus, S tags, lipoprotein D, and galactosidase. In some embodiments, a His tag is or comprises an amino acid sequence of Hn, wherein n is an integer between 2 and 10. Exemplary His tags include HHHHHH (SEQ ID NO:65) and MSYYHHHHHH (SEQ ID NO:66). In other embodiments, the fusion protein is free of tags such as protein purification tags, and is purified by a method not relying on affinity for a purification tag. In some embodiments, the fusion protein comprises no more than 1, 2, 3, 4, 5, 10, or 20 additional amino acids on one or both termini of a polypeptide of Table 1 or fusion protein of Table 2. [000206] Linkers or Spacers [000207] In some embodiments, a fusion protein comprises one or more linkers. In some embodiments, a linker is or comprises one or more amino acids. In some embodiments, a fusion protein comprises an cGAS polypeptide joined to a SUMO protein, or other Tag as disclosed herein by a linker. In some embodiments, a fusion protein comprises a first cGAS polypeptide and a SUMO protein, and at least one linker. In some embodiments, a fusion protein comprises a first cGAS polypeptide, a second cGAS polypeptide, a SUMO protein, and at least one linker. In some embodiments, the first cGAS polypeptide and the second cGAS polypeptide are joined by a linker. In some embodiments, the first cGAS polypeptide or the second cGAS polypeptide are joined to the SUMO protein by a linker. In some embodiments, the first cGAS polypeptide andAttorney Docket No: AF23853.P206WO the second cGAS polypeptide are joined by a first linker; and the first cGAS polypeptide or the second cGAS polypeptide are joined to the SUMO protein by a second linker. [000208] In some embodiments, a linker interposes a structure between two protein moieties. In some embodiments, the structure is or comprises an α-helix. In some embodiments, the structure is or comprises a β-strand. In some embodiments, the structure is or comprises a coil / bend. In some embodiments, the structure is or comprises a turn. In some embodiments, a linker decreases steric hindrance between two protein moieties joined by the linker. In some embodiments, a linker decreases unfavorable interactions between two protein moieties joined by the linker. In some embodiments, a linker comprises a mixture of glycine and serine residues. In some embodiments, the linker may additionally comprise threonine, proline, and / or alanine residues. In some embodiments, a linker is hydrophilic. In some embodiments, a linker is hydrophobic. In some embodiments, a linker increases the stability of the fusion protein containing the linker. [000209] In some embodiments, a linker does not interfere with the folding of an cGAS polypeptide to which it is joined. In some embodiments, a linker does not interfere with the enzymatic function or activity of an cGAS polypeptide to which it is joined. In some embodiments, a linker does not reduce the enzymatic activity of an cGAS polypeptide to which it is joined. In some embodiments, the effect of the linker is determined by comparing the polypeptide with the polypeptide joined to the linker. [000210] In some embodiments, a linker does not interfere with the folding of a SUMO protein to which it is joined. In some embodiments, a linker does not interfere with the ability of binding of a SUMO activating enzyme (SAE) to the SUMO protein to which it is joined. In some embodiments, a linker does not reduce the ability of binding of a SUMO activating enzyme (SAE) to the SUMO protein to which it is joined. In some embodiments, a linker does not eliminate the ability of a SUMO activating enzyme (SAE) to bind to the SUMO protein to which it is joined. In some embodiments the effect of the linker is determined by comparing the SUMO protein with the SUMO protein joined to the linker. [000211] In some embodiments, a linker comprises two or more amino acids. In some embodiments, a linker may be 3-100, 5-100, 10-100, 20-10030-100, 40-100, 50-100, 60-100, 70- 100, 80-100, 90-100, 5-55, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 3-10, 3-9, 3- 8, 3-7, 3-6, 3-5, 3-4, or 2-3 amino acids in length. In some embodiments, a linker comprisesAttorney Docket No: AF23853.P206WO between 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, or 10-15 amino acids. In some embodiments, the linker comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 amino acids. In some embodiments, a linker is or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids. [000212] In some embodiments, a linker is a flexible linker. Flexible linkers may be useful for joining domains that require a certain degree of movement or interaction and may include small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. Incorporation of Ser or Thr can also maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduce unfavorable interactions between the linker and the protein moieties. In some embodiments, a linker comprises small non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. In some embodiments, a linker is a Gly-Ser linker. [000213] In some embodiments, a linker is or comprises an amino acid sequence of AAA (SEQ ID NO:38). In some embodiments, a linker is or comprises an amino acid sequence of GGGGSSS (SEQ ID NO:37A). In some embodiments, a linker is or comprises a sequence of (GGGGS)n(SEQ ID NO:39), where n represents the number of repeating units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, a polypeptide linker may have an amino acid sequence that is or comprises GGGGSGGGGSGGGGS (SEQ ID NO:41) (i.e., (GGGGS)3 (SEQ ID NO: 41)) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:42) (i.e., (GGGGS)6 (SEQ ID NO: 42)). In some embodiments, a linker comprises one or more of Gly, Ser, Thr, Ala, Lys, and Glu. In some embodiments, a linker is or comprises KESGSVSSEQLAQFRSLD (SEQ ID NO:43). In some embodiments, a linker is or comprises EGKSSGSGSESKST (SEQ ID NO:44). In some embodiments, a linker is or comprises (Gly)n where n represents the number of repeating Gly residues and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, a linker is or comprises (Gly)6 (SEQ ID NO:40). In some embodiments, a linker is or comprises (Gly)8 (SEQ ID NO:46). In some embodiments, a linker is or comprises GSAGSAAGSGEF (SEQ ID NO:47). In some embodiments, a linker is or comprises an amino acid sequence of AAA (SEQ ID NO:38). [000214] In some embodiments, a linker is a rigid linker. Rigid linkers are useful to keep a fixed distance between domains and to maintain their independent functions. Rigid linkers may also be useful when a spatial separation of the domains is critical to preserve the stability orAttorney Docket No: AF23853.P206WO bioactivity of one or more components in the fusion. In some embodiments, a linker is or comprises (EAAAK)n (SEQ ID NO:48) where n represents the number of repeating EAAAK (SEQ ID NO: 48) units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, a linker is or comprises A(EAAAK)nA, (SEQ ID NO:49) where n represents the number of repeating EAAAK (SEQ ID NO: 48) units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, a linker is or comprises A(EAAAK)nA (SEQ ID NO: 108), where n represents the number of repeating EAAAK (SEQ ID NO: 48) units and is 2, 3, 4, or 5. In some embodiments, a linker is or comprises A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO:50). In some embodiments, a linker is or comprises [A(EAAAK)nA]m, (SEQ ID NO:51) wherein n is 2, 3, or 4 and m is 1 or 2. In some embodiments, a linker is or comprises AEAAAKEAAAKA (SEQ ID NO:52). [000215] In some embodiments a linker is or comprises (X-Pro)n with X designating any amino acid, where n represents the number of repeating X-Pro units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, a linker is or comprises (Ala-Pro)n, where n represents the number of repeating Ala-Pro units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, a linker is or comprises (Ala-Pro)n, where n represents the number of repeating Ala-Pro units and is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. [000216] In some embodiments a linker is or comprises (Lys-Pro)n, where n represents the number of repeating Lys-Pro units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, a linker is or comprises (Gln-Pro)n, where n represents the number of repeating Gln-Pro units is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the number of repeating Gln-Pro units is 7. [000217] In some embodiments, a linker is or comprises GAPGGGGGAAAAAGGGGGGAP (GAG linker, SEQ ID NO:58). In some embodiments a linker is or comprises GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP (GAG2 linker, SEQ ID NO:59). In some embodiments, a linker is or comprises GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGG GAP (GAG3 linker, SEQ ID NO:60). [000218] Suitable linkers or spacers also include those having an amino acid sequence of atAttorney Docket No: AF23853.P206WO least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to the above exemplary linkers. [000219] Additional linkers suitable for use with some embodiments may be found in U.S. Patent Publication No.2012 / 0232021, filed on March 2, 2012, and [Chen, 2013] the disclosures of which is hereby incorporated by reference in their entireties. [000220] Certain Definitions [000221] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included. [000222] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value. [000223] Agent: In general, the term “agent”, as used herein, may be used to refer to a compound or entity of any chemical class including, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, or combination or complex thereof. In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and / or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man- made in that it is designed, engineered, and / or produced through action of the hand of man and / or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identifyAttorney Docket No: AF23853.P206WO or characterize active agents within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and / or is substantially free of any polymer and / or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety. [000224] Amino acid: In its broadest sense, the term “amino acid”, as used herein, refers to any compound and / or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N–C(H)(R)–COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Non-standard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide. [000225] Associated with: Two entities are “associated” with one another, as that term is used herein, if the presence, level and / or form of one is correlated with that of the other. In some embodiments, two or more entities are physically “associated” with one another if they interact,Attorney Docket No: AF23853.P206WO directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another. In some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of affinity interactions, electrostatic interactions, hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof. [000226] Binding: It will be understood that the term “binding”, as used herein, typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts – including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system or cell). [000227] Derivative: As used herein, the term “derivative”, or grammatical equivalents thereof, refers to a structural analogue of a reference substance. That is, a “derivative” is a substance that shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. Such a substance would be said to be “derived from” said reference substance. In some embodiments, a derivative is a substance that can be generated from the reference substance by chemical manipulation. In some embodiments, a derivative is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. [000228] Domain: The term “domain” as used herein refers to a section or portion of an entity. In some embodiments, a “domain” is associated with a particular structural and / or functional feature of the entity so that, when the domain is physically separated from the rest of its parent entity, it substantially or entirely retains the particular structural and / or functional feature. Alternatively or additionally, a domain may be or include a portion of an entity that, when separated from that (parent) entity and linked with a different (recipient) entity, substantially retains and / or imparts on the recipient entity one or more structural and / or functional features thatAttorney Docket No: AF23853.P206WO characterized it in the parent entity. In some embodiments, a domain is a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a section of a polypeptide; in some such embodiments, a domain is characterized by a particular structural element (e.g., a particular amino acid sequence or sequence motif, α-helix character, β-sheet character, coiled-coil character, random coil character, etc.), and / or by a particular functional feature (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.). [000229] Endotoxin: The term “Endotoxin” refers to a toxic heat-stable lipopolysaccharide substance present in the outer membrane of gram-negative bacteria that is released from the cell upon lysis. Endotoxins can be generally acidic due to their high content of phosphate and carboxyl residues associated with the core polysaccharide, and can be highly hydrophobic due to the fatty acid content of the lipid-A region. The O-antigen region comprises large numbers of nonionic polysaccharides. [000230] Fragment: A “fragment” of a material or entity as described herein has a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment includes a discrete portion of the whole which discrete portion shares one or more functional characteristics found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a fragment of a polymer, e.g., a polypeptide or polysaccharide, comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) as found in the whole polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the monomeric units (e.g., residues) found in the whole polymer. The whole material or entity may in some embodiments be referred to as the “parent” of the whole. [000231] Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / orAttorney Docket No: AF23853.P206WO RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution. [000232] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percentAttorney Docket No: AF23853.P206WO identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. [000233] Improve, increase, inhibit or reduce: As used herein, the terms “improve”, “increase”, “inhibit’, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single subject) under otherwise comparable conditions absent presence of (e.g., prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment. [000234] Isolated: As used herein, the term “isolated”, or grammatical equivalents thereof, refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure", after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. To give but one example, in some embodiments, aAttorney Docket No: AF23853.P206WO biological polymer such as a polypeptide or polysaccharide that occurs in nature is considered to be "isolated" when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for instance, in some embodiments, a polypeptide or polysaccharide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated" polypeptide or polysaccharide. Alternatively or additionally, in some embodiments, a polypeptide or polysaccharide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide or polysaccharide to the extent that it has been separated from other components a) with which it is associated in nature; and / or b) with which it was associated when initially produced. [000235] Linker: As used herein, the term “linker” is used to refer to an entity that connects two or more elements to form a multi-element agent. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, wherein S1 and S2 may be the same or different and represent two domains associated with one another by the linker (L). In some embodiments, a polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. A variety of different linker elements that can appropriately be used when engineering polypeptides (e.g., fusion polypeptides) are known in the art (Holliger et al, 1993; Poljak, 1994). [000236] “Microorganism” refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell. Examples of microorganisms include bacteria, viruses, parasites, fungi, certain algae, protozoa, and yeast. In some embodiments, the microorganism is modified (“modified microorganism”) from its native state to produce one or more effectors or immune modulators. In certain embodiments, theAttorney Docket No: AF23853.P206WO modified microorganism is a modified bacterium. In some embodiments, the modified microorganism is a genetically engineered bacterium. In certain embodiments, the modified microorganism is a modified yeast. In other embodiments, the modified microorganism is a genetically engineered yeast. [000237] As used herein, the term “recombinant microorganism” refers to a microorganism, e.g., bacterial, yeast, or viral cell, or bacteria, yeast, or virus, that has been genetically modified from its native state. Thus, a “recombinant bacterial cell” or “recombinant bacteria” refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, a recombinant bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Recombinant bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, recombinant bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome. [000238] An “engineered microorganism” refers to a microorganism, e.g., bacterial, yeast, or viral cell, or bacteria, yeast, or virus, that has been genetically modified from its native state to perform a specific function. Thus, a “programmed or engineered bacterial cell” or “programmed or engineered bacteria” refers to a bacterial cell or bacteria that has been genetically modified from its native state to perform a specific function. In certain embodiments, the programmed or engineered bacterial cell has been modified to express one or more proteins, for example, one or more proteins that have a therapeutic activity or serve a therapeutic purpose. The programmed or engineered bacterial cell may additionally have the ability to stop growing or to destroy itself once the protein(s) of interest have been expressed. [000239] “Operably linked” refers a nucleic acid sequence, e.g., a gene encoding a fusion protein as disclosed herien, that is joined to a regulatory region sequence in a manner which allows expression of the nucleic acid sequence, e.g., acts in cis. A regulatory region is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5′ and 3′ untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns.Attorney Docket No: AF23853.P206WO [000240] An “inducible promoter” refers to a regulatory region that is operably linked to one or more genes, wherein expression of the gene(s) is increased in the presence of an inducer of said regulatory region. [000241] “Constitutive promoter” refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and / or to which it is operably linked. Constitutive promoters and variants are well known in the art and non-limiting examples of constitutive promoters are described herein and in International Patent Application PCT / US2017 / 013072, filed Jan. 11, 2017 and published as WO2017 / 123675, the contents of which is herein incorporated by reference in its entirety. In some embodiments, such promoters are active in vitro, e.g., under culture, expansion and / or manufacture conditions. [000242] As used herein, “stably maintained” or “stable” bacterium or virus is used to refer to a bacterial or viral host cell carrying non-native genetic material, e.g., encoding a fusion protein as disclosed herein, such that the non-native genetic material is retained, expressed, and propagated. The stable bacterium or virus is capable of survival and / or growth in vitro, e.g., in medium. For example, the stable bacterium or virus may be a genetically engineered bacterium comprising non-native genetic material encoding a fusion protein, in which the plasmid or chromosome carrying the non-native genetic material is stably maintained in the bacterium or virus, such that the fusion protein can be expressed in the bacterium or virus, and the bacterium or virus is capable of survival and / or growth in vitro. [000243] As used herein, a “non-native” or “heterologous” with respect to a nucleic acid sequence refers to a nucleic acid sequence not normally present in a microorganism, e.g., a heterologous sequence such as a sequence from a different species, strain, or substrain of bacteria or virus, or a sequence that is modified and / or mutated as compared to the unmodified sequence from bacteria or virus of the same subtype. In some embodiments, the non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence or a synthetic codon-optimized sequence. The non-native nucleic acid sequence may be a regulatory region, a promoter, a gene, and / or one or more genes in gene cassette. In some embodiments, “non-native” refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature, such as a nucleic acid encoding a fusion protein as disclosed herein. The non-native nucleic acid sequence may be present on a plasmid or chromosome. In some embodiments, the geneticallyAttorney Docket No: AF23853.P206WO engineered bacteria of the disclosure comprise a gene that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature. [000244] Protein: The term “Protein” refers to any of a group of complex organic macromolecules that contain carbon, hydrogen, oxygen, nitrogen, and usually sulfur and are composed principally of one or more chains of amino acids linked by peptide bounds. The protein may be of natural or recombinant origin. Proteins may be modified with non-amino acid moieties such as through glycosylation, pegylation, or conjugation with other chemical moieties. Examples of proteins include but are not limited to antibodies, clotting factors, enzymes, and peptide hormones. [000245] Host contaminant: The term: “Host contaminant” or “Host cell contaminant” refers to biomolecules that are produced by the cells in which the product of interest is grown. The term may include various classes of host contaminants, such as host proteins and host DNA. [000246] Host protein: “Host protein” or “Host cell protein” or “HCP” refers to proteins that are produced by the cells in which the product of interest is grown. Such proteins represent one class of contaminants that must be removed from the product of interest. [000247] Protein Preparation: the term “Protein preparation” refers to any aqueous or mostly aqueous solution containing a protein of interest, such as a cell-containing cell culture harvest, a (substantially) cell-free cell culture supernatant, or a solution containing the protein of interest from a stage of purification. [000248] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solutionAttorney Docket No: AF23853.P206WO or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces. [000249] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" applied to the carrier, diluent, or excipient used to formulate a composition as disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof. [000250] Polypeptide: The term “polypeptide”, as used herein, generally has its art- recognized meaning of a polymer of at least three amino acids, e.g., linked to each other by peptide bonds. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. [000251] Protein: As used herein, the term “protein” encompasses a polypeptide. Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by oneAttorney Docket No: AF23853.P206WO or more disulfide bonds or associated by other means. Polypeptides may contain l-amino acids, d-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and / or characteristic portions thereof. [000252] Recombinant: As used herein, the term “recombinant” is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and / or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and / or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc.). [000253] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, subject, population, sample, sequence or value of interest is compared with a reference or control agent, animal, subject, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing orAttorney Docket No: AF23853.P206WO determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control. [000254] EXAMPLES [000255] Example 1.1 Plasmid design [000256] The protein sequence encoding full-length mouse cGAS (mcGAS) was downloaded from Genbank (Accession Code: NP_775562). Codon optimization was performed and the mcGAS gene block was synthesized at IDT (Coralville, Iowa). The pET28A (+) plasmid was cut at NdeI and XhoI restriction enzyme cut sites and inserted the gene block sequence comprising a codon-optimized nucleic acid encoding a SUMO peptide and nucleic acid encoding the mcGAS full length gene along with overhangs into the plasmid backbone using Gibson Assembly. Bacterial transformation and plasmid propagation was done via electroporation on electrocompetent E. cloni EC10 cells. [000257] Example 1.2. E. coli strains and Bacterial Cell Culture [000258] Multiple bacterial strains were assessed to examine productivity and yield. The following strains were assessed: E. cloni EC10, which was used for transformation and plasmid propagation, as well as E. coli MG1655 (DE3), E. coli Bl21(DE3) and BL21(DE3) RIL (Agilent Inc.). The bacterial cell culture was performed using these strains similar to a procedure described in a previous study18. Briefly, agar plates containing the appropriate resistance marker were streaked for single colonies, which were then picked and seeded into 5ml modified M9 medium (minimal M9 salts supplemented with 0.8% glucose, 5 mM MgSO4, 0.1 mM CaCl2, and 0.01 mM ferrous sulfate) overnight at 37ºC. The overnight culture was seeded for a target OD600 of 0.05 in a 100ml modified M9 medium. This culture was shaken at 250 rpm at 37ºC and was induced after 6-8 hours at OD600 value of 0.6 to 0.8 with 0.1mM IPTG. Cultures were harvested once OD600 reached a value of 4 at 16-20 hours. The culture was spun at 4000g for 45 mins at 4ºC, the supernatant was filtered using a 0.2 µm filter (VWR LLC, PA) and both cell pellet and supernatant were stored separately for further processing at -80 ºC. [000259] Example 1.3. High-Performance Liquid Chromatography (HPLC)Attorney Docket No: AF23853.P206WO [000260] To estimate the concentration of 2’3’-cGAMP produced in bacteria culture, a HPLC standard curve was generated for various concentrations of 2’3’-cGAMP and its corresponding linear regression equation. The chromatography and peak analysis were done on a Shimadzu HPLC system and SPD-20A UV detector using the software LCsolution (version 1.25), respectively. The commercial standard of 2’3’-cGAMP was obtained from Chemie Tek (Indianapolis, US) and used without further purification. A 20mg / ml stock solution of 2’3’- cGAMP was prepared by dissolving the c-2’3’-cGAMP powder in distilled water and diluting them to produce standard concentrations (7.81, 15.62, 31.25, 62.5, 125, 500, and 1000μM). Each sample was injected with 20 μL volume per HPLC run (UV 256 nm). For the HPLC method, the mobile phases consisted of Acetonitrile (A) and 5 mM Ammonium Acetate (B). The flow rate was maintained at 1 ml / min and the initial composition was 1.5% A and 98.5% B. The concentration gradient for A was then linearly increased up to 10% A at 7.5 mins and to 30% A at 10 minutes. Flow composition returned to 1.5% A at 12.5 min. A 2’3’-cGAMP HPLC standard curve was prepared by plotting the 2’3’-cGAMP concentration vs. the corresponding peak areas (mAU x min). [000261] Example 1.4. Liquid Chromatography–Mass Spectrometry (LC-MS) [000262] To confirm the presence of 2’3’-cGAMP and quantitatively validate our findings for 2’3’-cGAMP productivities, LC-MS was performed using a previously published method19.2’3’- cGAMP from Chemie Tek (Indianapolis, US) was used as an internal standard at 10-100 μg / ml. Samples were analyzed for 2’3-cGAMP content on a Shimadzu HPLC with an autosampler set at 4 °C and connected to an AB Sciex QTRAP®4000 column. A volume of 10 μL was injected into a Biobasic®AX LC column (5 μm, 50 × 3 mm; ThermoFisher Scientific, MA). The mobile phase consisted of 100 mM ammonium carbonate (A) and 0.1% (v / v) formic acid in acetonitrile (B). The initial condition was 90% B, maintained for 0.5 min. The mobile phase was ramped linearly to 30% A from 0.5 min to 2.0 min, maintained at 30% A from 2.0 min to 3.5 min, ramped linearly to 90% B from 3.5 min to 3.6 min, and maintained at 90% B from 3.6 min to 5 min. The flow rate for samples was set to 0.6 ml / min. The mass spectrometer was operated in electrospray positive- ion (ESI) mode with the source temperature set at 500 °C. Declustering and collision-induced dissociation were achieved with nitrogen gas at a medium flow rate. Declustering and collision- induced dissociation were achieved with nitrogen gas. For each molecule, the MRM transition(s) (m / z) are mentioned in Table 4.Attorney Docket No: AF23853.P206WO [000263] Table 4. MRM transition(s) (m / z) parameters for 2'3'- cGAMP. MRM pair DP Collison Collision Collision CexpOptimized MW (Da) Energy 1 Energy 2 Energy 3 XIC (eV) (eV) (eV) (*10^5) (eV) 136 43 44 48 9 3 312 54 55 55 13 9 330 43 42 40 6 4 190 476 38 39 38 12 6 523 30 29 29 30 11 152 41 39 40 10 3.9 [000264] Example 1.5. Anion Exchange Chromatography (AXE) [000265] The present disclosure pertains to use of an efficient ion exchange chromatography step to separate 2’3’-cGAMP into a high-purity fraction using a single step. Towards this, the present disclosure used an anion exchange chromatography technique with an increasing salt concentration gradient20. Without wishing to be bound by theory, the chromatography method consisted of (i) Buffer A (20 mM Tris, pH 7.5) for loading and washing; and (ii) Buffer B (20 mM Tris, NaCl (50- 125 mM), pH 7.5). The filtered bacterial supernatant (obtained after centrifugation at 4000g for 45mins at 4ºC and filtered through 0.2 µm filter) was mixed with buffer A in a 1:5 volume ratio to reduce the conductivity of the load to 5 mS / cm. A HighQ® resin (Bio-Rad, CA) column (1 ml) was equilibrated with 25 column volumes (CV) of buffer A on an Akta® Explorer system (Shimadzu) and monitored using a UV detector (256 nm). The diluted supernatant (25 CV) was then loaded into the column. The column was washed over with buffer A (25 CV) to remove impurities. Incremental concentrations of NaCl in buffer B (50mM NaCl, 12.5 CV; 100mM NaCl, 10 CV) were applied to selectively elute 2’3’-cGAMP. All chromatography steps were carried out at a flow rate of 0.5 ml / min. [000266] Example 1.6. Lipopolysaccharide measurement and removal [000267] Amicon® Ultra 3kDa Molecular Weight Cut Off (MWCO) filters (MilliPore Sigma, MA) were used according to manufacturers’ instructions to reduce LPS concentration from chromatography concentrated eluates. Applicant measured the LPS concentrations at variousAttorney Docket No: AF23853.P206WO stages of the production and purification of 2’3’-cGAMP using the Pierce® Endotoxin kit (ThermoFisher, NJ). Standards for 1.0, 0.5, 0.25, and 0.1 EU / ml were prepared along with the standard curve; after which dilutions of samples (10x, 100x, 1000x) were prepared with endotoxin- free water provided with the kit. Following the manufacturer’s instructions, samples were prepared and LPS concentrations were measured for samples. Absorbance readings were taken at 405 nm and the readings which lie in the value range for the standard curve were noted. The actual concentrations of LPS in the samples were extrapolated using a linear regression equation generated from the standard curve. [000268] Example 1.7. Method of 2’3-cGAMP production and collection [000269] This Example outlines the method for 2’3-cGAMP production and collection from recombinant bacterial cells. In this Example, an exemplary fusion protein comprising mcGAS and SUMO is expressed by a bacterial cell and 2’3-cGAMP is secreted into the culture media, the culture media supernatant is filtered on 0.2 micro-M and the 2’3’-cGAMP is collected after Anion Exchange Chromatography (AXE) of the filtered supernatant. The collected elutant fraction from the AXE is substantially free from endotoxin contamination and endotoxin removal does not need to be performed. [000270] A bacterial cell is transformed with a nucleic acid encoding a fusion protein comprising cGAS fused to SUMO. In particular, a bacterial cell is transformed with an expression vector (e.g., plasmid) comprising a codon-optimized nucleic acid encoding a SUMO-hcGAS or SUMO-mcGAS fusion protein. Exemplary nucleic acids are disclosed in Table 2. It is envisioned that one of ordinary skill in the art can readily substitute the nucleic acids of SUMO-mcGAS or SUMO-hcGAS nucleic acid sequence with any of those disclosed in Table 2, or variants thereof (e.g., including the plasmids of SEQ ID NO: 32, 34, 71 and 73), or alternative tag proteins, as disclosed herein. [000271] Referring to FIGS. 1A-1C4, recombinant bacterial cells comprising a heterologous nucleic acid sequence encoding cGAS fused to SUMO are cultured in a defined culture media for a sufficient period of time for expression of cGAS, and production and secretion of 2’3-cGAMP into the culture media. The culture media is collected and centrifuged to separate the supernatant from the bacterial cell pellet. The supernatant is processed through anion exchange chromatography (AEX), and the elutant fractions comprising 2’3-cGAMP are collected. The collected fractions are concentrated and filtered through a 3kDa filtration. AsAttorney Docket No: AF23853.P206WO shown in FIGS. 2A-2E, 2’3-cGAMP is readily produced and is comparable to chemically synthesized 2’3-cGAMP. [000272] Example 1.8. Optimization of culture conditions and media for 2’3-cGAMP production [000273] 2’3-cGAMP yield was assessed in the presence of different culture media components, including the presence of Mn2+ or Mg2+ divalent cations (FIGS. 3A-3C), which unexpectedly decreased 2’3-cGAMP yield (FIG. 3E). Surprisingly, yield of 2’3-cGAMP production was dependent on specific culture conditions, with M9 and 2xYT producing highest 2’3-cGAMP yield as compared to in the presence of divalent cations Mg2+, Mn2+ and Zn2+, which negatively impacted 2’3-cGAMP yield (FIG.3E). This is surprising given Mn2+ has been reported to be an essential metal cofactor to activate the catalytic domain of cGAS and induce the catalytic synthesis of 2’3’-cGAMP (Zhao et al., Cell, 2020; 32(7); 108083), and that DNA- activated cGAS uses magnesium (Mg2+ ) as its catalytic cofactor to catalyze the conversion of ATP and GTP into 2’ 3’-cGAMP with 2’ 5’ and 3’ 5’-phosphodiester linkages (Gao et al.,2013b). [000274] cGAMP yield was assessed with different bacterial strains in the presence and absence of dsDNA inducer. E. Coli bacterial strains BL21(DE3) produced the highest yield of 2’3-cGAMP (FIG.3D). [000275] cGAMP yield was also assessed in different inducer (IPTG) concentrations (FIG. 3F), temperature (FIG.3G), optical density (OD) (FIG.3H) and plasmid concentrations (FIG. 3I). Interestingly, 2’3-cGAMP is produced at temperatures as low as 20 ºC (FIG.3G), therefore demonstrating that 2’3-cGAMP can be produced at high yields in an energy efficient manner without the need for high-temperature or high-energy bioreactors. [000276] Example 1.9. Optimization of expressed cGAS and cGAS modifications [000277] cGAMP yield was also assessed with variants of cGAS. In particular, the present disclosure compared the 2’3-cGAMP yield of wild-type mouse cGAS (wt-mcGAS) as compared to modified mouse cGAS variants, e.g., comprising the catalytic domain and has the immunomodulatory domain removed (mcGAS-ISD), a R222E, R241E modifications or where the plasmid comprises a pUC19 origin of replication (ori) (FIGS. 4A-4H). However, these specific modifications to mcGAS negatively impacted 2’3-cGAMP production (FIGS.4H-4J), demonstrating that modifications can be detrimental to 2’3-cGAMP production. [000278] Example 1.10. Yield of 2’3-cGAMP and minimal presence of endotoxinAttorney Docket No: AF23853.P206WO [000279] The cGAS produced 2’3-cGAMP was assessed at different stage of purification (FIG.5A-5E). FIG.5C shows high yield and high purity of 2’3-cGAMP harvested in the elutant fraction (100mM NaCl), with 80% of 2’3-cGAMP recovered from the elutant fraction (FIG.5B). Fractions 1-5 contained minimal amounts of 2’3-cGAMP (between 1.3-4.8%), with the greatest 2’3-cGAMP yield collected in the elutant fractions with either 50nM and 100nM NaCl Wash A + B fractions (38% and 42%, respectively) (FIG.5D). [000280] Analysis of LPS endotoxin demonstrated that the elutant fraction from 100nM NaCl had minimal LPS, which constituted 42% of the total yield (FIG. 5E). Together, as shown in FIGS.5B, 5C and 5E the present disclosure pertains to that expression of recombinant cGAS in a cell (e.g., bacterial cell), which can be used to produce 2’3-cGAMP, and which can be collected from the supernatant to produce a high yield of 2’3-cGAMP with minimal endotoxin contamination, and importantly, without the need for the extra method step to remove endotoxin from yielded 2’3-cGAMP. [000281] The present disclosure pertains to a method and composition for a high final yield of production of purified 2’3-cGAMP with < 20 EU / ml endotoxin. The present disclosure also pertains to the production of 2’3-cGAMP at a final yield of 58.8 ± 2.1 mg / L with < 20 EU / mL of endotoxin.Attorney Docket No: AF23853.P206WO References 1 Hopfner, K. P. & Hornung, V. Molecular mechanisms and cellular functions of cGAS-STING signalling. Nat Rev Mol Cell Biol 21, 501-521 (2020). https: / / doi.org:10.1038 / s41580-020-0244-x 2 Chen, C. & Xu, P. Cellular functions of cGAS-STING signaling. Trends Cell Biol (2022). https: / / doi.org:10.1016 / j.tcb.2022.11.001 3 Ritchie, C., Carozza, J. A. & Li, L. Biochemistry, Cell Biology, and Pathophysiology of the Innate Immune cGAS-cGAMP-STING Pathway. Annu Rev Biochem 91, 599-628 (2022). https: / / doi.org:10.1146 / annurev-biochem-040320-101629 4 Barber, G. N. STING: infection, inflammation and cancer. Nat Rev Immunol 15, 760-770 (2015). https: / / doi.org:10.1038 / nri3921 5 Ritchie, C., Cordova, A. F., Hess, G. T., Bassik, M. C. & Li, L. SLC19A1 Is an Importer of the Immunotransmitter cGAMP. Mol Cell 75, 372-381 e375 (2019). https: / / doi.org:10.1016 / j.molcel.2019.05.006 6 Carozza, J. A. et al. Extracellular cGAMP is a cancer cell-produced immunotransmitter involved in radiation-induced anti-cancer immunity. Nat Cancer 1, 184-196 (2020). https: / / doi.org:10.1038 / s43018-020-0028-4 7 Wu, Y. T. et al. Tumor-targeted delivery of a STING agonist improvescancer immunotherapy. Proc Natl Acad Sci U S A 119, e2214278119 (2022). https: / / doi.org:10.1073 / pnas.2214278119 8 Petrovic, M., Borchard, G. & Jordan, O. Considerations for the delivery of STING ligands in cancer immunotherapy. J Control Release 339, 235-247 (2021). https: / / doi.org:10.1016 / j.jconrel.2021.09.033 9 Gogoi, H., Mansouri, S. & Jin, L. The Age of Cyclic Dinucleotide Vaccine Adjuvants. Vaccines (Basel) 8 (2020). https: / / doi.org:10.3390 / vaccines8030453 10 An, X. et al. Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2. iScience 24, 103037 (2021). https: / / doi.org:10.1016 / j.isci.2021.103037 11 Bartsch, T., Becker, M., Rolf, J., Rosenthal, K. & Lutz, S. Biotechnological production of cyclic dinucleotides-Challenges and opportunities. Biotechnol Bioeng 119, 677- 684 (2022). https: / / doi.org:10.1002 / bit.28027Attorney Docket No: AF23853.P206WO 12 Gaffney, B. L., Veliath, E., Zhao, J. & Jones, R. A. One-flask syntheses of c-di- GMP and the [Rp,Rp] and [Rp,Sp] thiophosphate analogues. Org Lett 12, 3269-3271 (2010). https: / / doi.org:10.1021 / ol101236b 13 Becker, M., Nikel, P., Andexer, J. N., Lutz, S. & Rosenthal, K. A Multi-Enzyme Cascade Reaction for the Production of 2'3'-cGAMP. Biomolecules 11 (2021). https: / / doi.org:10.3390 / biom11040590 14 Becker, M., Lutz, S. & Rosenthal, K. Environmental Assessment of Enzyme Production and Purification. Molecules 26 (2021). https: / / doi.org:10.3390 / molecules26030573 15 Rolf, J., Siedentop, R., Lutz, S. & Rosenthal, K. Screening and Identification of Novel cGAS Homologues Using a Combination of in Vitro and In Vivo Protein Synthesis. Int J Mol Sci 21 (2019). https: / / doi.org:10.3390 / ijms21010105 16 Rosenthal, K. et al. Catalytic Promiscuity of cGAS: A Facile Enzymatic Synthesis of 2'-3'-Linked Cyclic Dinucleotides. Chembiochem 21, 3225-3228 (2020). https: / / doi.org:10.1002 / cbic.202000433 17 Lv, Y. et al. Highly Efficient Preparation of Cyclic Dinucleotides via Engineering of Dinucleotide Cyclases in Escherichia coli. Front Microbiol 10, 2111 (2019). https: / / doi.org:10.3389 / fmicb.2019.02111 18 Lv, Y. et al. Highly Efficient Preparation of Cyclic Dinucleotides via Engineering of Dinucleotide Cyclases in Escherichia coli. Frontiers in Microbiology 10 (2019). 19 Carozza, J. A. et al. Extracellular cGAMP is a cancer cell-produced immunotransmitter involved in radiation-induced anti-cancer immunity. Nat Cancer 1, 184-196 (2020). https: / / doi.org:10.1038 / s43018-020-0028-4 20 Holleufer, A. & Hartmann, R. A Highly Sensitive Anion Exchange Chromatography Method for Measuring cGAS Activity in vitro. Bio-protocol 8, e3055 (2018). https: / / doi.org:10.21769 / BioProtoc.3055 21. Zhao, et al., Mn2+ Directly Activates cGAS and Structural Analysis Suggests Mn2+ Induces a Noncanonical Catalytic Synthesis of 2′3′-cGAMP. Cell Reports, Volume 32, Issue 7, 2020, 108053. 22. Gao, P., et al. (2013b). Cyclic [G(2’,5’)pA(3’,5’)p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell 153, 1094–1107. 23. WO2019123414A1Attorney Docket No: AF23853.P206WO 4. CN108913737B 5. US20200149053 Equivalents [000282] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

Attorney Docket No: AF23853.P206WO CLAIMS 1. A nucleic acid sequence comprising a promoter operatively linked to a heterologous nucleotide sequence encoding a cyclic GMP–AMP synthase (cGAS) polypeptide, wherein the nucleic acid sequence comprises a codon optimized cGAS nucleic acid sequence selected from SEQ ID NO: 1 (mcGAS), SEQ ID NO: 2 (hcGAS), SEQ ID NO: 23 (hcGAS), or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

23.

2. The nucleic acid sequence of claim 1, wherein the nucleotide sequence also encodes a stabilizing peptide, wherein the expressed stabilizing peptide forms a fusion protein with the expressed cGAS polypeptide in any order.

3. The nucleic acid sequence of claim 2, wherein the stabilizing peptide is selected from the group consisting of a small ubiquitin modifying protein (SUMO) polypeptide, a maltose-binding protein (MBP), N-utilization substance (NusA), Thioredoxin (Trx), disulfide bond C (DsbC), glutathione S-transferase (GST), ubiquitin (Ub), fragments thereof, or combinations thereof.

4. The nucleic acid sequence of claim 2, wherein the stabilizing peptide comprises a small ubiquitin modifying protein (SUMO) polypeptide.

5. The nucleic acid sequence of claim 2, further comprising a nucleic acid sequence encoding a linker located between the nucleic sequence encoding the cGAS polypeptide and the stabilizing peptide.

6. The nucleic acid sequence of claim 1, wherein the promoter comprises an inducible promoter.

7. The nucleic acid sequence of claim 1, wherein the nucleic acid sequence is selected from the group consisting of: a nucleic acid comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 1, and a nucleicAttorney Docket No: AF23853.P206WO acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5 (co-mcGAS-SUMO); a nucleic acid comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO:

1. (SUMO-mcGAS); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 23 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO:

23. (SUMO-co1-hcGAS); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 23 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 23, and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO:

5. (co1-hcGAS-SUMO); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 2 (SUMO-co2-hcGAS); or a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 2, and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5 (co2-hcGAS-SUMO).

8. The nucleic acid of claim 1, wherein the nucleic acid is present in an expression vector.

9. A recombinant cell, wherein the recombinant cell expresses a heterologous nucleotide sequence encoding a cyclic GMP–AMP synthase (cGAS) polypeptide, wherein the nucleic acid sequence comprises a codon optimized cGAS nucleic acid sequence selected from SEQ ID NO: 1 (mcGAS), SEQ ID NO: 2 (hcGAS), SEQ ID NO: 23 (hcGAS), or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 23.Attorney Docket No: AF23853.P206WO 10. The recombinant cell of claim 9, wherein the nucleotide sequence also encodes a stabilizing peptide, wherein the expressed stabilizing peptide forms a fusion protein with the expressed cGAS polypeptide in any order.

11. The recombinant cell of claim 10, wherein the stabilizing peptide is selected from the group consisting of a small ubiquitin modifying protein (SUMO) polypeptide, a maltose-binding protein (MBP), N-utilization substance (NusA), Thioredoxin (Trx), disulfide bond C (DsbC), glutathione S-transferase (GST), ubiquitin (Ub), fragments thereof, or combinations thereof.

12. The recombinant cell of claim 11, wherein the stabilizing peptide comprises a small ubiquitin modifying protein (SUMO) polypeptide.

13. The recombinant cell of claim 9, wherein the nucleic acid sequence is selected from the group consisting of: a nucleic acid comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 1, and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5 (co-mcGAS-SUMO); a nucleic acid comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO:

1. (SUMO-mcGAS); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 23 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO:

23. (SUMO-co1-hcGAS); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 23 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 23,Attorney Docket No: AF23853.P206WO and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO:

5. (co1-hcGAS-SUMO); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 2 (SUMO-co2-hcGAS); or a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 2, and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5 (co2-hcGAS-SUMO).

14. The recombinant cell of claim 9, wherein the nucleic acid is present in an expression vector.

15. The recombinant cell of claim 9, wherein the cell is a bacterial cell.

16. The recombinant cell of claim 15, wherein the bacterial cell is E. coli.

17. The recombinant cell of claim 9, wherein the recombinant cell produces a yield of 2’3’- cGAMP which is at least 30% higher than the yield of 2’3’-cGAMP produced from a cell expressing a cGAS polypeptide encoded by a nucleic acid that is at least 98% identical to SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

25.

18. The recombinant cell of claim 9, wherein the recombinant cell produces a yield of 2’3’- cGAMP which is at least 30% higher than the yield of 2’3’-cGAMP produced from a cell expressing a cGAS polypeptide having an amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO:

24.

19. The recombinant cell of claim 9, wherein the recombinant cell produces a final yield of 2’3’- cGAMP which has less than 20EU / ml endotoxin.

20. A method of producing 2’3’ cyclic GMP–AMP (2’3’-cGAMP), said method comprising:Attorney Docket No: AF23853.P206WO (a) culturing a recombinant cell in a culture media, wherein the recombinant cell comprises a nucleotide sequence expressing a cyclic GMP–AMP synthase (cGAS) polypeptide, and wherein the culturing induces the expression and activation of the cGAS polypeptide; (b) collecting the culture media; (c) performing anion exchange chromatography (AEC) of the collected culture media; and (d) collecting the elutant from an AEC column, wherein the elutant comprises the 2’3’- cGAMP.

21. The method of claim 20, wherein the collecting step (d) comprises eluting the 2’3’-cGAMP from the AEC column.

22. The method of claim 20, wherein the method does not require protein purification of the cGAS polypeptide using protein affinity chromatography.

23. The method of claim 20, wherein the method does not require endotoxin removal from the elutant.

24. The method of claim 20, wherein the elutant comprises less than 20EU / ml endotoxin.

25. The method of claim 20, further comprising a step of (e) filtering the elutant.

26. The method of claim 20, wherein activation of the cGAS polypeptide comprises activation by double stranded DNA (dsDNA).Attorney Docket No: AF23853.P206WO 27. The method of claim 20, wherein the culture media is supplemented with at least one divalent cation, wherein the at least one divalent cation is selected from the group consisting of Zn, Mn, and Mg.

28. The method of claim 20, wherein the recombinant cell produces a yield of 2’3’-cGAMP which is at least 30% higher than the yield of 2’3’-cGAMP produced from a cell expressing a cGAS polypeptide encoded by a nucleic acid that is at least 98% identical to SEQ ID NO: 9 or SEQ ID NO:

10.

29. The method of claim 20, wherein the recombinant cell produces a yield of 2’3’-cGAMP which is at least 50% higher than the yield of 2’3’-cGAMP produced from a cell expressing a cGAS polypeptide encoded by a nucleic acid that is at least 98% identical to SEQ ID NO: 9 or SEQ ID NO:

10.

30. The method of claim 20, wherein the nucleic acid sequence comprises a codon optimized cGAS nucleic acid sequence selected from SEQ ID NO: 1 (mcGAS), SEQ ID NO: 2 (hcGAS), SEQ ID NO: 23 (hcGAS), or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

23.

31. The method of claim 20, wherein the nucleotide sequence also encodes a stabilizing peptide, wherein the expressed stabilizing peptide forms a fusion protein with the expressed cGAS polypeptide in any order.

32. The method of claim 31, wherein the stabilizing peptide is selected from the group consisting of a small ubiquitin modifying protein (SUMO) polypeptide, a maltose-binding protein (MBP), N- utilization substance (NusA), Thioredoxin (Trx), disulfide bond C (DsbC), glutathione S- transferase (GST), ubiquitin (Ub), fragments thereof, or combinations thereof.

33. The method of claim 31, wherein the stabilizing peptide comprises a small ubiquitin modifying protein (SUMO) polypeptide.Attorney Docket No: AF23853.P206WO 34. The method of claim 31, wherein the nucleic acid sequence is selected from the group consisting of: a nucleic acid comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 1, and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5 (co-mcGAS-SUMO); a nucleic acid comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO:

1. (SUMO-mcGAS); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 23 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO:

23. (SUMO-co1-hcGAS); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 23 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 23, and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO:

5. (co1-hcGAS-SUMO); a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5, and a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 2 (SUMO-co2-hcGAS); or a nucleic acid sequence comprising, in the following order, a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 2, and a nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence of at least 85% sequence identity to SEQ ID NO: 5 (co2-hcGAS-SUMO).

35. A fusion protein comprising a cyclic GMP–AMP synthase (cGAS) polypeptide and a stabilizing peptide.Attorney Docket No: AF23853.P206WO 36. The fusion protein of claim 35, wherein the cGAS polypeptide is produced by the expression of a nucleic acid sequence comprising a codon optimized cGAS nucleic acid sequence selected from SEQ ID NO: 1 (mcGAS), SEQ ID NO: 2 (hcGAS), SEQ ID NO: 23 (hcGAS), or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

23.

37. The fusion protein of claim 35, wherein the cGAS polypeptide comprises a sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 3, or SEQ ID NO:

4.

38. The fusion protein of claim 35, further comprising a linker located between the nucleic sequence encoding the cGAS polypeptide and the stabilizing peptide.

39. The fusion protein of claim 35, wherein the stabilizing peptide is selected from the group consisting of a small ubiquitin modifying protein (SUMO) polypeptide, a maltose-binding protein (MBP), N-utilization substance (NusA), Thioredoxin (Trx), disulfide bond C (DsbC), glutathione S-transferase (GST), ubiquitin (Ub), fragments thereof, or combinations thereof.

40. The fusion protein of claim 35, wherein the stabilizing peptide comprises a small ubiquitin modifying protein (SUMO) polypeptide.

41. The fusion protein of claim 40, wherein the SUMO polypeptide is produced by the expression of a nucleic acid sequence selected from SEQ ID NO: 5 or SEQ ID NO: 6, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 5 or SEQ ID NO:

6.

42. The fusion protein of claim 40, wherein the SUMO polypeptide comprises a sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 7, or SEQ ID NO: 8.

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