Compositions and methods for expression of il-1ra and il-18bp

Abstract

The present disclosure relates to modified viral genomes or self-replicating RNAs (srRNAs) and pharmaceutical compositions containing the same, as well as the use of such nucleic acid molecules and compositions for production of desired products in cell cultures or in a living body. Also provided are methods for modulating a pharmacodynamic effect in a subject in need thereof, as well as methods for preventing and / or treating various health conditions and diseases.

Description

COMPOSITIONS AND METHODS FOR EXPRESSION OF IL-IRA AND IL-18BPCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 504,564, filed on May 26, 2023. The disclosure of the above-referenced application is herein expressly incorporated by reference it its entirety, including any drawings.INCORPORATION OF THE SEQUENCE LISTING

[0002] The material in the accompanying Sequence Listing is hereby incorporated by reference into this application. The accompanying Sequence Listing XML file, named 058462_515001WO_Sequence_Listing.XML, was created on May 21, 2024, and is approximately 40,960 bytes in size.FIELD

[0003] The present disclosure relates to the field of immunology, and particularly relates to modified viral genomes or self-replicating RNAs (srRNAs) and pharmaceutical compositions containing the same, as well as the use of such nucleic acid molecules and compositions for production of desired products in cell cultures or in a living body. Also provided are methods for modulating a pharmacodynamic effect in a subject in need thereof, as well as methods for preventing and / or treating various health conditions and diseases.BACKGROUND

[0004] Autoimmune and inflammatory diseases are mediated by several pro-inflammatory cytokines. Blocking one or more of these cytokines may have a significant effect on the course and symptoms of these diseases. A number of therapeutic compositions and methods have developed for the treatment of these diseases. However, many of them are not satisfactory due to poor efficacy, side effects, and / or instability.

[0005] For example, interleukin-1 receptor antagonist protein (IL-IRA) can be used in many pharmaceutical compositions and therapeutic methods for the treatment of rheumatoid arthritis, an autoimmune disease in which interleukin 1 (IL-1) plays a key role, reducing inflammation and cartilage degradation associated with the disease. In another example,interleukin-18 binding protein (IL-18BP) is considered a potent IL-18 inhibitor. In particular, IL- 18BP reduces the severity of several experimental autoimmune diseases. Therefore, IL-18BP is believed to function as a natural anti-inflammatory and immunosuppressive molecule neutralizing the effects of high IL-18 levels during inflammation. Recombinant human IL-18BP has been used for a phase II clinical trial in Europe to treat adult-onset Still’s disease patients and a phase III clinical trial with the experimental drug IL-18BP in patients carrying a mutation of the NOD-like receptor C4 (NLRC4) gene characterized by severe, life-threatening systemic inflammation associated with extremely high levels of IL-18.

[0006] However, many such existing methods and compositions are associated with issues regarding stability and half-life of IL- IRA and IL-18BP, as well as the amount and rate of IL- 1RA and / or IL-18BP provided. As a result, improved compositions and methods for delivering IL-IRA and / or IL-18BP are desirable and would be useful in treating conditions and pathologies mediated by the interleukin- 1 and interleukin- 18 signaling, or aberrant inflammasome activation.

[0007] The disclosure provided here provides, inter alia, solutions to various problems existing with previous attempts to deliver IL-IRA and / or IL-18BP and offers improved methods for treatment of health conditions including autoimmune and inflammatory diseases.SUMMARY

[0008] The present disclosure relates generally to the development of immunotherapeutics, such as recombinant nucleic acids constructs and pharmaceutical compositions including the same for use in the prevention and management of various health conditions such as autoimmune, inflammatory, and cardiovascular diseases. In particular, as described in greater detail below, some embodiments of the disclosure provide nucleic acid constructs containing sequences that encode a modified genome or replicon, e.g., self-replicating RNA (srRNA), e.g., replicons, in which at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or replicon (e.g., srRNA) has been replaced by a coding sequence for a polypeptide construct comprising (a) a coding sequence for an interleukin- 1 receptor antagonist (IL-IRA) protein or a functional variant thereof; and (b) a coding sequence for an interleukin- 18 binding protein (IL-18BP) wherein the coding sequences for IL-IRA and IL-18BP are operably linked to one another, and wherein the polypeptide construct does not include a dimerization domain. Also disclosed are recombinant cells that have been engineeredto include one or more of the nucleic acid constructs disclosed herein, and pharmaceutical compositions including one or more of the following (a) a nucleic acid construct of the disclosure and / or (b) a recombinant cell of the disclosure. Further provided in particular aspects of the disclosure are compositions and methods for modulating at least one pharmacodynamic effect in a subject, and methods for preventing and / or treating various health conditions, including autoimmune, inflammatory, and cardiovascular diseases.

[0009] In one aspect of the disclosure, provided herein are nucleic acid constructs including a nucleic acid sequence encoding a modified alphavirus genome or replicon, e.g., selfreplicating RNA (srRNA), wherein at least a portion of the nucleic acid sequence encoding one or more viral structural proteins of the modified alphavirus genome or replicon (e.g., srRNA) has been replaced with a coding sequence for a polypeptide construct including: (a) a coding sequence for an interleukin- 1 receptor antagonist (IL- IRA) protein or a functional variant thereof; and (b) a coding sequence for an interleukin- 18 binding protein (IL-18BP) or functional variant thereof; wherein the coding sequences for IL-IRA and IL-18BP are operably linked to one another, and wherein the polypeptide construct does not include a dimerization domain.

[0010] Non-limiting exemplary embodiments of the nucleic acid constructs of the disclosure (for example, replicon constructs, e.g., srRNA constructs) can include one or more of the following features. In some embodiments, the polypeptide construct does not include a fragment crystallization region (Fc region) of an immunoglobulin. In some embodiments, the coding sequence for IL-IRA is N-terminally linked to the coding sequence for IL-18BP. In some embodiments, the coding sequence for IL-IRA is C-terminally linked to the second coding sequence for IL-18BP. In some embodiments, the coding sequences for IL-IRA and IL-18BP express proteins that are functional in a bioactivity assay. In some embodiments, the IL-18BP protein is the IL-18BP isoform a (IL-18BPa). In some embodiments, the IL-IRA protein and / or IL-18BP protein are from a mammalian subject. In some embodiments, the mammalian subject is a human subject.

[0011] In some embodiments of the disclosure, the coding sequences for IL-IRA and / or IL-18BP are independently optimized for one of more of the following: (a) enhancing RNA stability and / or expression level; (b) minimizing rare codon usage and / or secondary structures; and (c) facilitating better srRNA replication and RNA manufacturing process.

[0012] In some embodiments, the coding sequences for IL-IRA and IL-18BP in the nucleic acid constructs disclosed herein are operably linked to one another via a connector sequence encoding an autoproteolytic peptide and / or an internal ribosomal entry site (IRES). In some embodiments, the autoproteolytic peptide includes one or more autoproteolytic cleavage sequences from a calcium-dependent serine endoprotease (furin), a porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), or a combination thereof. In some embodiments, the internal ribosomal entry site (IRES) is from a Kaposi’s sarcoma-associated herpesvirus (KSHV) IRES, a hepatitis virus IRES, a Pestivirus IRES, a Cripavirus IRES, a Rhopalosiphum padi virus IRES, a fibroblast growth factor IRES, a platelet-derived growth factor IRES, a vascular endothelial growth factor IRES, an insulin-like growth factor IRES, a picomavirus IRES, an encephalomyocarditis virus (EMCV) IRES, a Pim-1 IRES, a p53 IRES, an Apaf-1 IRES, a TDP2 IRES, an L-myc IRES, and a c-myc IRES.

[0013] In some embodiments of the nucleic acid constructs disclosed herein, the modified viral genome or srRNA is devoid of a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. In some embodiments, the modified alphavirus genome or srRNA includes no nucleic acid sequence encoding viral structural proteins. In some embodiments, the coding sequences for IL-IRA and IL-18BP are operably linked to one another within a single open reading frame (i.e., in a polycistronic ORF).

[0014] In some embodiments, the nucleic acid sequence encoding the polypeptide construct is operably linked to a promoter sequence. In some embodiments, the promoter sequence is a subgenomic (sg) promoter. In some embodiments, the sg promoter sequence is a 26S subgenomic promoter. In some embodiments, the subgenomic promoter is heterologous relative to the remainder of the modified viral genome or srRNA. In some embodiments, the subgenomic promoter is an alphavirus subgenomic promoter.

[0015] In some embodiments of the disclosure, at least one nonstructural protein (nsP), or a portion thereof, of the modified viral genome or srRNA is heterologous relative to the remainder of the modified viral genome or srRNA. In some embodiments, the nucleic acid constructs disclosed herein further include a nucleic acid sequence encoding a heterologous nsP or a portionthereof. Tn some embodiments, the nucleic acid constructs disclosed herein further include one or more untranslated regions (UTRs). In some embodiments, at least one of the UTRs is a heterologous UTR.

[0016] In some embodiments of the disclosure, the modified alphavirus genome or srRNA is of an alphavirus belonging to the Venezuelan equine encephalitis virus / Eastem Equine Encephalitis virus (VEEV / EEEV) group, or the Semliki Forest virus (SFV) group, or the Sindbis virus (SINV) group. In some embodiments, the modified alphavirus genome or srRNA is of an alphavirus belonging to the BFV complex, EEEV complex, MIDV complex, NDUV, complex, SFV complex, VEEV complex, WEEV complex. In some embodiments, the alphavirus is Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O’Nyong-Nyong vims (ONNV), Ross River vims (RRV), Barmah Forest vims (BF), Getah vims (GET), Sagiyama vims (SAGV), Bebam vims (BEBV), Mayaro vims (MAYV), Una vims (UNAV), Sindbis vims (SINV), Aura vims (AURAV), Whataroa vims (WHAV), Babanki vims (B ABV), Kyzylagach vims (KYZV), Western equine encephalitis vims (WEEV), Highland J vims (HJV), Fort Morgan vims (FMV), Ndumu vims (NDUV), Madariaga vims (MADV), or Buggy Creek vims. In some embodiments, the alphavims is VEEV, EEEV, CHIKV, or SINV.

[0017] In some embodiments of the disclosure, the coding sequence for the polypeptide constmct includes, in 5’ to 3’ direction (i.e., in N-terminus to C-terminus direction of the polypeptide sequence): (a) (i) a coding sequence for IL- IRA, (ii) a connector sequence encoding an IRES, and (iii) a coding sequence for IL-18BP; (b) (i) a coding sequence for IL-IRA, (ii) a connector sequence encoding a P2A autoproteolytic peptide, and (iii) a coding sequence for IL- 18BP; (c) (i) a coding sequence for IL-18BP, (ii) a connector sequence encoding an IRES, and (iii) a coding sequence for IL- IRA; or (d) (i) a coding sequence for IL-18BP, (ii) a connector sequence encoding a P2A autoproteolytic peptide, and (iii) a coding sequence for IL- IRA.

[0018] In some embodiments of the disclosure, the polypeptide constmct includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 7-10.

[0019] In some embodiments, the coding sequence for the polypeptide construct includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 11-14. In some embodiments, the nucleic acid constructs of the disclosure include a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 15.

[0020] In one aspect, provided herein are recombinant cells including a nucleic acid construct as disclosed herein. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is an animal cell. In some embodiments, the animal cell is a vertebrate animal cell or an invertebrate animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is an insect cell. In some embodiments, the insect cell is a mosquito cell. In some embodiments, the recombinant cell is an immune cell. In some embodiments, the immune cell is a B cell, a monocyte, a natural killer (NK) cell, a natural killer T (NKT) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell (DC), a macrophage, a regulatory T cell, a helper T cell (TH), a cytotoxic T cell (Ten ), a memory T cell, a gamma delta (y5) T cell, a hematopoietic stem cell, or a hematopoietic stem cell progenitor. In some embodiments, the immune cell is a B cell, a T cell, a macrophage, or a dendritic cell (DC). Also provided, in a related aspect, are cell cultures that include at least one recombinant cell as disclosed herein and a culture medium.

[0021] In another aspect, provided herein are transgenic animals including a nucleic acid construct as described herein. In some embodiments, the transgenic animal is a vertebrate animal or an invertebrate animal. In some embodiments, the transgenic animal is a mammal. In some embodiments, the transgenic mammal is a non-human mammal. In some embodiments, the transgenic animal is an insect. In some embodiments, the transgenic insect is a transgenic mosquito.

[0022] In another aspect, provided herein are compositions including: a) a nucleic acid construct of the disclosure; and / or b) a recombinant cell of the disclosure. Non-limiting exemplary embodiments of the pharmaceutical compositions of the disclosure can include one ormore of the following features. In some embodiments, provided herein are compositions including a nucleic acid construct as disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, provided herein are compositions including a recombinant cell as disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, the composition is formulated with a delivery vehicle into a delivery system, wherein the delivery system includes a polymeric nanoparticle, a lipid-based nanoparticle (LNP), a liposome, a viral replicon particle (VRP), a physiologic buffer, a microsphere, an immune stimulating complex (ISCOM), a conjugate of bioactive ligand, or a combination of any thereof.

[0023] In some embodiments, the polymeric nanoparticle includes a cationic polymer, a non-cationic polymer, or a combination thereof. In some embodiments, the cationic polymer includes a naturally-derived cationic polymer. In some embodiments, the naturally-derived cationic polymer includes chitosan, gelatin, dextran, cellulose, cyclodextrin, or a combination thereof. In some embodiments, the cationic polymer includes a synthetic cationic polymer. In some embodiments, the synthetic cationic polymer includes a polyethyleneimine (PEI), poly-L- lysine (PLL), a poly(amino acid) (PAA), a poly(amidoamine) (PAMAM), a poly(cystamine bisacrylamide-co-4-amino-l-butanol) (pABOL), a poly(amino-co-ester) (PAE), poly(2-N,N- dimethylaminoethylmethacrylate, a poly(beta-amino ester) (PBAE), an imidazole-containing polymer, a tertiary-amine containing polymer, poly(2-(dimethylamino)ethyl methacrylate), poly- N-(2-hydroxy-propyl)methacrylamide, a polyamidoamine dendrimer, a cationic glycopolymer, or derivatives thereof.

[0024] In some embodiments, the non-cationic polymer is negatively-charged (i.e., anionic) or electronically neutral. In some embodiments, the non-cationic polymer includes a polyethylene glycol (PEG), a polyester (e.g., polylactic acid (PLA), poly (lactic-co-glycolic acid) (PLGA), poly glycolic acid (PGA), polycaprolactone (PCL)), and polysarcosine (pSar), or derivatives thereof. In some embodiments, the polymer is water-soluble and / or biodegradable.

[0025] In some embodiments of the disclosure, the polymeric nanoparticle includes one or more of the following: poly-(y-L-glutamylglutamine) (PGGA), poly-(Y-L-aspartylglutamine) (PGAA), poly-L-lactic acid (PLLA), poly-(lactic acid-co-glycolic acid) (PLGA), polyalkylcyanoacrylate (PACA), polyanhydrides, polyhydroxyacids, polypropylfumerate, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, [N-(2-hydroxypropyl)methacrylamide] (HPMA) copolymer, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polyurea, polyamine polyepsilon-caprolactone (PCL), and copolymers thereof.

[0026] In some embodiments, the LNP delivery system includes a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid. In some embodiments, the lipid is present in mass ratio of lipid to RNA from about 100: 1 to about 4: 1. In some embodiments, the lipid-based nanoparticles have an average diameter of about 25 nm to about 1000 nm. In some embodiments, the composition as disclosure herein is formulated as a biotherapeutic.

[0027] In another aspect, provided herein are methods for modulating a pharmacodynamic effect in a subject in need thereof, the method includes administering to the subject a composition including: (a) a nucleic acid construct of the disclosure; (b) a recombinant cell of the disclosure; and / or c) a pharmaceutical composition of the disclosure. In some embodiments, the pharmacodynamic effect includes one or more of the following: immunogenicity effect, a biomarker response, a therapeutic effect, a prophylactic effect, a desired effect, an undesired effect, an adverse effect, and effect in a disease model. In some embodiments, the pharmacodynamic effect includes eliciting an immune response in the subject.

[0028] In yet another aspect, provided herein are methods for improving / prolonging kinetics of expression of IL-IRA and / or IL-18BP; or for enhancing the endogenous expression of IL- IRA and / or IL-18BP in a subject, the method including administering to the subject a composition including: (a) a nucleic acid construct of the disclosure; (b) a recombinant cell of the disclosure; and / or (c) a pharmaceutical composition of any one of the disclosure.

[0029] In yet another aspect, provided herein are methods for preventing and / or treating a health condition in a subject in need thereof, the method includes prophylactically or therapeutically administering to the subject a composition including: (a) a nucleic acid construct of the disclosure; (b) a recombinant cell of the disclosure; and / or (c) a pharmaceutical composition of any one of the disclosure. In some embodiments, the administered composition elicits pharmacodynamic effect. In some embodiments, the pharmacodynamic effect includes eliciting an immune response in the subject.

[0030] Non-limiting exemplary embodiments of the methods of the disclosure can include one or more of the following features. In some embodiments, the health condition is anautoimmune disease, an inflammatory disease, and a cardiovascular disease. In some embodiments, the subject has or is suspected of having a health condition associated with an autoimmune disease, an inflammatory disease, and a cardiovascular disease. In some embodiments, the administered composition elicits an immune response in the subject. In some embodiments, the administered composition modulates production of one or more pro- inflammatory molecules in the subject. In some embodiments, the one or more pro-inflammatory molecules includes interleukin-1 alpha (IFNoc), inteleukin-1 beta (IFNP), interleukin- 18 (IL-18), interleukin-6 (IL-6), interferon gamma (ZFNy), cytokines, TNF-a, GM-CSF, and MIPla, granzyme B, granzyme A, perforin, or a combination of any thereof. In some embodiments, the composition is administered to the subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapies. In some embodiments, the at least one additional therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery.

[0031] In yet another aspect, provided herein are methods for improving / prolonging kinetics of expression of IL-IRA and / or IL-18BP; or for enhancing the endogenous expression of IL-IRA and / or IL-18BP in a subject, the method including administering to the subject a composition including: (a) a nucleic acid construct of the disclosure; (b) a recombinant cell of the disclosure; and / or (c) a pharmaceutical composition of any one of the disclosure.

[0032] In yet another aspect, provided herein are kits for eliciting a pharmacodynamic response, eliciting an immune response, and / or for the prevention and / or treatment of a health condition, the kit including: (a) a nucleic acid construct of the disclosure; (b) a recombinant cell of the disclosure; and / or (c) a pharmaceutical composition of the disclosure.

[0033] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

[0034] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 is graphical representation of a non-limiting example of expression vector containing a modified alphavirus genome as described herein. In this example, an EEEV-based vector contains a bigenic expression cassette for expression of IL-IRA and IL-18BP. The coding sequences for IL-IRA and IL-18BP are linked to one another via a connector sequence encoding an internal ribosomal entry site (IRES).

[0036] FIG. 2 is graphical illustrations of an exemplary EEEV srRNA designs in accordance with some non-limiting embodiments of the disclosure, in which the sequence encoding the modified EEEV genome was incorporated into a plasmid DNA vector, which also included coding sequences for an exemplary polypeptide construct including a coding sequence for an IL- IRA protein, a coding sequence for an IL-18BP protein, and an internal ribosomal entry site (IRES).

[0037] FIG. 3 graphically summarizes the results of ELISA experiments performed to measure in vitro protein expression of IL-18BP in monogenic and bigenic expression cassettes. In these experiments, culture supernatants from BHK-21 cells transfected with each srRNA construct were used to measure the protein concentration of secreted IL-18BP.

[0038] FIG 4 graphically summarizes the results of experiments performed to measure in vitro protein levels of bioactive IL-18BP in monogenic and bigenic expression cassette. In these experiments, culture supernatants from BHK-21 cells transfected with each srRNA construct were used to measure bioactivity of IL-18BP by inhibiting IL- 18 signaling on reporter cells expressing the cognate IL-18 receptor.

[0039] FIG. 5 is a graphical representation of the results from ELISA experiments performed to measure in vitro protein expression of IL- IRA in bigenic expression cassettes. In these experiments, culture supernatants from BHK-21 cells transfected with each srRNA construct were used to measure the concentration of secreted IL- IRA.

[0040] FIG. 6 is a graphical representation of the results from ELISA experiments performed to measure in vitro protein levels of bioactive IL-IRA in bigenic expression cassettes. In these experiments, culture supernatants from BHK-21 cells transfected with each srRNA construct were used to measure bioactivity of IL-IRA as measured via inhibition of recombinant IL-ip signaling on reporter cells expressing the cognate receptor.

[0041] FIG. 7 is a graphical representation of the ratios of in vitro protein levels ofbioactive IL-IRA to ELISA IL-IRA expressed from bigenic expression cassettes. In these experiments, culture supernatants from BHK-21 cells transfected with each srRNA construct were used to measure the concentration of secreted IL- IRA.

[0042] FIG. 8 is a graphical representation of the results from ELISA experiments performed to measure in vitro protein expression of IL-IRA in seventeen multigenic expression cassettes to evaluate the ordinality of constituent components. Here, the multigenic expression cassettes contain IL-IRA and one or more additional polypeptides. Also see Table 2 for a more detailed description of the expression cassettes and alphavirus vectors used in these experiments. Culture supernatants from BHK-21 cells transfected with each srRNA construct were used to measure the concentration of secreted IL- IRA.

[0043] FIG. 9 is a graphical representation of the results from ELISA experiments performed to measure in vitro protein levels of bioactive IL-IRA in the multigenic expression cassettes of FIG. 8. In these experiments, culture supernatants from BHK-21 cells transfected with each srRNA construct were used to measure bioactivity of IL-IRA as measured via inhibition of recombinant IL-10 signaling on reporter cells expressing the cognate receptor.

[0044] FIG. 10 is a graphical representation of the in vitro protein expression of IL- IRA in different alphavirus vectors as determined by ELISA. Also see Table 1 for a more detailed description of the alphavirus vectors used in these experiments. In these experiments, culture supernatants from BHK-21 cells transfected with each srRNA construct were used to measure the concentration of secreted IL- IRA.

[0045] FIG. 11 is a graphical representation of the in vitro protein levels of bioactive IL- 1RA in the modified alphavirus vectors described in FIG. 10. In these experiments, culture supernatants from BHK-21 cells transfected with each srRNA construct were used to measure bioactivity of IL-IRA as measured via inhibition of recombinant IL-10 signaling on reporter cells expressing the cognate receptor.

[0046] FIG. 12 graphically summarizes the results of ELISA experiments performed to measure in vitro protein expression of IL-18BP in different alphavirus vectors. Culture supernatants from BHK-21 cells transfected with each srRNA construct were used to measure the concentration of secreted IL-18BP.

[0047] FIG. 13 graphically summarizes in vitro protein levels of bioactive IL-18BP in themodified alphavirus vectors described in FIG. 12. Culture supernatants from BHK-21 cells transfected with each srRNA construct were used to measure bioactivity of IL-18BP by inhibiting IL- 18 signaling on reporter cells expressing the cognate IL- 18 receptor.

[0048] FIG. 14 graphically summarizes the results from in vivo experiments performed to evaluate a panel of modified alphavirus vectors for in vivo expression of IL- IRA. Serum from mice administered with a single dose of a srRNA, encapsulated in an LNP, either encoding both IL-IRA and IL-18BP (bigenic) or IL-IRA alone as a control (monogenic) were used to measure the concentration of secreted IL-IRA and compared to vehicle-treated animals as a control.

[0049] FIG. 15 summarizes the results from in vivo experiments performed to evaluate in vivo expression of IL-18BP from the modified alphavirus vectors described in FIG. 14 with the exception of the monogenic being a srRNA encoding IL-18BP alone, formulated in an LNP, and used as a control.

[0050] FIG. 16 is a graphical representation of in vivo expression of IL-IRA in serum of animals from a bigenic EEEV vector formulated with two different delivery vehicles, which are (i) LNP and (ii) polymeric nanoparticle, in accordance with some non-limiting embodiments of the disclosure. In these experiments, unformulated alphavirus vector and delivery vehicle only were used as controls.

[0051] FIG. 17 is a graphical representation of in vivo expression of IL-18BP in the formulations described in FIG. 16. In these experiments, unformulated alphavirus vector and delivery vehicle only were used as controls.

[0052] FIG. 18 graphically summarizes the results of in vivo pharmacokinetic studies performed to demonstrate that srRNA constructs expressing IL- IRA and IL-18BP (e.g, in LNP formulated vectors) as described herein can extend the pharmacokinetics of a target protein (for instance, a recombinant IL-IRA) that has a short half-life. FIG. 18A: Schematic of an in vivo study design measuring serum levels of human IL- IRA after administration of a recombinant protein version administered at t=0 hours (Top) or an LNP -formulated srRNA encoding ILIRN (gene for human IL-IRA) along with IL-18BPa, administered 96 hours prior to achieve Tmax (Bottom). FIG. 18B: Serum levels of human IL- IRA were measured using a standard ELISA at 2 and 8 hours post administration of a recombinant protein version of IL-IRA or an srRNA- encoded IL- IRA.DETAILED DESCRIPTION OF THE DISCLOSURE

[0053] Provided herein are, inter alia, viral expression systems including self-replicating RNAs (srRNAs) based on RNA viruses (e.g., alphaviruses) with superior expression potential which are suitable for expressing heterologous molecules such as, for example, therapeutic polypeptides, in recombinant cells. For example, some embodiments of the disclosure relate generally to nucleic acid constructs expressing (i) an interleukin-1 receptor antagonist (IL-IRA) protein and or a functional variant thereof, and (ii) an interleukin- 18 binding protein (IL-18BP) or functional variant thereof, for the purposes of therapeutic treatment of human health conditions or diseases, such as, for example, autoimmune diseases, inflammatory diseases, and cardiovascular diseases. These constructs address the problem with treatment modalities, such as recombinant cytokine administration, due to the issue of dose-limiting toxi cities that have been previously demonstrated. In some embodiments, provided herein are gene expression systems with superior expression potential which are suitable for expressing a coding sequence for an IL- 1RA or a functional variant thereof, and a IL-18BP or functional variant thereof, in recombinant cells. For example, some embodiments of the disclosure relate to nucleic acid constructs such as, e.g. expression constructs and vectors, containing a modified genome or srRNA of an alphavirus in which at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced by a coding sequence for a polypeptide construct comprising (a) a coding sequence for an interleukin-1 receptor antagonist (IL-IRA) protein or a functional variant thereof and (b) a coding sequence for an interleukin- 18 binding protein (IL-18BP) or functional variant thereof, wherein the coding sequences for IL- 1RA and IL-18BP are operably linked to one another. In some embodiments, the polypeptide construct does not comprise a dimerization domain. This is because the presence of a dimerization domain in the polypeptide constructs as disclosed herein is believed to likely result in undesirable properties, for example, changes in protein structure, reduced stability, unstable protein folding, loss of function, gain of undesired signaling function, etc. Further provided are recombinant cells and transgenic animals that are genetically engineered to include one or more of the nucleic acid constructs disclosed herein. Biomaterials and recombinant products derived from such recombinant cells are also within the scope of the application. Also provided are compositions and methods useful for (i) modulating pharmacodynamics effect, (ii)improving / prolonging kinetics of expression of IL-IRA and / or IL-18BP in a subject, (iii) or for enhancing the endogenous expression of IL-IRA and / or IL-18BP in a subject, and / or (iv) for preventing and / or treating a health condition in a subject in need thereof.

[0054] As described in greater detail below, IL-1B and IL-18 are inflammatory cytokines that are cleaved into their bioactive forms upon activation of the inflammasome. Both cytokines are downstream mediators of inflammasome activation, and can act synergistically to promote inflammation. Antagonism of both IL-1 and IL- 18 signaling can thus act as a downstream inhibitor of the inflammasome, a central mediator of several autoimmune and inflammatory diseases (both of the immune system and other conditions such as cardiovascular disease). Both cytokines have endogenous antagonists, IL- IRA and IL-18BP, respectively. IL- IRA can also block IL-1A function, as both IL-1 A and IL-1B share the same receptor, IL-1R1, the target of IL-IRA. Inflammasome activation of caspase-1 results in cleavage of pro-IL-lB and pro-IL-18 into their active forms. A number of existing assets that target specific aspects of this pathway (e.g., Glibenclamide, curcumin, MCC950, BHB, Resveratrol, Pralnacasan, Belnacasan, Rilonacept, P2D7KK, LY2189102, Gevokizumab, Canakinumab, and GSK-1070806) are being used for the treatment of a limited number of autoimmune and autoinflammatory diseases, such as cryopyrin-associated periodic syndrome (CAPS), deficiency of the interleukin-1 receptor antagonist (DIRA), pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA syndrome), familial Mediterranean fever (FMF), and mevalonate kinase deficiency (MKD). Co-expression of these two endogenous antagonists, IL-IRA and IL-18BP, can help reduce inflammation associated with inflammasome activation in several autoimmune and inflammatory indications where single agents may be limiting. As discussed above, due to the short half-life of IL- IRA in vivo (commercially available as a recombinant protein under anakinra / Kineret™), which is less than 4 hours in humans and even lower in rodents, strategies to increase its half-life have been explored. Although some of these strategies have shown success, administration of recombinant IL-IRA still requires frequent dosing to maintain achieve therapeutic levels of the encoded protein(s). As described in greater detail below, the use of srRNA platform is expected to surpass what has been observed with these existing methodologies as it may continue to express protein for weeks. Additionally, various modifications of the recombinant proteins, such as addition of an Fc region of an immunoglobulin (even if humanized), can be immunogenic and lead to anti-drug antibodies (ADA) with repeat dosing in a chronic disease setting. The compositions and methods disclosed herein provide an approach that allows for endogenous expression of IL-IRA and IL-18BP with no additional motifs that can result in anti -drug antibodies.

[0055] While the combination of multiple cytokines into a single expression vector have been previously proposed, the ability to express multiple functional cytokines in a single srRNA vector requires much more than just simply to assemble individual cytokine-coding sequences in random orders. In addition, since protein expression does not always correlate with functional protein product, the experimental results presented herein demonstrate that the combination of IL1-RA with IL-18BP in a srRNA vector is not a readily solved problem in the art of srRNA vector design.

[0056] The disclosure provided here provides, inter alia, solutions to these various problems existing with previous attempts to deliver IL-IRA and / or IL-18BP and thereby offers improved methods for treatment of health conditions including autoimmune and inflammatory diseases in, but not limited to, immunological and cardiovascular diseases. As illustrated in Examples 2-4 below, srRNA vectors capable of expressing both functional IL-IRA and functional IL-18BP can be generated by using the compositions and methods disclosed herein.DEFINITIONS

[0057] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0058] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, comprising mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.

[0059] The terms “administration” and “administering”, as used herein, refer to thedelivery of a bioactive composition or formulation by an administration route comprising, but not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering.

[0060] The terms “cell”, “cell culture”, and “cell line” refer not only to the particular subject cell, cell culture, or cell line but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the original cell, cell culture, or cell line.

[0061] The term “construct” refers to a recombinant molecule, e.g., recombinant nucleic acid or polypeptide, including one or more isolated nucleic acid sequences or amino acid sequences from heterologous sources. For example, polypeptide constructs can be chimeric polypeptide molecules in which two or more amino acid sequences of different origin are operably linked to one another in a single polypeptide construct. Similarly, nucleic acid constructs can be chimeric nucleic acid molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule. Thus, representative nucleic acid constructs can include any recombinant nucleic acid molecules, linear or circular, single stranded or double stranded DNA or RNA nucleic acid molecules, derived from any source, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid sequences have been operably linked. Two or more nucleic acid constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors.

[0062] In some embodiments of the disclosure, the one or more nucleic acid constructsmay be incorporated (e. ., inserted) within a single nucleic acid molecule, such as a single vector, or can be incorporated (e.g., inserted) within two or more separate nucleic acid molecules, such as two or more separate vectors. The term “vector” is used herein to refer to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. Thus, the term “vector” encompasses both DNA-based vectors and RNA-based vectors. The term “vector” includes cloning vectors and expression vectors, as well as viral vectors and integrating vectors. An “expression vector” is a vector that includes a regulatory region, thereby capable of expressing DNA sequences and fragments in vitro, ex vivo, and / or in vivo. In some embodiments, a vector may include sequences that direct autonomous replication in a cell such as, for example a plasmid (DNA-based vector) or a self-replicating RNA vector. In some embodiments, a vector may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. In some embodiments, the vector of the disclosure can be single-stranded vector (e.g., ssDNA or ssRNA). In some embodiments, the vector of the disclosure can be double-stranded vector (e.g., dsDNA or dsRNA). In some embodiments, a vector is a gene delivery vector. In some embodiments, a vector is used as a gene delivery vehicle to transfer a gene into a cell. In some embodiments, the vector of the disclosure is a self-replicating RNA (srRNA) vector.

[0063] In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and / or elements to facilitate stable integration of the construct into the genome of a cell. As described above, two or more constructs can be incorporated within a single nucleic acid molecule, such as a single vector, or can be incorporated within two or more separate nucleic acid molecules, such as two or more separate vectors. An “expression construct” generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in a cell. For the practice of the present disclosure, compositions and methods for preparing and using constructs and cells are known to one skilled in the art.

[0064] The term “effective amount”, “therapeutically effective amount”, or“pharmaceutically effective amount” of a composition of the disclosure, e.g., nucleic acid constructs (e.g., srRNA constructs), recombinant cells, and / or pharmaceutical compositions, generally refers to an amount sufficient for the composition to accomplish a stated purpose relative to the absence of the composition (e.g., achieve the effect for which it is administered, stimulate an immune response, prevent or treat a disease, or reduce one or more symptoms of a disease, disorder, infection, or health condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20thEdition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0065] The term “naked” as used herein when referencing nucleic acids that are substantially free of other macromolecules, such as lipids, polymers, and proteins. A “naked” nucleic acid, such as a self-replicating RNA, is not formulated with other macromolecules to improve cellular uptake. Accordingly, a naked nucleic acid is not encapsulated in, absorbed on, or bound to a liposome, a microparticle, a nanoparticle, a cationic emulsion, and the like.

[0066] The term “operably linked”, as used herein, denotes a physical or functional linkage between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion. For example, the term “operably linked” when used in context of the nucleic acid molecules described herein or the coding sequences and promoter sequences in a nucleic acid molecule means that the coding sequences and promoter sequences are in-frame and in proper spatial and distance away to permit the effects of the respective binding by transcription factors or RNA polymerase on transcription. It should be understood that operably linked elements may be contiguous or non-contiguous (e.g., linked to one another through a linker). In the context of polypeptide constructs, “operably linked” refers to a physical linkage (e.g., directly or indirectly linked) between amino acid sequences (e.g.,different segments, portions, regions, or domains) to provide for a described activity of the constructs. Operably linked segments, portions, regions, and domains of the polypeptides or nucleic acid molecules disclosed herein may be contiguous or non-contiguous (e.g., linked to one another through a linker).

[0067] The term “portion” as used herein refers to a fraction. With respect to a particular structure such as a polynucleotide sequence or an amino acid sequence or protein the term “portion” thereof may designate a continuous or a discontinuous fraction of said structure. For example, a portion of an amino acid sequence comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of the amino acids of said amino acid sequence. In addition or alternatively, if the portion is a discontinuous fraction, said discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or more parts of a structure (e.g., domains of a protein), each part being a continuous element of the structure. For example, a discontinuous fraction of an amino acid sequence may be composed of 2, 3, 4, 5, 6, 7, 8, or more, for example not more than 4 parts of said amino acid sequence, wherein each part comprises at least 1, at least 2, at least 3, at least 4, at least 5 continuous amino acids, at least 10 continuous amino acids, at least 20 continuous amino acids, or at least 30 continuous amino acids of the amino acid sequence.

[0068] The term “percent identity,” as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g., the NCBI website at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the complement of a query sequence. This definition includes sequence comparison performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. This definition also includes sequences that have deletionsand / or additions, as well as those that have substitutions. Sequence identity can be calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux etal., Nucleic Acids Res (1984) 12:387), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol (1990) 215:403). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof. Additional methodologies that can suitably be utilized to determine similarity or identity amino acid sequences include those relying on position-specific structure-scoring matrix (P3SM) that incorporates structure-prediction scores from Rosetta, as well as those based on a length-normalized edit distance as described previously in, e.g., Setcliff et a , Cell Host & Microbe 23(6), May 2018.

[0069] The term “pharmaceutically acceptable excipient” as used herein refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration of a compound(s) of interest to a subject. As such, “pharmaceutically acceptable excipient” can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds e.g., antibiotics and additional therapeutic agents) can also be incorporated into the compositions.

[0070] The term “recombinant” when used with reference to a cell, a nucleic acid, a protein, or a vector, indicates that the cell, nucleic acid, protein or vector has been altered or produced through human intervention such as, for example, has been modified by or is the result of laboratory methods. Thus, for example, recombinant proteins and nucleic acids include proteins and nucleic acids produced by laboratory methods. Recombinant proteins can include amino acid residues not found within the native (non-recombinant or wild-type) form of theprotein or can be include amino acid residues that have been modified, e.g, labeled. The term can include any modifications to the peptide, protein, or nucleic acid sequence. Such modifications may include the following: any chemical modifications of the peptide, protein or nucleic acid sequence, including of one or more amino acids, deoxyribonucleotides, or ribonucleotides; addition, deletion, and / or substitution of one or more of amino acids in the peptide or protein; creation of a fusion protein, e.g., a fusion protein comprising an antibody fragment; and addition, deletion, and / or substitution of one or more of nucleic acids in the nucleic acid sequence. The term ’’recombinant” when used in reference to a cell is not intended to include naturally-occurring cells but encompass cells that have been engineered / modified to include or express a polypeptide or nucleic acid that would not be present in the cell if it was not engineered / modified.

[0071] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human individuals) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a health condition of interest (e.g., autoimmune disease, inflammatory disease, or cardiovascular disease) and / or one or more symptoms of the health condition. The subject can also be an individual who is diagnosed with a risk of the health condition of interest at the time of diagnosis or later. The term “non-human animals” includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, livestock, domesticated animals and pets, non-human primates, and other mammals, such as e.g., sheep, dogs, cats, cows, chickens, and non-mammals, such as amphibians, reptiles, etc.

[0072] It is understood that aspects and embodiments of the disclosure described herein include “comprising”, “consisting”, and “consisting essentially of’ aspects and embodiments. As used herein, “comprising" is synonymous with "including", "containing", or "characterized by", and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of' excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of steps of a method, isunderstood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0073] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0074] Certain ranges are presented herein with numerical values being preceded by the term “about” which, as used herein, has its ordinary meaning of approximate. The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.

[0075] Where a range of values is provided, it is understood by one having ordinary skill in the art that all ranges disclosed herein encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to”, “at least”, “greater than”, “less than”, and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled inthe art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0076] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass homologous and / or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In some embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human. The term “gene” is also intended to include variants thereof.

[0077] Headings, e.g., (a), (b), (i) etc., are presented merely for ease of reading the specification and claims. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0078] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.IL-IRA

[0079] The interleukin-1 receptor antagonist protein (IL-IRA) is a member of the interleukin 1 cytokine family. IL1RA protein is secreted by various types of cells including immune cells, epithelial cells, and adipocytes, and is a natural inhibitor of the pro-inflammatory effect of IL-1 beta (IL1B) and IL-1 alpha (ILIA).

[0080] In human, IL-IRA acts as a natural inhibitor of IL-1 receptor. IL-IRA protein has been used to suppress biological activities caused by interleukin 1 alpha and beta (IL-1A, IL-1B). It binds to cell membrane bound IL-1 receptor (IL-1R) and prevents IL-1 from binding to the same IL-1R, thereby preventing IL-1 from sending a signal to that cell. IL-IRA inhibits the activities of IL-1 alpha (ILIA) and IL-1 beta (IL1B), and modulates a variety of IL-1 related immune and inflammatory responses.

[0081] In human, IL-IRA is encoded by the IL1RN gene. KINERET® — Anakinra, which is a recombinant non-glycosylated version of the human IL- IRA, is approved for the treatment of rheumatoid arthritis (RA). It acts as a competitive inhibitor of the pro-inflammatory cytokines IL-1 alpha and IL-1 beta, which are released at inflammatory sites by immune cells and by local tissue cells. KINERET® was approved for the treatment of rheumatoid arthritis (RA) and cryopyrin-associated periodic syndrome (CAPS) in Europe and for the severest form of CAPS, namely chronic infantile neurological cutaneous and articular syndrome (CINCA) in the USA. Anakinra has a short half-life of 4-6 h and therefore common posology requirements are daily subcutaneous injections of 100 mg / day for RA and 1-2 mg / kg / day for CINCA.IL-18BP

[0082] Interleukin- 18 binding protein (IL-18BP) is a natural antagonist of IL-18 which is associated with arthritis and with many other inflammatory and / or autoimmune diseases. It has been reported that IL-18BP binds to the IL-18 ligand with a high affinity, and this high-affinity binding of 1L-18BP sufficiently blocks the interaction of IL-18 with the IL-I 8R0. ligand-binding chain on the cell surface and eventually inhibits the IL-18 signaling pathway Therefore, IL- 18BP is considered a potent IL-18 inhibitor. In particular, IL-18BP reduced the severity of several experimental autoimmune diseases. Therefore, IL-18BP is believed to function as a natural anti-inflammatory and immunosuppressive molecule neutralizing the effects of high TL- 18 levels during inflammation. IL-18BP is specifically induced by IFNy as part of a negativefeedback loop that regulates the induction of JFNy by IL- 18.

[0083] IL-18BP has 4 isoforms (a, b, c, and d) in humans and 2 isoforms (c and d) in mice. The human isoforms a and c as well as mouse isoforms c and d can bind to the receptor-binding site of IL-18 to inhibit IL-18 at equimolar ratio. In human, IL-18BP isoform a (IL-18BPa) has the greatest affinity for IL-18 (e.g., higher than isoform c). Isoforms b and d lack IL-18 neutralization activity. IL-18BP binds to TL-18 with high affinity to block the IL-18 and IL-18R complex formation and subsequently inhibits IL- 18 activation. Under normal conditions, there is enough naturally occurring IL-18BP to keep a low level of free IL-18. However, in patients with certain inflammatory diseases and high level of IL-18 could cause the IL-18 / IL-18BP balance disruption, leading to an increase in free and active IL- 18, which in turn results in pathological inflammation. This imbalance of IL-18 / IL-18BP can be associated with increased disease severity. It has been reported that the absence of IL-18BP caused imbalance of IL-18 / IL-18BP and excessive NK cell activation by high level IL- 18 resulted in uncontrolled killing of human hepatocytes. IL-18 / IL-18BP imbalance is highly linked to immunologically mediated diseases, especially diseases that have a pathological role of IFNy, such as macrophage activated syndrome (MAS).

[0084] The effects of IL-18 have been implicated in many diseases including autoimmune diseases, cardiovascular diseases, neurovascular EC damage, infrarenal aortic occlusion- mediated renal injury, hemophagocytic lymphohistiocytosis, and inflammatory bowel disease in clinical and animal studies. IL-18BP and anti-IL-18 antibody have been used to neutralize IL- 18 and treat these diseases. At least 33 disease models have been reported where inhibition of IL-18 activity by administration of either neutralizing anti-IL-18 antibodies or IL-18BP resulted in a reduction of disease severity. Recently, recombinant human IL-18BP (Tadekinig Alfa) has been used for a phase II clinical trial in Europe to treat adult-onset Still’s disease patients and a phase III clinical trial with the experimental drug IL-18BP in patients carrying a mutation of the NOD- like receptor C4 (NLRC4) gene characterized by severe, life-threatening systemic inflammation associated with extremely high levels of IL-18.Self-replicating RNA

[0085] As will be appreciated by the skilled artisan, the term “self-replicating RNA” (srRNA) refers to RNA molecule that contains all of the genetic information required fordirecting its own amplification or self-replication within a permissive cell. Therefore, srRNA is sometimes also referred to as “self-amplifying RNA” (saRNA). In some embodiments, the srRNA is a “replicon,” which can be a linear or circular section of DNA or RNA which replicates sequentially as a unit. Non-limiting examples of replicons include “replicon RNA” or “RNA replicon.” To direct its own replication, the srRNA generally (1) encodes polymerase, replicase, or other proteins which may interact with viral or host cell-derived proteins, nucleic acids or ribonucleoproteins to catalyze the RNA amplification process; and (2) contain c / .s-acting RNA sequences required for replication and transcription of the subgenomic RNA. These sequences may be bound during the process of replication to its self-encoded proteins, or nonself-encoded cell-derived proteins, nucleic acids or ribonucleoproteins, or complexes between any of these components. In some embodiments of the disclosure, the replicon, e.g., srRNA, is derived from a Madariaga virus (MADV). In some embodiments of the disclosure, a MADV srRNA construct (e.g., srRNA, saRNA, or RNA replicon molecule) generally contains the following elements: 5' viral or defective-interfering RNA sequence(s) required in cis for replication, sequences coding for biologically active alphavirus non-structural proteins (e.g., nsPl, nsP2, nsP3, and nsP4), a subgenomic promoter (sg) for the subgenomic RNA (sgRNA), 3' viral sequences required in cis for replication, and optionally a polyadenylate tract (poly(A)). In some instances, a subgenomic promoter (sg) that directs expression of a heterologous sequence can be included in the srRNA construct of the disclosure.

[0086] Further, the term srRNA molecule (e.g, srRNA, saRNA, or RNA replicon molecule) generally refers to a molecule of positive polarity, or “message” sense, and the srRNA may be of length different from that of any known, naturally-occurring alphavirus. In some embodiments of the present disclosure, the srRNA does not contain at least a portion of the coding sequence for one or more of the alphavirus structural proteins; and / or sequences encoding structural genes can be substituted with heterologous sequences. In those instances, where the srRNA is to be packaged into a recombinant alphavirus particle, it can contain one or more sequences, so-called packaging signals, which serve to initiate interactions with alphavirus structural proteins that lead to particle formation.

[0087] Nucleic acid molecules of the present disclosure can be nucleic acid molecules of any length, including nucleic acid molecules that are generally between about 2 kb and 50 kb inlength, for example between about 5 kb and about 40 kb, between about 5 kb and about 30 kb, between about 5 kb and about 20 kb, or between about 10 kb and about 50 kb, for example between about 15 kb to 30 kb, between about 20 kb and about 50 kb, between about 20 kb and about 40 kb, between about 5 kb and about 25 kb, or between about 30 kb and about 50 kb. In some embodiments, the nucleic acid molecules are at least 6 kb in length. In some embodiments, the nucleic acid molecules are between about 6 kb and about 20 kb. In some embodiments of the disclosure. The replicon constructs (e.g., srRNA constructs) of the disclosure generally have a length of at least about 2 kb. For example, the srRNA can have a length of at least about 2 kb, at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb or more than 12 kb. In some embodiments, the srRNA can have a length of about 4 kb to about 20 kb, about 4 kb to about 18 kb, about 5 kb to about 16 kb, about 6 kb to about 14 kb, about 7 kb to about 12 kb, about 8 kb to about 16 kb, about 9 kb to about 14 kb, about 10 kb to about 18 kb, about 11 kb to about 16 kb, about 5 kb to about 18 kb, about 6 kb to about 20 kb, about 5 kb to about 10 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb to about 11 kb. In some embodiments, the srRNA can have a length of about 6 kb to about 14 kb. In some embodiments, the srRNA can have a length of about 6 kb to about 16 kb.COMPOSITIONS OF THE DISCLOSURE

[0088] As described in greater detail below, one aspect of the present disclosure relates to nucleic acid constructs sequences that encode a modified alphavirus genome or srRNA where at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced by an coding sequence for a polypeptide construct comprising (a) a coding sequence for an IL- IRA protein or a functional variant thereof; and (b) a coding sequence for an IL-18BP or functional variant thereof; wherein the coding sequences for IL-IRA and IL-18BP are operably linked to one another, and wherein thepolypeptide construct does not include a dimerization domain. Also provided are recombinant cells and cell cultures that have been engineered to include a nucleic acid construct as disclosed herein.A. Nucleic acid constructs

[0089] As described in greater detail below, one aspect of the present disclosure relates to nucleic acid constructs including a nucleic acid sequence encoding a modified alphavirus genome or replicon (e.g., srRNA) where at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or replicon, e.g., srRNA, has been replaced by an coding sequence for a polypeptide construct comprising (a) a coding sequence for an TL-1RA protein or a functional variant thereof; and (b) a coding sequence for an IL-18BP or functional variant thereof; wherein the coding sequences for IL-IRA and IL-18BP are operably linked to one another, and wherein the polypeptide construct does not include a dimerization domain. In some embodiments, the sequence encoding a srRNA construct can be operably linked, e.g., placed under the control of elements required for expression (e.g., promoter sequences), which allow expression of the srRNA construct in a host cell, in a subject, or in an ex-vivo cell -free expression system.

[0090] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. A nucleic acid molecule can be double-stranded or single-stranded (e.g., a sense strand or an antisense strand). A nucleic acid molecule may contain unconventional or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence” as used herein interchangeably refer to the sequence of a polynucleotide molecule. The nomenclature for nucleotide bases as set forth in 37 CFR §1.822 is used herein.

[0091] Nucleic acid molecules of the present disclosure can be of any length, including for example, between about 1.5 Kb and about 50 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.

[0092] Non-limiting exemplary embodiments of the nucleic acid constructs (e.g., srRNA constructs) of the disclosure can include one or more of the following features. In some embodiments, due to the dimerizing ability of immunoglobin Fc region, the polypeptide constructs of the present disclosure do not include a fragment crystallization region (Fc region) of an immunoglobulin. In some embodiments, the coding sequence for IL- IRA is N-terminally linked to the coding sequence for IL-18BP. In some embodiments, the coding sequence for IL- 1RA is C-terminally linked to the second coding sequence for IL-18BP. In some embodiments, the coding sequences for IL- IRA and / or IL-18BP express proteins that are functional in a bioactivity assay. In some embodiments, the IL-18BP protein of the present disclosure is the IL- 18BP isoform a (IL-18BPa). In some embodiments, the IL-IRA protein and / or IL-18BP protein of the present disclosure are from a mammalian subject. In some embodiments, the mammalian subject is a human subject.

[0093] In some embodiments of the disclosure, the coding sequence for IL- IRA and / or IL- 18BP is optimized for one of more desired characteristics. In some embodiments, the coding sequences for IL-IRA and / or IL-18BP are independently optimized for one or more of the following: (a) enhancing RNA stability, (b) enhancing expression level, (c) minimizing rare codon usage, (c) minimizing secondary structures, (d) facilitating better srRNA replication, and (e) facilitating better RNA manufacturing process.

[0094] In some embodiments, the modified alphavirus genome or replicon, e.g., srRNA vector, is devoid of at least a portion of the nucleic acid sequence encoding one or more of the viral structural proteins CP, El, E2, E3, and 6K of the alphavirus genome or srRNA vector. In some embodiments, the modified alphavirus genome or srRNA vector is devoid of a portion of or the entire sequence encoding CP. In some embodiments, the modified alphavirus genome or srRNA vector is devoid of a portion of or the entire sequence encoding EL In some embodiments, the modified alphavirus genome or srRNA vector is devoid of a portion of or the entire sequence encoding E2. In some embodiments, the modified alphavirus genome or srRNA vector is devoid of a portion of or the entire sequence encoding E3. In some embodiments, the modified alphavirus genome or srRNA vector is devoid of a portion of or the entire sequence encoding 6K. In some embodiments, the modified alphavirus genome or srRNA vector is devoid of a portion of or the entire sequence encoding a combination of CP, El, E2, E3, and 6K. Insome embodiments of the disclosure, the coding sequence for nonstructural proteins nsPl , nsP2, nsP3, and nsP4 of the modified alphavirus genome or srRNA vector is present, however at least a portion of or the entire sequence encoding one or more structural proteins (e.g., CP, El, E2, E3, and 6K) of the modified alphavirus genome or srRNA vector is absent.

[0095] In some embodiments, the modified alphavirus genome or srRNA vector is devoid of a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. The skilled artisan will understand that a substantial portion of a nucleic acid sequence encoding a viral structural polypeptide can include enough of the nucleic acid sequence encoding the viral structural polypeptide to afford putative identification of that polypeptide, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (see, for example, in “Basic Local Alignment Search Tool”; Altschul SF et al., J. Mol. Biol. 215:403-410, 1993). Accordingly, a substantial portion of a nucleotide sequence comprises enough of the sequence to afford specific identification and / or isolation of a nucleic acid fragment comprising the sequence. For example, a substantial portion of a nucleic acid sequence can include at least about 20%, for example, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of the full-length nucleic acid sequence.

[0096] In some embodiments, the modified alphavirus genome or srRNA vector of the present disclosure is devoid of the entire sequence encoding viral structural proteins, e.g., the modified alphavirus genome or srRNA vector includes no nucleic acid sequence encoding the viral structural proteins.

[0097] The nucleic acid constructs of the disclosure further include a coding sequence for a polypeptide construct that replaces at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA. In principle, the nucleic acid constructs disclosed herein can generally include any number of coding sequences for a polypeptide construct. In some embodiments, the nucleic acid constructs disclosed herein can include at least one, at least two, at least three, at least four, at least five, or at least six coding sequences for polypeptide constructs. A coding sequence for a polypeptide construct can be a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and / or translation of the coding sequences in a cell, in vivo and / orex vivo. The coding sequence for a polypeptide construct can be inserted into a vector for targeting to a desired host cell and / or into a subject or individual. Accordingly, in some embodiments, the term “coding sequence for a polypeptide construct” can be used interchangeably with the term “expression construct.” In some embodiments, a coding sequence for a polypeptide construct can be a nucleic acid construct that includes a gene encoding a protein or functional RNA operably linked to regulatory elements such as, for example, a promoter and / or a termination signal, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the gene.

[0098] The nucleic acid constructs described herein include a coding sequence for an interleukin-1 receptor antagonist (IL-IRA) protein or a functional variant thereof, and a coding sequence for an interleukin- 18 binding protein (IL-18BP) or functional variant thereof, wherein the coding sequences for IL- IRA and IL-18BP are operably linked to one another, and wherein the polypeptide construct does not comprise a dimerization domain. In some embodiments, the nucleic acid constructs encode IL-IRA and IL-18BP polypeptides that are able to elicit a pharmacodynamics effect in a subject. The functional variants of IL- IRA and IL-18BP can encompass coding sequences for polypeptides having an amino acid sequence that is the same or essentially the same as that of the reference protein (e.g., IL-IRA and IL-18BP) except having at least one amino acid modified, for example, deleted, inserted, or replaced, respectively. The amino acid replacement may be a conservative amino acid substitution, preferably at a non- essential amino acid residue in the protein. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). A variant of a protein may have an amino acid sequence at least about 80%, 90%, 95%, or 99%, preferably at least about 90%, more preferably at least about 95%, identical to the amino acid sequence of the protein. Preferably, a variant is a functional variant ofa protein that retains the same or essentially the same function as the protein. The terms “variant”, when used in reference to a nucleic acid sequence, refer to a nucleic acid sequence that differs by one or more nucleotides from another, usually related nucleotide acid sequence. As such, the term “variant” can refer to a change of one or more nucleotides of a reference nucleic acid which includes the insertion of one or more new nucleotides, deletion of one or more nucleotides, and substitution of one or more existing nucleotides. A variant can also include a point mutation, multiple mutation, single nucleotide polymorphism (SNP), deletion, insertion, and translocation. Thus, variants of the coding sequences described herein include nucleic acids that encode polypeptides that can be, for example, full length, mutated, truncated, inactivated, peptide / epitopes or combinations thereof of IL-IRA and IL-18BP.

[0099] The full-length amino acid sequence of the human IL- IRA is set forth in SEQ ID NO: 1 (177 aa) as follows:MEICRGLRSHLITLLLFLFHSETICRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAG YLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSEN RKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFY FQEDE

[0100] In some embodiments, the coding sequence for the IL-IRA protein in the nucleic acid constructs described herein encodes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid constructs of the disclosure include a nucleic acid sequence encoding an IL-IRA protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NO: 1. In some embodiments, the coding sequence for the IL-IRA protein encodes smaller portions of the amino acid sequence of SEQ ID NO: 1. These smaller portions can include at least 8, 10, 12, 14, 16, 18, 20, 30 or more amino acids of SEQ ID NO: 1.

[0101] In some embodiments, the coding sequence for a polypeptide construct in the nucleic acids described herein encodes a variant of the IL-IRA protein.

[0102] As described supra, the nucleic acid constructs described herein also include coding sequences for IL-18BP or a functional variant thereof. In some embodiments, the nucleic acid constructs described herein also include coding sequences for IL-18BP isoform a (IL-18BPa) or a functional variant thereof

[0103] The full-length amino acid sequence of the human IL-18BPa, along with its signal sequence, is set forth in SEQ ID NO: 2 (194 aa) as follows:MTMRHNWTPDLSPLWVLLLCAHVVTLLVRATPVSQTTTAATASVRSTKDPCPSQPP VFPAAKQCPALEVTWPEVEVPLNGTLSLSCVACSRFPNFSILYWLGNGSFIEHLPGRL WEGSTSRERGSTGTQLCKALVLEQLTPALHSTNFSCVLVDPEQVVQRHVVLAQLWA GLRATLPPTQEALPS SHS SPQQQG

[0104] In some embodiments, the coding sequence for the IL-18BP in the nucleic acid constructs described herein encodes the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid constructs of the disclosure include a nucleic acid sequence encoding an IL-18BP protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NO: 2. In some embodiments, the coding sequence for the IL-18BP encodes smaller portions of the amino acid sequence of SEQ ID NO: 2. These smaller portions can include at least 8, 10, 12, 14, 16, 18, 20, 30 or more amino acids of SEQ ID NO: 2.

[0105] In some embodiments, the coding sequence for a polypeptide construct in the nucleic acids described herein encodes a variant of the IL-18BP.

[0106] In some embodiments, the coding sequence for the IL-IRA protein or functional variant thereof, and IL-18BP or functional variant thereof includes a coding sequence for a single polypeptide (e.g., monogenic). In some embodiments, the coding sequence for the IL- IRA protein or functional variant thereof, and IL-18BP or functional variant thereof includes coding sequences for a plurality of polypeptides, e.g., multigenic (e.g., bigenic or trigenic). In some embodiments, each of the coding sequences for the IL- IRA protein or functional variant thereof, and IL-18BP or functional variant thereof is operably linked to a separate promoter sequence. In some embodiments, the coding sequences of the IL- IRA protein or functional variant thereof, and IL-18BP or functional variant thereof are operably linked to one another within a single open reading frame (e.g., in a polycistronic ORF). In some embodiments, the nucleic acid sequence encoding the polypeptide construct is operably linked to a promoter sequence. In some embodiments, the coding sequence of the polycistronic ORF is operably linked to a promoter sequence. In some embodiments, the promoter sequence is a subgenomic (sg) promoter. In some embodiments, the sg promoter sequence is a 26S subgenomic promoter. In some embodiments,the subgenomic promoter is heterologous relative to the remainder of the modified viral genome or srRNA. In some embodiments, the subgenomic promoter is an alphavirus subgenomic promoter.

[0107] In some embodiments of the disclosure, at least one nonstructural protein (nsP), or a portion thereof, of the modified viral genome or srRNA is heterologous relative to the remainder of the modified viral genome or srRNA. In some embodiments, the nucleic acid constructs disclosed herein further include a nucleic acid sequence encoding a heterologous nsP or a portion thereof. In some embodiments, the nucleic acid constructs disclosed herein further include one or more untranslated regions (UTRs). In some embodiments, at least one of the UTRs is a heterologous UTR.

[0108] In some embodiments of the methods described herein, the recombinant alphavirus srRNA is of a virus belonging to the Alphavirus genus of the Togaviridae family. In some embodiments of the disclosure, the modified alphavirus genome or srRNA is of an alphavirus belonging to the Venezuelan equine encephalitis virus / Eastem Equine Encephalitis virus (VEEV / EEEV) group, or the Semliki Forest virus (SFV) group, or the Sindbis virus (SINV) group. In some embodiments, the modified alphavirus genome or srRNA is of an alphavirus belonging to the BFV complex, EEEV complex, MIDV complex, NDUV, complex, SFV complex, VEEV complex, WEEV complex. In some embodiments, the alphavirus is Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O’Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu virus (NDUV), Madariaga virus (MADV), or Buggy Creek virus. In some embodiments, the alphavirus is VEEV, EEEV, CHIKV, or SINV. In some embodiments, the alphavirus is VEEV. In some embodiments, the alphavirus is EEEV. In some embodiments, the alphavirus is Western Equine Encephalitis virus (WEEV). In some embodiments, the alphavirus is CHIKV. In some embodiments, the alphavirus is SINV.

[0109] In some embodiments, the alphavirus is Chikungunya virus (CHIKV). Non-limiting examples of CHIKV strains suitable for the compositions and methods of the disclosure include CHIKV S27, CHIKV LR2006-OPY-1, CHIKV YO 123223, CHIKV DRDE, CHIKV 37997, CHIKV 99653, CHIKV Ag41855, and Nagpur (India) 653496 strain. Virulent and avirulent CHIKV strains are both suitable. Additional examples of CHIKV strains suitable for the compositions and methods of the disclosure include but are not limited to those described in Afreen etal. Microbiol. Immunol. 2014, 58:688-696, Lanciotti and Lambert ASTMH 2016, 94(4):800-803 and Langsjoen etal. mBio. 2018, 9(2):e02449-17. In some embodiments, the modified CHIKV genome or replicon RNA (e.g., self-replicating RNA) is derived from CHIKV strain S27. In some embodiments, the modified CHIKV genome or replicon RNA is derived from CHIKV strain DRDE. In some embodiments, the modified CHIKV genome or replicon RNA is derived from CHIKV strain DRDE-06. In some embodiments, the modified CHIKV genome or replicon RNA is derived from CHIKV strain DRDE-07.

[0110] In some embodiments, the alphavirus is Eastern Equine Encephalitis virus (EEEV). Non-limiting examples of EEEV strains suitable for the compositions and methods of the disclosure include EEEV 792138, 783372, BeAn5122, BeAr300851, BeAr436087, C-49, FL91- 4679, FL93-939, GML903836, MP-9, PE6, and V105-00210. Virulent and avirulent EEEV strains are both suitable. Additional suitable EEEV strains include, but are not limited to those described in the Virus Pathogen Resource website (ViPR; which is publicly available at www.viprbrc.org / brc / vipr_genome_search. spg?method=SubmitForm&blockId=868&decorator= toga). In some embodiments, the modified EEEV genome or replicon RNA (e.g., self-replicating RNA) is derived from EEEV strain FL93-939.[0U1] In some embodiments, the alphavirus is Sindbis virus (SINV). In some embodiments, the modified genome or RNA replicon (e.g., self-replicating RNA) is of a SINV strain. Non-limiting examples of SINV strains suitable for the compositions and methods of the disclosure include SINV strain AR339, AR86, and Girdwood. Examples of SINV strains suitable for the compositions and methods of the disclosure include, but are not limited to those described in Sammels et al. J. Gen. Virol. 1999, 80(3):739-748, Lundstrbm and Pfeffer Vector Borne Zoonotic Dis. 2010, 10(9): 889-907, Sigei etal. Arch, of Virol. 2018, 163:2465-2469 and Ling et al. J. Virol. 2019, 93:e00620-19. Additional suitable SINV strains include, but are not limited tothose described in the Virus Pathogen Resource website (ViPR; which is publicly available at www.viprbrc.org / brc / vipr_genome_search. spg?method=SubmitForm&blockId=868&decorator= toga). Virulent and avirulent SINV strains are both suitable. In some embodiments, the modified genome or RNA replicon is of a SINV strain Girdwood. In some embodiments, the modified genome or RNA replicon is of a SINV strain AR86. In some embodiments, the modified SINV genome or replicon RNA is derived from SINV strain Girdwood. In some embodiments, the modified SINV genome or replicon RNA is derived from SINV strain AR86. In some embodiments, the at least one heterologous nsP or portion thereof of the modified genome or RNA replicon is derived from a SINV strain AR86. In some embodiments, the at least one heterologous nsP or portion thereof is nsPl, nsP3, nsP4, or a portion of any thereof, or a combination of any of the foregoing. In some embodiments, the modified genome or RNA replicon is of a SINV strain AR86.

[0112] In some embodiments, the alphavirus is Western Equine Encephalitis virus (WEEV). Non-limiting examples of WEEV strains suitable for the compositions and methods of the disclosure include WEEV California, McMillan, IMP181, Imperial, Imperialist, IMPR441, 71V-1658, AG80-646, BFS932, COA592, EP-6, E1416, BFS1703, BFS2005, BSF3060, BSF09997, CHLV53, KERN5547, 85452NM, Montana-64, S8-122, and TBT-235. Additional examples of WEEV strains suitable for the compositions and methods of the disclosure include 5614, 93A27, 93A30, 93A38, 93A79, B628(C1 15), CBA87, CNTR34, CO921356, Fleming, Lake43, PV012357A, PV02808A, PV72102, R02PV001807A, R02PV002957B, R02PV003422B, R05PV003422B, R0PV003814A and R0PV00384A. Virulent and avirulent WEEV strains are both suitable. Additional suitable WEEV strains include, but are not limited to those described in Bergren NA et al., J. Virol. 88(16): 9260-9267, Aug 2014, and in the Virus Pathogen Resource website (ViPR; which is publicly available at https: / / www.viprbrc.org / brc / vipr_genome_search. spg?method=SubmitForm&blockId=57240&d ecorator=toga). In some embodiments, the modified WEEV genome or srRNA is derived from WEEV strain Imperial. In some embodiments, the modified WEEV genome or srRNA is derived from WEEV strain McMillan.

[0113] In some embodiments, the alphavirus is Madariaga virus (MADV), formerly referred to as South American Eastern Equine Encephalitis virus (SA EEEV). Non-limitingexamples of MADV strains suitable for the compositions and methods of the disclosure include ArgLL, ArgB, BeAn-5122, ArgM, 24443 (TR59), 25714 (BG60), BeAr 18205, 900188 (PA62), BeAr 81828, BeAr 126650, 68U231, 77U1104 (PE70), 75V1496, BeAr 300851, 75U40, and El Delirio (Arrigo NC et al., supra 2010). Additional examples of MADV strains suitable for the compositions and methods of the disclosure include 76V25343, 77U1 (BR77), BeAr 348998, IVICPan57151, B eAn416361, 903836 (PA84), BeAr436087, 435731 (PA86), C49 (CO92), PE- 0.0155-96 (0.0155), PE-3.0815-96 (3.0815), PE-16.0050-98 (16.0050), PE-18.0140-99 (18.0140), and PE-18.0172-99 (18.0172) (Arrigo NC et al., supra 2010). Additional suitable MADV strains include, but are not limited to those described in Arrigo NC et al., supra 2010, and in the Virus Pathogen Resource website (ViPR; which is publicly available at www.viprbrc.org / brc / vipr genome_search.spg?method=SubmitForm&blockId=868&decorator= toga). In some embodiments, the modified MADV genome or srRNA is derived from MADV strain BeAr300851.

[0114] In some embodiments of the disclosure, the coding sequence for the polypeptide construct includes, in 5’ to 3’ direction (i.e., in N-terminus to C-terminus direction of the polypeptide sequence): (a) (i) a coding sequence for IL- IRA, (ii) a connector sequence encoding an IRES, and (iii) a coding sequence for IL-18BP; (b) (i) a coding sequence for IL-IRA, (ii) a connector sequence encoding a P2A autoproteolytic peptide, and (iii) a coding sequence for IL- 18BP; (c) (i) a coding sequence for IL-18BP, (ii) a connector sequence encoding an IRES, and (iii) a coding sequence for IL- IRA; or (d) (i) a coding sequence for IL-18BP, (ii) a connector sequence encoding a P2A autoproteolytic peptide, and (iii) a coding sequence for IL- IRA.

[0115] In some embodiments of the disclosure, the polypeptide construct includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 7-10. In some embodiments, the polypeptide construct includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the polypeptide construct includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 8. In some embodiments, the polypeptide construct includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the polypeptide construct includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0116] In some embodiments, the polypeptide construct includes an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 7, and further wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues in the amino acid sequence is substituted by a different amino acid. In some embodiments, the polypeptide construct includes an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 8, and further wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues in the amino acid sequence is substituted by a different amino acid. In some embodiments, the polypeptide construct includes an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 9, and further wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues in the amino acid sequence is substituted by a different amino acid. In some embodiments, the polypeptide construct includes an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 10, and further wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues in the amino acid sequence is substituted by a different amino acid.

[0117] In some embodiments, the polypeptide construct includes an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the polypeptide construct includes an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the polypeptide construct includes an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the polypeptide construct includes an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0118] In some embodiments of the disclosure, the coding sequence for the polypeptide construct includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 11-14. In some embodiments, the coding sequence for the polypeptide construct includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the coding sequence for the polypeptide construct includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the coding sequence for the polypeptide construct includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the coding sequence for the polypeptide construct includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

[0119] In some embodiments, the coding sequence for the polypeptide construct includes a nucleic acid sequence having 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 11, and further wherein one, two, three, four, five, six, seven, eight, nine, or ten nucleotides in the nucleic acid sequence is substituted by a different nucleotide. In some embodiments, the coding sequence for the polypeptide construct includes an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, and further wherein one, two, three, four, five, six, seven, eight, nine, or ten nucleotides in the nucleic acid sequence is substituted by a different nucleotide. In some embodiments, the coding sequence for the polypeptide construct includes a nucleic acid sequence having 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 13, and further wherein one, two, three, four, five, six, seven, eight, nine, or ten nucleotides in the nucleic acid sequence is substituted by a different nucleotide. In some embodiments, the coding sequence for the polypeptide construct includes a nucleic acid sequence having 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14, and further wherein one, two, three, four, five, six, seven, eight, nine, or ten nucleotides in the nucleic acid sequence is substituted by a differentnucleotide.

[0120] In some embodiments, the coding sequence for the polypeptide construct includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 15 In some embodiments, the coding sequence for the polypeptide construct includes a nucleic acid sequence having 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 15, and further wherein one, two, three, four, five, six, seven, eight, nine, or ten nucleotides in the nucleic acid sequence is substituted by a different nucleotide. In some embodiments, the coding sequence for the polypeptide construct includes a nucleic acid sequence having 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 15.

[0121] In some embodiments, the coding sequences for IL-IRA and IL-18BP in the nucleic acid constructs disclosed herein are operably linked to one another via one or more connector sequences. In some embodiments, the length and amino acid composition of the connector sequences can be optimized to vary the orientation, flexibility, and / or proximity of the polypeptides relative to one another to achieve a desired activity or property of the polypeptide construct. In some embodiments, a connector sequence includes coding sequence for an autoproteolytic peptide and / or an internal ribosomal entry site (IRES). First identified in the foot- and-mouth disease virus (FMDV), a member of the picornavirus group, several autoproteolytic peptides have been subsequently identified such as, for example, “2A like” peptides from equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), and their activities in proteolytic cleavage have been shown in various ex vitro, in vitro, ex vivo, and in vivo eukaryotic systems. As such, the concept of autoproteolytic peptides is available to one of skill in the art with many naturally-occurring autoprotease systems have been identified. Well studied autoprotease systems are e.g. viral proteases, developmental proteins (e.g., HetR, Hedgehog proteins), Rum A autoprotease domain, UmuD, etc.). Non -limiting examples of autoproteolytic peptides suitable for the compositions and methods of the present disclosure include one or more autoproteolytic cleavage sequences from a calcium-dependent serine endoprotease (furin), a porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), or acombination thereof. In some embodiments, the autoproteolytic peptide includes a P2A sequence.

[0122] In some embodiments, the coding sequences for IL-IRA protein or functional variant thereof, and IL- IBP or functional variant thereof are operably linked to one another by a coding sequence for one or more an internal ribosomal entry sites (IRES). An IRES or “internal ribosome entry site” is a sequence located between polycistronic genes that permits the production of the expression product originating from the second gene by internal initiation of the translation of the bi-cistronic mRNA. It promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene. See, e.g., Jackson etal., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1 (10):985- 1000. In some embodiments, the IRES can be a viral IRES, a cellular IRES, or an artificial IRES. Examples of IRES generally employed by those of skill in the art include those described in U.S. Pat. No. 6,692,736. In some embodiments, the IRES is selected from a Kaposi’s sarcoma-associated herpesvirus (KSHV) IRES, a hepatitis virus IRES, a Pestivirus IRES, a Cripavirus IRES, a Rhopalosiphum padi virus IRES, a fibroblast growth factor IRES, a platelet-derived growth factor IRES, a vascular endothelial growth factor IRES, an insulin-like growth factor IRES, a picomavirus IRES, an encephalomyocarditis virus (EMCV) IRES, a Pim-1 IRES, a p53 IRES, an Apaf-1 IRES, a TDP2 IRES, an L-myc IRES, and a c-myc IRES. In some embodiments, the IRES is from EMCV.

[0123] One of skill in the art will appreciate that different configurations of coding sequences for IL-IRA protein or functional variant thereof, IL-1BP or functional variant thereof, and IRES can be employed as long as expression and / or bioactivity of IL-IRA protein or functional variant thereof and IL-1BP or functional variant thereof is adequately maintained. These sequences will typically be configured and operably linked so that the polypeptide encoded by the gene of interest can be released from the ribosome and other sequences after self- cleavage or ribosomal skipping.

[0124] Methodologies and techniques for operably linking two or more sequences of DNA together are familiar to one of ordinary skill in the art, and such methods have been described in many books for standard molecular biological manipulation (see, for example, Maniatis et al.,“Molecular Cloning: A Laboratory Manual” 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Gibson et al., Nature Methods 6:343-45, 2009).

[0125] As described in greater detail in Examples 1-3 below, IL-IRA and IL-18BP expression and bioactivity were measured from different monogenic and bigenic constructs in different modified alphavirus vectors. Exemplary configurations of the nucleic acid constructs described herein are shown in Table 1 below.

[0126] In some embodiments, the coding sequence of the IL-IRA protein and / or IL-1BP is redesigned, refactored, and / or optimized for a desired property, such as increased stability, potency, and expression e.g., translation efficiency), which in turns can maximize the impact of producing, delivering, and administering the biotherapeutic IL-IRA protein and / or IL-1BP. For example, in some embodiments, the coding sequence of the IL- IRA protein and / or IL- IBP is optimized for expression at a level higher than the expression level of a reference coding sequence, for example, 20% higher, 30% higher, 40% higher, 50% higher, 60% higher, 70% higher, 80% higher, 90% higher, or 95% higher than a reference coding sequence. In some embodiments, the reference coding sequence is a wild-type non-optimized sequence. In some embodiments, the coding sequence of the IL-IRA protein and / or IL-1BP is optimized for one or more of the following: (a) enhancing RNA stability, (b) enhancing expression level, (c) minimizing rare codon usage, (c) minimizing secondary structures, (d) facilitating better srRNA replication, and (e) facilitating better RNA manufacturing process.

[0127] With respect to sequence-optimization of nucleotide sequences, degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, the nucleic acid constructs of the present disclosure may also have any base sequence that has been changed from any polynucleotide sequence disclosed herein by substitution in accordance with degeneracy of the genetic code. References describing codon usage are readily publicly available. In some embodiments, polynucleotide sequence variants can be produced for a variety of reasons, e.g., to optimize expression for a particular host (e.g., changing codon usage in the alphavirus mRNA to those preferred by other organisms such as human, non-human primates, hamster, mice, or monkey). Accordingly, in some embodiments, the coding sequence is optimized for expressionin a target host cell through the use of codons optimized for expression. The techniques for the construction of synthetic nucleic acid sequences encoding genes using preferred codons optimal for host cell expression may be determined by computational methods analyzing the commonality of codon usage for encoding native proteins of the host cell genome and their relative abundance by techniques known in the art. Various codon usage databases (e.g., www.kazusa.or.jp / codon) may be used for generation of codon optimized sequences in mammalian cell environments. Furthermore, a variety of software tools are available to convert sequences from one organism to the optimal codon usage for a different host organism such as the JCat Codon Optimization Tool (www.jcat.de), Integrated DNA Technologies (IDT) Codon Optimization Tool (https: / / www.idtdna.com / CodonOpt) or the Optimizer online codon optimization tool (http: / / genomes.urv.es / OPTIMIZER). Such synthetic sequences may be constructed by techniques known in the art for the construction of synthetic nucleic acid molecules and may be obtained from a variety of commercial vendors.

[0128] Accordingly, in some embodiments, the coding sequence of the IL-IRA protein and / or IL-1BP is optimized for expression at a level higher than the expression level of a reference coding sequence, such as, for example, a coding sequence that has not been codon- optimized. In some embodiments, the codon-optimized sequence of the IL-IRA protein and / or IL-1BP results in an increased expression level by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% compared to a reference coding sequence that has not been codon-optimized. In some embodiments, the codon- optimized sequence of the IL-IRA protein and / or IL-1BP results in an increased expression level by at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold compared to a reference coding sequence that has not been codon-optimized.

[0129] In some embodiments, the coding sequence of the IL-IRA protein and / or IL-1BP is optimized for enhanced RNA stability and / or expression. The stability of RNA generally relates to the “half-life” of RNA. “Half-life” relates to the period of time which is needed to eliminate half of the activity, amount, or number of molecules. In the context of the present disclosure, the half-life of an RNA is indicative for the stability of said RNA. The half-life of RNA may influence the “duration of expression” of the RNA. Additional information regarding principles, strategies, and methods for use in enhancing RNA stability can be found at, for example, LeppekK. et al., Nature Communications, 22 Mar 2022, 13(1): 1536 .

[0130] In some embodiments, the nucleic acid constructs of the disclosure include a nucleic acid sequence encoding a polypeptide construct of the disclosure, wherein the nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0131] In some embodiments, the nucleic acid constructs of the disclosure include a n nucleic acid sequence encoding a polypeptide construct having 100% sequence identity to the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, wherein one, two, three, four, five, or more nucleotides of the nucleic acid sequence may be substituted by a different nucleotide.

[0132] Nucleic acid sequences having a high degree of sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to a sequence of a modified alphavirus genome or srRNA of interest can be identified and / or isolated by using the sequences identified herein (e.g., SEQ ID NO: 1) or any others as they are known in the art, by genome sequence analysis, hybridization, and / or PCR with degenerate primers or gene-specific primers from sequences identified in the respective alphavirus genome or srRNA.

[0133] The molecular techniques and methods by which these new nucleic acid constructs were assembled and characterized are described more fully in the Examples of the present application. In some embodiments, the nucleic acid molecules are recombinant nucleic acid molecules. As used herein, the term recombinant means any molecule (e.g. DNA, RNA, polypeptide), that is, or results, however indirect, from human manipulation. As non-limiting examples, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector. As non-limiting examples, a recombinant nucleic acid molecule: 1) has been synthesized or modified in vitro, for example, using chemical or enzymatic techniques (for example, by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification(including, e.g., methylation), or recombination (including homologous and site-specific recombination) of nucleic acid molecules; 2) includes conjoined nucleotide sequences that are not conjoined in nature; 3) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleotide sequence; and / or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleotide sequence.

[0134] In some embodiments, the nucleic acid molecules disclosed herein are produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis. Nucleic acid molecules as disclosed herein include natural nucleic acid molecules and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which one or more nucleotide residues have been inserted, deleted, and / or substituted, in such a manner that such modifications provide the desired property in effecting a biological activity as described herein.

[0135] A nucleic acid molecule, including a variant of a naturally-occurring nucleic acid sequence, can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., In: Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). The sequence of a nucleic acid molecule can be modified with respect to a naturally-occurring sequence from which it is derived using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as but not limited to site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and / or mutagenesis of selected regions of a nucleic acid sequence, recombinational cloning, and chemical synthesis, including chemical synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules, and combinations thereof. Nucleic acid molecule homologs can be selected from a mixture of modified nucleic acid molecules by screening for the function of the protein or the srRNA encoded by the nucleic acid molecule and / or by hybridization with a wild-type gene or fragment thereof, or by PCR using primers having homology to a target or wild-type nucleic acid molecule or sequence.B. Recombinant cells

[0136] The nucleic acid constructs of the present disclosure can be introduced into a host cell to produce a recombinant cell containing the nucleic acid molecule. Accordingly, prokaryotic or eukaryotic cells that contain a nucleic acid construct encoding a modified alphavirus genome or srRNA as described herein are also features of the disclosure. In a related aspect, some embodiments disclosed herein relate to methods of transforming a cell which includes introducing into a host cell, such as an animal cell, a nucleic acid construct as provided herein, and then selecting or screening for a transformed cell. Introduction of the nucleic acid constructs of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)- mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like.

[0137] In one aspect, some embodiments of the disclosure relate to recombinant cells, for example, recombinant animal cells that include a nucleic acid construct described herein. The nucleic acid construct can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for a stable or transient expression. Accordingly, in some embodiments of the disclosure, the nucleic acid construct is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid construct is stably integrated into the genome of the recombinant cell. Stable integration can be completed using classical random genomic recombination techniques or with more precise genome editing techniques such as using guide RNA directed CRISPR / Cas9 or TALEN genome editing. In some embodiments, the nucleic acid construct present in the recombinant host cell as a mini-circle expression vector for a stable or transient expression.

[0138] Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule. Accordingly, in some embodiments, host cells can be genetically engineered (e.g., transduced or transformed or transfected) with at least one nucleic acid molecule.

[0139] Suitable host cells for cloning or expression of the polypeptides of interest asdescribed herein include prokaryotic or eukaryotic cells described herein. Accordingly, in some embodiments, the recombinant cell is a prokaryotic cell, such as the bacterium E. coli, or a eukaryotic cell, such as an insect cell (c. ., a mosquito cell or a S£21 cell), or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). In some embodiments, the cell is in vivo, for example, a recombinant cell in a living body, e.g., cell of a transgenic subject. In some embodiments, the subject is a vertebrate animal or an invertebrate animal. In some embodiments, the subject is an insect. In some embodiments, the subject is a mammalian subject. In some embodiments, the cell is ex vivo, e.g., has been extracted, as an individual cell or as part of an organ or tissue, from a living body or organism for a treatment or procedure, and then returned to the living body or organism. In some embodiments, the cell is in vitro, e.g., is obtained from a repository. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate animal cell or an invertebrate animal cell. In some embodiments, the recombinant cell is a mammalian cell. Non-limiting examples of recombinant cells suitable for the methods and compositions of the disclosure include monkey kidney CV1 cells transformed by SV40 (e.g., COS-7 cells), human embryonic kidney cells (e.g., HEK 293 or HEK 293 cells) or derivative cells thereof (e.g., BHK-21 or BHK-570 cells), baby hamster kidney cells (BHK), mouse sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 cells), human cervical carcinoma cells (e.g., HeLa cells), canine kidney cells (MDCK cells), buffalo rat liver cells (e.g., BRL 3A cells), human lung cell (e.g, W138 cells), human liver cell (e.g, Hep G2 cells), mouse mammary tumor (e.g., MMT 060562 cells), TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cell (e.g., Vero cells), human A549 cells, human cervix cells, human CHME5 cells, human PER.C6 cells, NSO murine myeloma cells, human epidermoid larynx cells, human fibroblast cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocyte cells, human macrophage cells, human RAW 264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells.

[0140] In some embodiments, the recombinant cell is selected from the group consisting of African green monkey kidney cell (Vero cell), baby hamster kidney (BHK) cell, Chinese hamster ovary cell (CHO cell), human A549 cell, human cervix cell, human CHME5 cell, humanepidermoid larynx cell, human fibroblast cell, human HEK-293 cell, human HeLa cell, human HepG2 cell, human HUH-7 cell, human MRC-5 cell, human muscle cell, mouse 3T3 cell, mouse connective tissue cell, mouse muscle cell, and rabbit kidney cell.

[0141] In some embodiments, the recombinant cell is an immune cell. In some embodiments, the immune cell is a B cell, a monocyte, a natural killer (NK) cell, a natural killer T (NKT) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell (DC), a macrophage, a regulatory T cell, a helper T cell (Tn), a cytotoxic T cell (TCTL), a memory T cell, a gamma delta (yo) T cell, a hematopoietic stem cell, or a hematopoietic stem cell progenitor. In some embodiments, the immune cell is a B cell, a T cell, a macrophage, or a dendritic cell (DC). In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a T cell.

[0142] In some embodiments, the recombinant cell is a cell derived from a cell described above (i.e., a derivative cell of an original cell described herein) such as, for example, a cell that is either expanded from a clone of the original cell, an engineered version of the original cell, or a reclassification of the original cell after it has undergone extensive passaging, or has been passaged through another host.

[0143] In some embodiments, the recombinant cell is an insect cell, e.g., cell of an insect cell line. In some embodiments, the recombinant cell is a Sf21 cell. Additional suitable insect cell lines include, but are not limited to, cell lines established from insect orders Diptera, Lepidoptera and Hemiptera, and can be derived from different tissue sources. In some embodiments, the recombinant cell is a cell of a lepidopteran insect cell line. In the past few decades, the availability of lepidopteran insect cell lines has increased at about 50 lines per decade. More information regarding available lepidopteran insect cell lines can be found in, e.g., Lynn D.E., Available lepidopteran insect cell lines. Methods Mol Biol. 2007;388: 117-38, which is herein incorporated by reference. In some embodiments, the recombinant cell is a mosquito cell, e.g., a cell of mosquito species within Anopheles (An.), Culex (Cx.) m A des (Stegomyia) (Ae.) genera. Exemplary mosquito cell lines suitable for the compositions and methods described herein include cell lines from the following mosquito species: Aedes aegypti, Aedes albopictus, Aedes pseudoscutellaris, Aedes triseriatus, Aedes vexans, Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Culex quinquefasciatus, Culex theileri, Culextritaeniorhynchus, Culex bitaeniorhynchus, and Toxorhynchites amboinensis. Suitable mosquito cell lines include, but are not limited to, CCL-125, Aag-2, RML-12, C6 / 26, C6 / 36, C7-10, AP- 61, A t. GRIP-1, A t. GRIP-2, UM-AVE1, Mos.55, SualB, 4a-3B, Mos.43, MSQ43, and LSB- AA695BB. In some embodiments, the mosquito cell is a cell of a C6 / 26 cell line.

[0144] In another aspect, provided herein are cell cultures including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art. C. Transgenic animals

[0145] Also provided, in another aspect, are transgenic animals including a nucleic acid construct as described herein (e.g., vector, replicon, or srRNA molecule). In some embodiments, the transgenic animal is a vertebrate animal or an invertebrate animal. In some embodiments, the transgenic animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the transgenic animal is a mammal. In some embodiments, the transgenic mammalian is a non-human mammal. Generally, transgenic animals of the present disclosure can be any non-human animal known in the art. In some embodiments, the non-human animals of the disclosure are non-human primates. Other animal species suitable for the compositions and methods of the disclosure include animals that are (i) suitable for transgenesis and (ii) capable of rearranging immunoglobulin gene segments to produce an antibody response. Examples of such species include but are not limited to mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep and cows. Additional examples of non-human animals suitable for the compositions and methods of the disclosure can include, without limitation, laboratory animals (e.g., mice, rats, hamsters, gerbils, guinea pigs, etc.), livestock (e.g., horses, cattle, pigs, sheep, goats, ducks, geese, chickens, etc. , domesticated animals and pets (e.g. cats, dogs, etc.), non-human primates (e.g., apes, chimpanzees, orangutans, monkeys, etc.), fish, amphibians (e.g., frogs, salamanders, etc.), reptiles (e.g., snakes, lizards, etc.), and other animals (e.g., foxes, weasels, rabbits, mink, beavers, ermines, otters, sable, seals, coyotes, chinchillas, deer, muskrats, possums, etc.).

[0146] In some embodiments, the transgenic animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the transgenic animals of the present disclosure are chimeric transgenic animals. In some embodiments, the transgenic animals of the present disclosure are transgenic animals with germ cells and somatic cells containing one or more (e.g., one or more, two or more, three or more, four or more, etc.) nucleic acid constructs of the present disclosure. In some embodiments, the one or more nucleic acid constructs are stably integrated into the genome of the transgenic animals. In some embodiments, the genomes of the transgenic animals of the present disclosure can comprise any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more copies of the one or more nucleic acid constructs of the present disclosure.

[0147] Approaches and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector mediated DNA transfer into early embryos and sperm-mediated transgenesis, adenovirus mediated introduction of DNA into animal sperm (e.g., in pig), retroviral vectors (e.g., avian species), somatic cell nuclear transfer (e.g., in goats). The state of the art in the preparation of transgenic domestic farm animals is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298. In some embodiments, the transgenic non-human host animals of the disclosure are prepared using standard methods known in the art for introducing exogenous nucleic acid into the genome of a non-human animal. In some embodiments, the transgenic animals of the disclosure can be generated using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR / Cas genome editing, or DNA- guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the transgenic animals of the disclosure can be made using transgenic microinjection technology and do not require the use of homologous recombination technology and thus are considered to be easier to prepare and select than approaches using homologous recombination. In some embodiments, the transgenic animal produces a polypeptide construct as described herein.

[0148] The transgenic non-human host animals of the disclosure are prepared using standard methods known in the art for introducing exogenous nucleic acid into the genome of a non-human animal. In some embodiments, the non-human animals of the disclosure are non- human primates. Other animal species suitable for the compositions and methods of thedisclosure include animals that are (i) suitable for transgenesis and (ii) capable of rearranging immunoglobulin gene segments to produce an antibody response. Examples of such species include but are not limited to mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep and cows. Approaches and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector mediated DNA transfer into early embryos and sperm-mediated transgenesis, adenovirus mediated introduction of DNA into animal sperm (e.g., in pig), retroviral vectors (e.g., avian species), somatic cell nuclear transfer (e.g., in goats). The state of the art in the preparation of transgenic domestic farm animals is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298.

[0149] In some embodiments, the animal is a vertebrate animal or an invertebrate animal. In some embodiments, the animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the animal is a mammalian subject. In some embodiments, the mammalian animal is a non-human animal. In some embodiments, the mammalian animal is a non-human primate. In some embodiments, the transgenic animals of the disclosure can be made using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR / Cas genome editing, or DNA-guided endonuclease genome editing with NgAgo (Natrojiobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the transgenic animals of the disclosure may be made using transgenic microinjection technology and do not require the use of homologous recombination technology and thus are considered to be easier to prepare and select than approaches using homologous recombination. In another aspect, provided herein are methods for producing a polypeptide of interest, wherein the methods include (i) rearing a transgenic animal as disclosed herein; or (ii) culturing a recombinant cell including a nucleic acid construct as disclosed herein under conditions wherein the transgenic animal or the recombinant cell produces the polypeptide construct encoded by the nucleic acid construct disclosed herein. In another aspect, provided herein are methods for producing a polypeptide of interest in a subject, wherein the methods include administering to the subject a nucleic acid construct as disclosed herein. In some embodiments, the subject is vertebrate animal or an invertebrate animal. In some embodiments, the subject is an insect. In some embodiments, the insect is a mosquito. In someembodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a non-human subject. In some embodiments, the mammalian subject is a human subject.D. Pharmaceutical compositions

[0150] The nucleic acid constructs (for example, replicon constructs, e.g., srRNA constructs) and recombinant cells of the disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally include one or more of the nucleic acid constructs (for example, replicon constructs, e.g., srRNA constructs) and recombinant cells described and provided herein, and a pharmaceutically acceptable excipient, e.g., carrier. In some embodiments, the compositions of the disclosure are formulated for the prevention, treatment, or management of a health condition such as an autoimmune disease, an inflammatory disease, or a cardiovascular disease. For example, the compositions of the disclosure can be formulated as a prophylactic composition, a therapeutic composition, or a pharmaceutical composition comprising a pharmaceutically acceptable excipient, or a mixture thereof. In some embodiments, the compositions of the present application are formulated for use as an adjuvant.

[0151] Accordingly, in one aspect, provided herein are pharmaceutical compositions including a pharmaceutically acceptable excipient and: (a) a nucleic acid construct of the disclosure; and / or (b) a recombinant cell of the disclosure.

[0152] Non-limiting exemplary embodiments of the pharmaceutical compositions of the disclosure can include one or more of the following features. In some embodiments, provided herein are compositions including a nucleic acid construct as disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, provided herein are compositions including a recombinant cell as disclosed herein and a pharmaceutically acceptable excipient.

[0153] In some embodiments, the nucleic acid constructs of the disclosure (e.g., a vectors or srRNA molecules) can be used in a naked form or formulated with a delivery vehicle. Exemplary delivery vehicles suitable for the compositions and methods of the disclosure include, but are not limited to liposomes e.g., neutral or anionic liposomes), microspheres, immune stimulating complexes (ISCOMS), lipid-based nanoparticles (LNP), solid lipid nanoparticles (SLN), polyplexes, polymer nanoparticles, viral replicon particles (VRPs), or conjugated with bioactive ligands, which can facilitate delivery and / or enhance the immune response. Thesecompounds are readily available to one skilled in the art; for example, see Liposomes: A Practical Approach, RCP New Ed, IRL press (1990). Adjuvants other than liposomes and the like are also used and are known in the art. Adjuvants may protect the antigen (e.g., nucleic acid constructs, vectors, srRNA molecules) from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. An appropriate selection can be made by those skilled in the art, for example, from those described below.

[0154] The composition of the disclosure can be formulated in a format to be compatible with its intended route of administration, such as liposome, lipid-based nanoparticle (LNP), a polymer nanoparticle, a polyplex, viral replicon particle (VRP), microsphere, immune stimulating complex (ISCOM), conjugate of bioactive ligand, or a combination of any thereof. Accordingly, in some embodiments, the compositions of the disclosure can be formulated in a liposome.

[0155] In some embodiments, the compositions of the disclosure that formulated in a lipid- based nanoparticle (LNP). Exemplary types of lipids suitable for the delivery systems described herein include cationic lipids, ionizable cationic lipids, anionic lipids, neutral lipids, and combinations thereof.

[0156] In some embodiments, the LNP of the disclosure can include one or more ionizable lipids. Exemplary ionizable lipids suitable for the compositions and methods of the disclosure includes those described in PCT publications WO2020252589A1 and W02021000041A1, and Love K.T. etal., Proc Natl Acad Sci USA, Feb. 2, 2010 107 (5) 1864-1869, which are incorporated by reference herein in their entirety.

[0157] Accordingly, in some embodiments, the LNP of the disclosure includes one or more lipid compounds described in Love K.T. et al., 2010 supra, such as C16-96, C14-110, and C12- 200. In some embodiments, the LNP includes an ionizable cationic lipid selected from the group consisting of ALC-0315, C12-200, LN16, MC3, MD1, SM-102, and a combination of any thereof. In some embodiments, the LNP of the disclosure includes C12-200.

[0158] In some embodiments, the LNP of the disclosure includes one or more cationic lipids. Suitable cationic lipids include, but are not limited to, 98N12-5, C 12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1.

[0159] In some embodiments, the LNP of the disclosure includes one or more neutral lipids. As described above, neural lipids, also known as “structural lipids” or “helper lipids” can also be incorporated into lipid formulations and lipid particles in some embodiments. The lipid formulations and lipid particles can include one or more structural lipids at about 10 to 40 Mol% of the composition. Suitable structural lipids support the formation of particles during manufacture. Structural lipids refer to any one of a number of lipid species that exist in either in an anionic, uncharged or neutral zwitterionic form at physiological pH. Representative structural lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, diacylphosphatidylglycerols, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.

[0160] Exemplary structural lipids include zwitterionic lipids, for example, di stearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl- phosphatidy ethanol amine (SOPE), and 1,2-dielaidoyl- sn-glycero-3-phophoethanolamine (trans DOPE).

[0161] In another embodiment, the structural lipid can be any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerols such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyolphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups joined to neutral lipids. Other suitable structural lipids include glycolipids (e.g., monosialoganglioside GM1).

[0162] Non-limiting neutral lipids suitable for the compositions and methods of the disclosure include DPSC, DPPC, POPC, DOPE, and SM. In some embodiments, the LNP of the disclosure includes one or more ionizable lipid compounds described in PCT publications WO2020252589A1 and W02021000041A1, which are incorporated by reference herein in theirentirety.

[0163] In some embodiments, the LNP of the disclosure includes at least one lipid selected from the group consisting of C 12-200, C14-PEG2000, DOPE, DMG-PEG2000, DSPC, DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE- DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).

[0164] In some embodiments where the delivery systems described herein include an LNP, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 100: 1 to about 3: 1, about 70: 1 to 10:1, or 16: 1 to 4: 1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 16: 1 to 4: 1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 20: 1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 8: 1. In some embodiments, the lipid- based nanoparticles (LNPs) have an average diameter of less than about 1000 nm, about 500 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 75 nm, about 50 nm, or about 25 nm. In some embodiments, the LNPs have an average diameter ranging from about 70 nm to 100 nm. In some embodiments, the LNPs have an average diameter ranging from about 88 nm to about 92 nm, from 82 nm to about 86 nm, or from about 80 nm to about 95 nm.

[0165] Stabilizing agents can be included in lipid formulations embodiments to ensure integrity of the mixtures. Stabilizing agents are a class of molecules which disrupt or help form the hydrophobic-hydrophilic interactions among molecules. Suitable Stabilizing agents include, but are not limited to, polysorbate 80 (also known as Tween 80, 1UPAC name 2-[2-[3,4-bis(2- hydroxyethoxy)oxolan-2-yl]-2- (2-hydroxy ethoxy )ethoxy]ethyl octadec-9-enoate), Myrj52 (Polyoxyethylene (40) stearate), and Brij™ S10 (Polyoxyethylene (10) stearyl ether). Polyethylene glycol conjugated lipids may also be used. The stabilizing agents may be used alone or in combinations with each other.

[0166] In some embodiments, the stabilizing agents comprises about .1 to 3 Mol% of the overall lipid mixture. In some embodiments, the stabilizing agents comprise about 0.5 to 2.5 Mol% of the overall lipid mixture. In some embodiments, the stabilizing agent is present at greater than 2.5 Mol%. In some embodiments the stabilizing agent is present at 5 Mol%. In some embodiments the stabilizing agent is present at 10 Mol%. In some embodiments, the stabilizing agent is about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, and so forth. In other embodiments, the stabilizing agent is 2.6-10 Mol% of the lipid mixture. In other embodiments, the stabilizing agents is present at greater than 10 Mol% of the lipid mixture.

[0167] Steroids can also be included in the lipid compositions for certain applications, and lipid particles made therefrom include sterols, such as cholesterol and phytosterol.Polymer nanoparticle

[0168] In some embodiments, the compositions of the disclosure can be formulated in a polymer nanoparticle. In some embodiments, the polymeric nanoparticle includes a cationic polymer, a non-cationic polymer, or a combination thereof. In some embodiments, the cationic polymer includes a naturally-derived cationic polymer. In some embodiments, the naturally- derived cationic polymer includes chitosan, gelatin, dextran, cellulose, cyclodextrin, or a combination thereof. In some embodiments, the cationic polymer includes a synthetic cationic polymer. In some embodiments, the synthetic cationic polymer includes a polyethyleneimine (PEI), poly-L-lysine (PLL), a poly(amino acid) (PAA), a poly(amidoamine) (PAMAM), a poly(cystamine bisacrylamide-co-4-amino-l-butanol) (pABOL), a poly(amino-co-ester) (PAE), poly(2-N,N-dimethylaminoethylmethacrylate, a poly(beta-amino ester) (PBAE), an imidazole- containing polymer, a tertiary-amine containing polymer, poly(2-(dimethylamino)ethyl methacrylate), poly-N-(2-hydroxy-propyl)methacrylamide, a polyamidoamine dendrimer, a cationic glycopolymer, or derivatives thereof.

[0169] In some embodiments, the non-cationic polymer is negatively -charged (i.e., anionic) or electronically neutral. In some embodiments, the non-cationic polymer includes a polyethylene glycol (PEG), a polyester (e.g., polylactic acid (PLA), poly (lactic-co-glycolic acid) (PLGA), poly glycolic acid (PGA), polycaprolactone (PCL)), and polysarcosine (pSar), or derivatives thereof. In some embodiments, the polymer is water-soluble and / or biodegradable.

[0170] In some embodiments of the disclosure, the polymeric nanoparticle includes one or more of the following: poly-(y-L-glutamylglutamine) (PGGA), poly-(y-L-aspartylglutamine) (PGAA), poly-L-lactic acid (PLLA), poly-(lactic acid-co-glycolic acid) (PLGA), polyalkylcyanoacrylate (PACA), poly anhydrides, polyhydroxy acids, polypropylfumerate, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, [N-(2- hydroxypropyl)methacrylamide] (HPMA) copolymer, polyvinyl alcohol, polyurethane,polyphosphazene, polyacrylate, polyurea, polyamine polyepsilon-caprolactone (PCL), and copolymers thereof.Lipid-based nanoparticle (LNP)

[0171] In some embodiments, the compositions of the disclosure can be formulated in a lipid-based nanoparticle (LNP). For example, the nucleic acid constructs of the disclosure can be delivered to a cell or a subject by a lipid-based nanoparticle (LNP). LNP are generally less immunogenic than viral particles. While many humans have preexisting immunity to viral particles there is no pre-existing immunity to LNP. In addition, adaptive immune response against LNP is unlikely to occur which enables repeat dosing of LNP.

[0172] The lipids suitable for the compositions and methods described herein can be cationic lipids, ionizable cationic lipids, anionic lipids, or neutral lipids.

[0173] In some embodiments, the LNP of the disclosure can include one or more ionizable lipids. As used herein, the term "ionizable lipid" refers to a lipid that is cationic or becomes ionizable (protonated) as the pH is lowered below the pKa of the ionizable group of the lipid, but is more neutral at higher pH values. At pH values below the pKa, the lipid is then able to associate with negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term "ionizable lipid" includes lipids that assume a positive charge on pH decrease from physiological pH, and any of a number of lipid species that carry a net positive charge at a selective pH, such as physiological pH. Permanently cationic lipids such as DOTMA have proven too toxic for clinical use. The ionizable lipid can be present in lipid formulations according to other embodiments, preferably in a ratio of about 30 to about 70 Mol%, in some embodiments, about 30 Mol%, in other embodiments, about 40 Mol%, in other embodiments, about 45 Mol% in other embodiments, about 47.5 Mol% in other embodiments, about 50 Mol%, in still other embodiments, and about 60 Mol% in yet others (“Mol%” means the percentage of the total moles that is of a particular component), the LNP of the disclosure can include DODMA, or 1 ,2- dioleyloxy-3-dimethylaminopropane, which is an ionizable lipid, as is DLin-MC3-DMA or 0- (Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N- dimethylamino) (“MC3”).

[0174] Exemplary ionizable lipids suitable for the compositions and methods of the disclosure includes those described in PCT publications WO2020252589A1 and W02021000041A1, U.S. Patent Nos. 8,450,298 and 10,844,028, and Love K.T. etal., Proc NatlAcad Sci USA, Feb. 2, 2010 107 (5) 1864-1869, all of which are hereby incorporated by reference in their entirety. Accordingly, in some embodiments, the LNP of the disclosure includes one or more lipid compounds described in Love K.T. et al., 2010 supra, such as Cl 6-96, C14-110, and C12-200. In some embodiments, the LNP includes an ionizable cationic lipid selected from the group consisting of ALC-0315, C12-200, LN16, MC3, MD1, SM-102, and a combination of any thereof. In some embodiments, the LNP of the disclosure includes Cl 2-200. The structure of C 12-200 lipid is known in the art and described in, e.g., U.S. Patent Nos. 8,450,298 and 10,844,028, which are hereby incorporated by reference in their entirety. In some embodiments the C12-200 is combined with cholesterol, C14-PEG2000, and DOPE. In some embodiments, the C12-200 is combined with DSPC and DMG-PEG2000.

[0175] In some embodiments, the LNP of the disclosure includes one or more cationic lipids. Suitable cationic lipids include, but are not limited to, 98N12-5, C12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. In some embodiments, the LNP of the disclosure includes one or more neutral lipids. Non-limiting neutral lipids suitable for the compositions and methods of the disclosure include DPSC, DPPC, POPC, DOPE, and SM. In some embodiments, the LNP of the disclosure includes one or more ionizable lipid compounds described in PCT publications WO2020252589A1 and W02021000041A1, which are hereby incorporated by reference in their entirety.

[0176] A number of other lipids or combination of lipids that are known in the art can be used to produce an LNP. Non-limiting examples of lipids suitable for use to produce LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP- DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Non-limiting examples of cationic lipids include 98N12-5, C12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, 7C1, and a combination of any thereof. Non-limiting examples of neutral lipids include DPSC, DPPC, POPC, DOPE, and SM. Non-limiting examples of PEG-modified lipids include PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0177] In some embodiments, the LNP of the disclosure includes at least one lipid selected from the group consisting of C12-200, C14-PEG2000, DOPE, DMG-PEG2000, DSPC, DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP cholesterol, GAP-DMORIE DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). In some embodiments the C12-200 is combined with cholesterol, C14-PEG2000, and DOPE. In some embodiments, the Cl 2-200 is combined with DSPC and DMG-PEG2000.

[0178] In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 100:1 to about 3: 1, about 70: 1 to 10: 1, or 16: 1 to 4: 1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 16:1 to 4: 1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 20: 1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 8: 1. In some embodiments, the lipid-based nanoparticles have an average diameter of less than about 1000 nm, about 500 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 75 nm, about 50 nm, or about 25 nm. In some embodiments, the LNPs have an average diameter ranging from about 70 nm to 100 nm. In some embodiments, the LNPs have an average diameter ranging from about 88 nm to about 92 nm, from 82 nm to about 86 nm, or from about 80 nm to about 95 nm.

[0179] As described above, neural lipids, also known as “structural lipids” or “helper lipids” can also be incorporated into lipid formulations and lipid particles in some embodiments. The lipid formulations and lipid particles can include one or more structural lipids at about 10 to 40 Mol% of the composition. Suitable structural lipids support the formation of particles during manufacture. Structural lipids refer to any one of a number of lipid species that exist in either in an anionic, uncharged or neutral zwitterionic form at physiological pH. Representative structural lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, diacylphosphatidylglycerols, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.

[0180] Exemplary structural lipids include zwitterionic lipids, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanol amine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1 -stearoyl-2-oleoyl- phosphatidy ethanol amine (SOPE), and 1 ,2-di elaidoyl-sn-glycero-3- phophoethanolamine (trans DOPE).

[0181] In another embodiment, the structural lipid can be any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerols such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyolphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups joined to neutral lipids. Other suitable structural lipids include glycolipids (e.g., monosialoganglioside GM1).

[0182] Stabilizing agents can be included in lipid formulations embodiments to ensure integrity of the mixtures. Stabilizing agents are a class of molecules which disrupt or help form the hydrophobic-hydrophilic interactions among molecules. Suitable Stabilizing agents include, but are not limited to, polysorbate 80 (also known as Tween 80, 1UPAC name 2-[2-[3,4-bis(2- hydroxyethoxy)oxolan-2-yl]-2- (2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate), Myrj52 (Polyoxyethylene (40) stearate), and Brij™ S10 (Polyoxyethylene (10) stearyl ether). Polyethylene glycol conjugated lipids may also be used. The stabilizing agents may be used alone or in combinations with each other.

[0183] In some embodiments, the stabilizing agents comprises about .1 to 3 Mol% of the overall lipid mixture. In some embodiments, the stabilizing agents comprise about 0.5 to 2.5 Mol% of the overall lipid mixture. In some embodiments, the stabilizing agent is present at greater than 2.5Mol%. In some embodiments the stabilizing agent is present at 5 Mol%. In some embodiments the stabilizing agent is present at 10 Mol%. In some embodiments, the stabilizing agent is about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, and so forth. In other embodiments, the stabilizing agent is 2.6-10 Mol% of the lipid mixture. In other embodiments, the stabilizing agents is present at greater than 10 Mol% of the lipid mixture.

[0184] Steroids can also be included in the lipid compositions for certain applications, and lipid particles made therefrom include sterols, such as cholesterol and phytosterol.

[0185] In some embodiments, the immunogenic compositions are substantially non- immunogenic or minimally immunogenic (e.g. compositions that minimally stimulate an immune response in a subject. In some embodiments, the non-immunogenic or minimallyimmunogenic compositions are formulated as a biotherapeutic. In some embodiments, the pharmaceutical compositions are formulated for one or more of intranasal administration, transdermal administration, intraperitoneal administration, intramuscular administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, cardiac administration and oral administration.

[0186] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™. (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS), tris (tromethamine), and HEPES. In these cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage, and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be generally to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sucrose, trehalose, and / or sodium chloride in the composition. In some embodiments, the composition comprises tris and sucrose. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0187] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by fdtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basicdispersion medium and the required other ingredients from those enumerated above.

[0188] In some embodiments, the pharmaceutical compositions are formulated for one or more of intranasal administration, transdermal administration, intrathecal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, intraocular administration, rectal administration, and oral administration.

[0189] In some embodiments, the pharmaceutical compositions of the disclosure are formulated for inhalation, such as an aerosol, spray, mist, liquid, or powder. Administration by inhalation may be in the form of either dry powders or aerosol formulations, which are inhaled by a subject (e.g., a patient) either through use of an inhalation device, e.g., a microspray, a pressurized metered dose inhaler, or nebulizer.METHODS OF THE DISCLOSURE

[0190] Administration of any one of the therapeutic compositions described herein, e.g., nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions, can be used for modulating at least one pharmacodynamics effect in a subject, or can be used in the treatment of relevant health conditions, such as autoimmune diseases and / or inflammatory diseases.

[0191] In some embodiments, the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as described herein can be useful for modulating, e.g., eliciting or suppressing a pharmacodynamic effect in a subject in need thereof. In some embodiments, the pharmacodynamic effect includes eliciting an immune response in the subject. Non-limiting examples of pharmacodynamic effect include immunogenicity effects, biomarker responses, therapeutic effects, prophylactic effects, desired effects, undesired effects, adverse effects, and effects in a disease model.

[0192] Accordingly, one aspect of the disclosure relates to methods for modulating a pharmacodynamic effect in a subject in need thereof, the methods include administering to the subject a composition including one or more of the following: (a) a nucleic acid construct as described herein; (c) a recombinant cell as described herein; and (c) a pharmaceutical composition as described herein. In some embodiments, the pharmacodynamic effect includes one or more of the following: immunogenicity effect, a biomarker response, a therapeutic effect,a prophylactic effect, a desired effect, an undesired effect, an adverse effect, and effect in a disease model. In some embodiments, the pharmacodynamic effect includes eliciting an immune response in the subj ect.

[0193] In some embodiments, the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as described herein can be used in a method for improving and / or prolonging kinetics of expression of IL-IRA and / or IL-18BP in a subject. In this instance, the use of a srRNA-based expression system allows for the production of IL-IRA and / or IL-18BP for a longer period of time compared to an expression system relying on administration of mRNAs or recombinant proteins. Thus, the use of a srRNA-based expression system results in an effective longer "half-life" of IL-IRA and IL-18BP in the subject, as compared to direct administration of mRNAs or recombinant proteins. In a related aspect, the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as described herein can be used in a method for enhancing the endogenous expression of IL-IRA and / or IL-18BP in a subject. In some embodiments, the methods include administering to the subject a composition including one or more of the following: (a) a nucleic acid construct as described herein; (c) a recombinant cell as described herein; and (c) a pharmaceutical composition as described herein.

[0194] In some embodiments, the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as described herein can be incorporated into therapeutic agents for use in methods of treating a subject who has, who is suspected of having, or who may be at high risk for developing one or more relevant health conditions or diseases (e g., autoimmune disease). Exemplary health conditions or diseases can include, without limitation, immune diseases, autoimmune diseases, inflammatory diseases). In some embodiments, the subject is a patient under the care of a physician.

[0195] Accordingly, in another aspect, provided herein are methods for preventing or treating a health condition in a subject, the methods include prophylactically or therapeutically administering to the subject a composition including one or more of the following: (a) a replicon, e.g., self-replicating RNA construct (srRNA) as described herein; (b) a nucleic acid as described herein; (c) a recombinant cell as described herein; and (d) a pharmaceutical composition as described herein. In some embodiments, the administered composition elicits an immune response in the subject. In some embodiments, the administered composition extends thepharmacokinetics of a protein that has a short half-life in vivo. In some embodiments, the protein with a short half-life in vivo is an endogenous protein. In some embodiments, the protein with a short half-life in vivo is an exogenous protein. In some embodiments, the administered composition induces production of one or more pro-inflammatory molecules in the subject. In some embodiments, the one or more pro-inflammatory molecules includes interleukin- 1 alpha (IFNoc), inteleukin-1 beta (IFNP), interleukin- 18 (IL-18), interleukin-6 (IL-6), interferon gamma (IFNy), cytokines, TNF-a, GM-CSF, and MIPla, granzyme B, granzyme A, perforin, or a combination of any thereof. In some embodiments, the subject has been previously treated with one or more therapies and has developed at least a partial resistance to said one or more therapies.

[0196] In some embodiments, the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of the disclosure can be useful in the treatment and / or prevention of immune diseases, autoimmune diseases, or inflammatory diseases.

[0197] Non-limiting examples of inflammatory suitable for the methods of the disclosure include inflammatory diseases such as asthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly, and rheumatoid arthritis. In one aspect, provided herein are methods for inducing a pharmacodynamic effect in a subject, the methods include administering to the subject a composition including one or more of the following: (a) a nucleic acid construct as described herein; (b) a recombinant cell as described herein; and (b) a pharmaceutical composition as described herein. In some embodiments, the pharmacodynamic effect includes eliciting an immune response in the subject.

[0198] Examples of autoimmune diseases suitable for the methods of the disclosure include, but are not limited to, rheumatoid arthritis, osteoarthritis, Still’s disease, Familiar Mediterranean Fever, systemic sclerosis, multiple sclerosis, ankylosing spondylitis, systemic lupus erythematosus, Sjogren's syndrome, diabetic retinopathy, diabetic vasculopathy, diabetic neuralgia, insulitis, psoriasis, alopecia greata, warm and cold autoimmune hemolytic anemia (AIHA), pernicious anemia, acute inflammatory diseases, autoimmune adrenalitis, chronic inflammatory demyelinating polyneuropathy (CIDP), Lambert-Eaton syndrome, lichen sclerosis, Lyme disease, Graves disease, Behcet's disease, Meniere's disease, reactive arthritis (Reiter's syndrome), Churg-Strauss syndrome, Cogan syndrome, CREST syndrome, pemphigus vulgarisand pemphigus foliaceus, bullous pemphigoid, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, pancreatitis, peritonitis, psoriatic arthritis, rheumatic fever, sarcoidosis, Sjbrgensen syndrome, scleroderma, celiac disease, stiff-man syndrome, Takayasu arteritis, transient gluten intolerance, autoimmune uveitis, vitiligo, polychondritis, dermatitis herpetiformis (DH) or Duhring's disease, fibromyalgia, Goodpasture syndrome, Guillain-Barre syndrome, Hashimoto’s thyroiditis, autoimmune hepatitis, inflammatory bowel disease (IBD), Crohn's disease, colitis ulcerosa, myasthenia gravis, immune complex disorders, glomerulonephritis, polyarteritis nodosa, anti-phospholipid syndrome, polyglandular autoimmune syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), urticaria, autoimmune infertility juvenile rheumatoid arthritis, sarcoidosis, and autoimmune cardiomyopathy.

[0199] Non limiting examples of cardiovascular diseases suitable for the methods of the disclosure include blood pressure, hypertension, dyslipidemia, diabetes, coronary artery disease, stroke, pulmonary embolism, peripheral artery disease (PAD), serum cholesterol, and serum homocysteine or platelet function.

[0200] As described above, administration of any one of the therapeutic compositions described herein, e.g., nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions, can be used for inducing at least one pharmacodynamic effect in a subject. In some embodiments, the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions described herein are analyzed for their capacity to confer at least one pharmacodynamic effect is carried out in vivo or ex vivo. Examples of pharmacodynamic effects that can be analyzed include: immunogenicity effect (e.g., eliciting an immune response in vivo), a biomarker response, a therapeutic effect, a prophylactic effect, a desired effect, an undesired effect, an adverse effect, and effect in a disease model. In some embodiments, the assessment of pharmacodynamic effects includes assessing induction of an immune response in vivo. In some embodiments, the assessment of pharmacodynamic effects includes assessing induction of cytokine pathways that can potentiate an immune response and prevent angiogenesis and metastasis.

[0201] In some embodiments, the disclosed composition is formulated to be compatible with its intended route of administration. For example, the nucleic acid constructs e.g, srRNAconstructs), recombinant cells, and / or pharmaceutical compositions of the disclosure may be given orally or by inhalation, but it is more likely that they will be administered through a parenteral route. Examples of parenteral routes of administration include, for example, intramuscular, intratumoral, intraocular, intravenous, intranodal, intradermal, subcutaneous, transdermal (topical), transmucosal, intravaginal, and rectal administration. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered intratumorally or via cardiac administration. Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, phosphates, tris, sucrose and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as mono- and / or di-basic sodium phosphate, hydrochloric acid or sodium hydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5). The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0202] Dosage, toxicity and therapeutic efficacy of such subject nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of the disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are generally suitable. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0203] For example, the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed andthe route of administration utilized. For any compound used in the method of the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (e.g., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0204] The therapeutic compositions described herein, e.g., nucleic acid constructs, e.g., srRNA constructs, recombinant cells, and / or pharmaceutical compositions, can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the subject multivalent polypeptides and multivalent antibodies of the disclosure can include a single treatment or, can include a series of treatments. In some embodiments, the compositions are administered every 8 hours for five days, followed by a rest period of 2 to 14 days, e.g., 9 days, followed by an additional five days of administration every 8 hours. With regard to nucleic acid constructs (for example, replicon constructs, e.g., srRNA constructs), the therapeutically effective amount of a nucleic acid construct of the disclosure (e.g., an effective dosage) depends on the nucleic acid construct selected.

[0205] As discussed supra, a therapeutically effective amount includes an amount of a therapeutic composition that is sufficient to promote a particular effect when administered to a subject, such as one who has, is suspected of having, or is at risk for a health condition, e.g., an autoimmune disease, an inflammatory disease, or a cardiovascular disease. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease.

[0206] The efficacy of a treatment including a disclosed therapeutic composition for thetreatment of health condition or disease can be determined by the skilled clinician. However, a treatment is considered effective treatment if at least any one or all of the signs or symptoms of disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the health condition or disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a health condition or disease in a subject or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the health condition or disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the health condition or disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0207] In some embodiments, the nucleic acid constructs (for example, replicon constructs, e.g., srRNA constructs), recombinant cells, and / or pharmaceutical compositions of the disclosure can be administered to a subject in a composition having a pharmaceutically acceptable carrier and in an amount effective to stimulate an immune response. Generally, a subject can be immunized through an initial series of injections (or administration through one of the other routes described below) and subsequently given boosters to increase the protection afforded by the original series of administrations. The initial series of injections and the subsequent boosters are administered in such doses and over such a period of time as is necessary to stimulate an immune response in a subject. In some embodiments, the administered composition results in an increased production of interferon in the subject. In some embodiments of the disclosed methods, the subject is a mammal. In some embodiments, the mammal is a human subject.

[0208] When the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions are suitably protected, as described above, they may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the individual's diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0209] In some embodiments, the nucleic acid constructs of the disclosure can be delivered to a cell or a subject by a lipid-based nanoparticle (LNP). LNP are generally less immunogenic than viral particles. While many humans have preexisting immunity to viral particles there is no pre-existing immunity to LNP. In addition, adaptive immune response against LNP is unlikely to occur which enables repeat dosing of LNP.Additional therapies

[0210] In some embodiments, a composition according to the present disclosure is administered to the subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapies (e. ., second therapy). In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy or surgery. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy is administered at the same time as the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in rotation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.KITS

[0211] Also provided herein are various kits for the practice of a method described herein as well as written instructions for making and using the same. In particular, some embodiments of the disclosure provide kits for modulating (e.g., inducing, eliciting, or suppressing) a pharmacodynamic effect. Some embodiments of the disclosure provide kits for eliciting an immune response in a subject. Some other embodiments relate to kits for the prevention of a health condition, e.g., an autoimmune disease, in a subject in need thereof. Some other embodiments relate to kits for methods of treating a health condition, e.g., an autoimmune disease, in a subject in need thereof. For example, provided herein, in some embodiments, arekits that include one or more of the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as provided and described herein, as well as written instructions for making and using the same.

[0212] In some embodiments, the kits of the disclosure further include one or more means useful for the administration of any one of the provided nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions to a subject. For example, in some embodiments, the kits of the disclosure further include one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer any one of the provided nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions to a subject. In some embodiments, a kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the other kit components for a desired purpose, e.g., for diagnosing, preventing, or treating a condition in a subject in need thereof.

[0213] Any of the above-described kits can further include one or more additional reagents, where such additional reagents can be selected from: dilution buffers, reconstitution solutions, wash buffers, control reagents, control expression vectors, negative controls, positive controls, reagents suitable for in vitro production of the provided nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of the disclosure.

[0214] In some embodiments, the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container. Accordingly, in some embodiments of the disclosure, the kit includes one or more of the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as provided and described herein in one container (e.g., in a sterile glass or plastic vial) and a further therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0215] In another embodiment, the kit includes a combination of the compositions described herein, including one or more nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of the disclosure in combination with one or more further therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.

[0216] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device (e.g., an injection device or catheter) for performing suchadministration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above containing one or more nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of the disclosure.

[0217] In some embodiments, the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container.

[0218] In some embodiments, a kit can further include instructions for using the components of the kit to practice the methods disclosed herein. For example, the kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the disclosure may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, over-dosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer / distributor information and intellectual property information.

[0219] The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof e.g., associated with the packaging or subpackaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0220] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0221] No admission is made that any reference cited herein constitutes prior art. Thediscussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0222] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.

[0223] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.EXAMPLES

[0224] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology . New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY : Wiley -Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed ). New York, NY: Cold Spring Harbor Laboratory Press;Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY : Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression inMammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

[0225] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.EXAMPLE 1Construction of modified alphavirus vectors

[0226] This Example describes the experiments performed to construct base alphavirus vectors (e.g., without a heterologous gene) that were subsequently used for construction of vectors that express a gene or genes of interest (e.g., (i) IL-IRA protein or a functional variant thereof and / or (ii) IL-18BP or functional variant thereof).

[0227] The base EEEV vector (i.e. without a heterologous gene of interest) was constructed as follows: The base EEEV vector was synthesized de novo in four ~4 kb parts (Twist Bioscience) from a reference sequence (Genbank EFl 51502) with several modifications. Silent mutations G301A, A3550C, G4516A, G5725A, G7399A mutations were incorporated to eliminate restriction enzyme cut sites. A unique restriction enzyme cut site (Spel, 5’-A’CTAG,T- 3’) was incorporated in place of the coding sequence of the native EEEV structural genes (where the 5’ A matches the location of the structural polyprotein ATG start codon, and the 3’ T matches the location of the structural polyprotein stop codon TAA). A 5’ adaptor sequence (5’- CTGGAGACGTGGAGGAGAACCCTGGACCT-3’; SEQ ID NO: 3) was inserted upstream of the Spel site, and a 3’ adaptor sequence (5’-GACCGCTACGCCCCAATGACCCGACCAGC-3’; SEQ ID NO: 4) was inserted downstream of the Spe site for subsequent Gibson Assembly® procedures (Gibson et al., Nat. Methods 6, 343-345, 2009). A bacteriophage T7 RNA polymerase promoter (5’-TAATACGACTCACTATAG-3’; SEQ ID NO: 5) was included upstream of the EEEV genome sequence, and downstream contained a poly(A) sequence followed by a Sapl site, which cuts upstream of the recognition site. Immediately downstream of the Sap\ site is a T7 terminator sequence (5’-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3’; SEQ ID NO: 6) followed by a unique restriction enzyme cut site (Notl, 5’-GC’GGCC,GC-3’).The parts were combined in a five-piece Gibson Assembly® reaction: a linearized pYL backbone and the four synthesized fragments to result in the EEEV base vector.

[0228] Other base srRNA vectors (VEEV, CHIKV, and SINV) were assembled similarly to the construction of the base EEEV vector as described above.

[0229] Construction of vectors containing heterologous genes was carried out as follows: the empty base vector was linearized by Spel digestion. The IL-1RN and IL-18BP genes were codon optimized / refactored for human expression in silica and along with an encephalomyocarditis virus (EMCV) IRES were synthesized de novo (IDT). The synthetic products were amplified using primers which added either 5’ and 3’ adaptor sequences to the ends of the genes, or primers which added P2A sequences and / or sequences of homology to neighboring gene inserts. The digestion product and PCR products were combined by Gibson Assembly® procedure to result in the final vectors. A summary of the modified alphavirus vectors prepared in these experiments is presented in Table 1 below. The amino acid sequences of the gene cassettes are also presented in the Sequence Listing.

[0230] TABLE 1 :EXAMPLE 2In vitro evaluation of modified alphavirus vectors

[0231] This Example describes the results of in vitro experiments performed to evaluate expression levels of the synthetic srRNA constructs described in Example 1 above, and to investigate any differential behavior thereof (e.g., replication and protein expression).

[0232] In vitro transcription'. RNA was prepared by in vitro transcription from a Sapl- linearized plasmid template with bacteriophage T7 RNA polymerase with either a 5’ ARC A cap (HiScribe™ T7 ARCA mRNA Kit, NEB) or by uncapped transcription (HiScribe™ T7 High Yield RNA Synthesis Kit, NEB) followed by addition of a 5’ cap 1 (Vaccinia Capping System, mRNA Cap 2'-O-Methyltransferase, NEB). RNA is then purified using phenol / chloroform extraction, or column purification (Monarch® RNA Cleanup Kit, NEB). RNA concentration is determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0233] Replication'. RNA was transformed by electroporation into BHK-21 or Vero cells (e. , 4D-Nucleofector™, Lonza). At 15-22 hours following transformation, the cells were fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained using a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons) to quantify the frequency of dsRNA+ cells by fluorescence flow cytometry.

[0234] Protein expression by ELISA : Human IL-IRA and IL-18BP were detected from electroporated BHK-21 cells with monogenic or multigenic srRNA constructs. Supernatants were harvested at approximately 24 and 48 hours after transfection and assayed with Human IL- Ira / IL-1F3 DuoSet ELISA (R&D Systems, cat# DY280) or Human IL-18 Bpa DuoSet ELISA (R&D Systems, cat# DY119).

[0235] FIGS. 3 and 5 summarize the results of the ELISA experiments performed to measure in vitro expression of IL-18BP and IL-IRA in monogenic and / or bigenic expression cassettes. IL-18BP expression was observed from the monogenic and all the bigenic expression cassettes at 24 and 48 hours post-transformation (see, e.g., FIG. 3), with the highest expression level observed from the IL1RA-IRES-IL18BP cassette. IL- IRA expression was observed in all bigenic cassettes at 24 and 48 hours post-transformation (see, e.g., FIG. 5), with the highest expression level observed from the IL18BP-IRES-IL1RA cassette.

[0236] Bioactivity assay. Functional human IL-IRA and IL-18BP were detected fromelectroporated BHK-21 cells with monogenic or multigenic srRNA constructs. Supernatants were harvested at approximately 24 and 48 hours after transfection. IL- IRA was assayed using IL-ip Reporter HEK 293 Cells (Invivogen, cat# hkb-illbv2) by pre-incubating cells with dilutions of BHK-21 supernatants, then incubating cells with 1 ng / mL IL- 1 P and developing the signal as per the manufacturer’s protocol. IL-18BP was assayed using IL-18 Reporter HEK 293 Cells (Invivogen, cat# hkb-hmill8) by preparing dilutions of BHK-21 supernatants into 200 pg / ml final IL- 18 then incubating and developing the signal as per the manufacturer’s protocol.

[0237] FIGS. 4 and 6 summarize the results of the experiments performed to measure in vitro expression of bioactive IL-18BP and bioactive IL-IRA in monogenic and / or bigenic expression cassettes. Bioactive IL-18BP expression was observed from the monogenic and all the bigenic expression cassettes at 24 and 48 hours post-transformation (see, e.g., FIG. 4), with the highest expression level observed from the IL1RA-IRES-IL18BP cassette. Bioactive IL-IRA expression was observed in all bigenic cassettes at 24 and 48 hours post-transformation (see, e.g., FIG. 6), with the highest expression level observed from the IL18BP-IRES-IL1RA cassette.

[0238] FIG. 7 shows the ratio of in vitro bioactive IL-IRA to ELISA IL-IRA bigenic expression cassettes expressed from bigenic expression cassettes. Notably, when the amino acid sequence of IL1-RA is C-terminally linked to a P2A sequence (in the case of the IL1RA-P2A- IL18BP cassette), the bioactivity of IL-IRA was disproportionately lower than the total IL-IRA protein measured. Since protein expression does not always correlate with functional protein product, the experiments results presented here shows that the combination of IL1-RA with IL- 18BP in a srRNA vector is not a readily solved problem in the art of srRNA vector design.EXAMPLE 3In vitro evaluation of modified alphavirus vectors

[0239] This Example describes the results of in vitro experiments performed to evaluate IL-IRA expression and bioactivity levels of the synthetic srRNA multigenic constructs that encode IL-IRA and one or more additional polypeptides, and to investigate any differential behavior thereof (e.g., replication and protein expression). In these experiments, IL-IRA protein expression and bioactivity were measured from different multigenic constructs having different ordinalities of genes. A summary of the nucleic acid constructs used in these experiments are presented in Table 2 below:

[0240] TABLE 2: Exemplary multigenic constructs having different ordinalities of the following components: IL- 12 subunit p35 (P35), IL- 12 subunit p40 (p40), IL- IRA, P2A, and IRES.

[0241] In vitro transcription'. RNA was prepared by in vitro transcription from a Sap - linearized plasmid template with bacteriophage T7 polymerase with a 5’ ARCA cap (HiScribe™ T7 ARCA mRNA Kit, NEB). RNA was then purified using phenol / chloroform extraction, or column purification (Monarch® RNA Cleanup Kit, NEB). RNA concentration was determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0242] Replication: RNA was transformed by electroporation into BHK-21 or Vero cells (e.g., 4D-Nucleofector™ Lonza). At 15-22 hours following transformation, the cells were fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained using a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons) toquantify the frequency of dsRNA+ cells and the mean fluorescence intensity (MFI) of dsRNA in individual cells by fluorescence flow cytometry.

[0243] Protein expression by ELISA : Human IL-IRA was detected from electroporated BHK-21 cells with 500 ng of srRNA multigenic constructs. Supernatants were harvested at approximately 24 and 48 hours after transfection and assayed with Human IL-lra / IL-lF3 DuoSet ELISA from RnD Systems (cat# DY280).

[0244] Bioactivity assay. Functional human IL-IRA was detected from electroporated BHK-21 cells with 500 ng of srRNA multigenic constructs. Supernatants were harvested at approximately 24 and 48 hours after transfection and assayed using IL- 10 Reporter HEK 293 Cells (Invivogen, cat# hkb-illbv2) pre-incubated with 4 ng / mL ZL10 (1 ng / mL final) and supernatants from transfected BHK cells as per the manufacturer’s protocol.

[0245] Evaluation of ordinality of genes'. Results of IL-lRA-detecting ELISA experiments as measured from transfected BHK-21 cells are shown in FIG. 8. Seventeen multigenic constructs having different ordinalities of IL-12 subunit p35, IL-12 subunit p40, IL-IRA, P2A, and IRES, were tested in order to determine which configuration of genes in the constructs yielded the most robust in vitro expression of IL-IRA. The Y-axis shows IL-IRA concentration in ng / mL. In these experiments, IL-IRA expression was quantified from all multigenic cassettes at 24 and 48 hours post-transformation.

[0246] FIG. 9 summarizes the results of the experiments performed to measure in vitro expression of bioactive IL-IRA in the multigenic expression cassettes described in Table 2 and FIG. 8 above. Bioactivity of expressed IL-IRA was quantified from all multigenic cassettes at 24 and 48 hours post-transformation.

[0247] Similar to the differences observed in the relative IL-IRA expression and bioactivity from bigenic vectors containing IL-IRA and IL-18BP (FIGS. 5-7), the bioactivity of IL-IRA does not always correspond to the magnitude of IL-IRA expression. This further shows that that the combination of IL-IRA with one or more additional polypeptides is not a readily solved problem in the art of srRNA vector design.EXAMPLE 4In vivo evaluation of modified alphavirus vectors

[0248] This Example describes the results of in vivo experiments performed to evaluate the srRNA constructs described herein (e.g., both unformulated and LNP formulated vectors).

[0249] In these experiments, synthetic srRNA constructs derived from various alphavirus strains were designed and subsequently evaluated.

[0250] Mice and injections'.

[0251] BALB / c mice were purchased from Charles River Labs, Envigo, or Jackson Laboratories. On day of dosing, between 0.01-40 pg of material was injected intramuscularly either into one or split into both quadricep muscles. Vectors were administered either unformulated in saline, LNP formulated, or polymer formulated. Animals were monitored for body weight and other general observations throughout the course of the study. For pharmacokinetic studies, animals were dosed on Day 0 only. Serum was collected on Day 3 for expression analysis.

[0252] LNP formulation'. srRNA was formulated in lipid nanoparticles using a microfluidics mixer and analyzed for particle size, poly dispersity using dynamic light scattering, and encapsulation efficiency using a dye exclusion assay (Ribogreen). Lipids were suspended in ethanol. Each srRNA was suspended in 100 mM NaOAc pH 4.0 at a concentration of 82 pg / ml and is mixed at a flow rate of 3 : 1 (aqueous: organic).

[0253] Polymer formulation:

[0254] srRNA is formulated into polymeric complexes (polyplexes) by mixing equal volumes of anionic components (srRNA and shielding components [e.g. PEG]) with cationic components (nitrogen containing ionized polymers). Mixing at small scale is accomplished with pipette mixing followed by optional brief vortexing. For larger scale, microfluidic mixing can be utilized. Complexes are characterized for size and poly dispersity using dynamic light scattering, and srRNA encapsulation efficiency is measured with a dye exclusion assay (Ribogreen). Particle properties can be optimized by varying polymer to RNA weight ratios as well as through buffer selection and mixing conditions. Buffers are typically in the pH range of 6.5-7.5 and include low salt buffers (e.g. 10-20 mM Tris / His) for non-shi elded polyplexes or standard buffers like PBS for PEGylated / shi elded polyplexes.

[0255] Protein expression by ELISA : To measure the serum concentrations of IL- IRA and IL-18BP, ELISA analysis was performed using Human IL- Ira ELISA Kit (Abeam, cat #ab211650) and Human IL-18BP ELISA Kit (Invitrogen, cat # EHILI 8BP) as per manufacturer’s protocol. Sera was pooled prior to measurement from animals injected with the unformulated srRNA group due to low volume.

[0256] FIGS. 14 and 15 summarize the results of the ELISA experiments performed to measure in vivo expression of IL-IRA and IL-18BP in different modified alphavirus vectors that were each formulated in an LNP delivery system. IL- IRA was detected in the sera of animals 3 days following administration of monogenic and most bigenic vectors (FIG. 14). The expression levels were below the detection limit of the assay for the SINV vectors, with the highest expression observed from the LNP -formulated bigenic EEEV vector. IL-18BP expression was observed 3 days following administration of the LNP-formulated bigenic EEEV vector, while the expression levels for the other bigenic vectors were below detection limit of the assay (FIG. 15).

[0257] FIGS. 16 and 17 summarize the results of the ELISA experiments performed to measure in vivo expression of IL-IRA and IL-18BP from a bigenic EEEV vector formulated with two different delivery vehicles, which were (i) LNP and (ii) polymeric nanoparticle. IL- 1RA expression was observed at 3 and 7 days following administration of LNP-formulated vector, and expression was detected at 7 days following administration of polymer-formulated vector (FIG. 16). IL-18BP expression was observed at 3 and 7 days following administration of LNP-formulated vector, and expression was detected at 3 and 7 days following administration of polymer-formulated vector (FIG. 17). These data demonstrate in practice that a bigenic srRNA vector encoding IL-IRA and IL-18BP can be formulated in multiple ways to express these proteins in vivo.Example 5 srRNA constructs expressing IL-IRA and IL-18BP extends the pharmacokinetics of a target protein that has a short half-life

[0258] This Example describes the results of in vivo experiments performed to demonstrate that srRNA constructs expressing IL-IRA and IL-18BP (e.g., in LNP formulated vectors) as described herein can extend the pharmacokinetics of a target protein (IL- IRA) that has a short half-life.

[0259] In these experiments, as described in FIGS. 18A-18B, mice were administered human IL-IRA recombinant protein at t=0 or LNP-formulated srRNA-encoding IL-IRA and IL-18BP 96 hours prior to achieve Tmax for srRNA-based protein expression. Human IL-IRA levels were measured at t=2 and 8 hours by standard serum ELISA. As shown in FIG. 18, srRNA-based expression of IL-IRA and IL-18BP shows less decay in systemic protein levels versus recombinant protein.

[0260] It was reported previously that systemic levels of human IL-IRA are rapidly reduced from 2 to 8 hours in mice administered with a recombinant protein due to its short halflife. However, in the present studies, mice administered with an LNP-formulated sRNA coencoding IL-IRA and IL-18BP 4 days prior to achieve maximal protein expression from this platform, show stable levels of encoded human IL-IRA at 2 and 8 hours. Thus, systemic expression of a protein with a short half-life, such as human IL-IRA, can be extended with a srRNA-based approach, due to the longer kinetics of protein expression from this technology.

[0261] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A nucleic acid construct comprising a nucleic acid sequence encoding a modified alphavirus genome or self-replicating RNA (srRNA), wherein at least a portion of the nucleic acid sequence encoding one or more viral structural proteins of the modified alphavirus genome or srRNA has been replaced with a coding sequence for a polypeptide construct comprising: a) a coding sequence for an interleukin-1 receptor antagonist (IL-IRA) protein or a functional variant thereof; and b) a coding sequence for an interleukin- 18 binding protein (IL-18BP) or functional variant thereof; wherein the coding sequences for IL- IRA and IL-18BP are operably linked to one another, and wherein the polypeptide construct does not comprise a dimerization domain.

2. The nucleic acid construct of Claim 1, wherein the polypeptide construct does not comprise a fragment crystallization region (Fc region) of an immunoglobulin.

3. The nucleic acid construct of any one of Claims 1 to 2, wherein the coding sequence for IL- 1RA is N-terminally linked to the coding sequence for IL-18BP.

4. The nucleic acid construct of any one of Claims 1 to 2, wherein the coding sequence for IL- 1RA is C-terminally linked to the second coding sequence for IL-18BP.

5. The nucleic acid construct of any one of Claims 1 to 4, wherein the coding sequences for IL-IRA and IL-18BP express proteins that are functional in a bioactivity assay.

6. The nucleic acid construct of any one of Claims 1 to 5, wherein the IL-18BP protein is the IL-18BP isoform a (IL-18BPa).

7. The nucleic acid construct of any one of Claims 1 to 6, wherein the IL-IRA protein and / or IL-18BP protein are from a mammalian subject.

8. The nucleic acid of Claim 7, wherein the mammalian subject is a human subject.

9. The nucleic acid construct of any one of Claims 1 to 8, wherein the coding sequence for IL- 1RA and / or IL-18BP is optimized for one of more of the following:a) enhancing RNA stability and / or expression level; b) minimizing rare codon usage and / or secondary structures; and c) facilitating better srRNA replication and RNA manufacturing process.

10. The nucleic acid construct of any one of Claims 1 to 9, wherein the coding sequences for IL-IRA and IL-18BP are operably linked to one another via a connector sequence encoding an autoproteolytic peptide and / or an internal ribosomal entry site (IRES).

11. The nucleic acid construct of Claim 10, wherein the autoproteolytic peptide comprises one or more autoproteolytic cleavage sequences from a calcium-dependent serine endoprotease (furin), a porcine teschovirus-1 2 A (P2A), a foot-and-mouth disease virus (FMDV) 2 A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), or a combination thereof.

12. The nucleic acid construct of Claim 11, wherein the internal ribosomal entry site (IRES) is from a Kaposi’s sarcoma-associated herpesvirus (KSHV) IRES, a hepatitis virus IRES, a Pestivirus IRES, a Cripavirus IRES, a Rhopalosiphum padi virus IRES, a fibroblast growth factor IRES, a platelet-derived growth factor IRES, a vascular endothelial growth factor IRES, an insulin-like growth factor IRES, a picornavirus IRES, an encephalomyocarditis virus (EMCV) IRES, a Pim-1 IRES, a p53 IRES, an Apaf-1 IRES, a TDP2 IRES, an L-myc IRES, and a c-myc IRES.

13. The nucleic acid construct of any one of Claims 1 to 12, wherein the modified viral genome or srRNA is devoid of a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins.

14. The nucleic acid construct of any one of Claims 1 to 12, wherein the modified alphavirus genome or srRNA comprises no nucleic acid sequence encoding viral structural proteins.

15. The nucleic acid construct of any one of Claims 1 to 14, wherein the coding sequences for IL-IRA and IL-18BP are operably linked to one another within a single open reading frame i.e., in a polycistronic ORF).

16. The nucleic acid construct of any one of Claims 1 to 15, wherein the nucleic acid sequence encoding the polypeptide construct is operably linked to a promoter sequence.

17. The nucleic acid construct of Claim 16, wherein the promoter sequence is a subgenomic (sg) promoter.

18. The nucleic acid construct of Claim 17, wherein the sg promoter sequence is a 26S subgenomic promoter.

19. The nucleic acid construct of any one of Claims 17 to 18, wherein the subgenomic promoter is heterologous relative to the remainder of the modified viral genome or srRNA.

20. The nucleic acid construct of any one of Claims 17 to 19, wherein the subgenomic promoter is an alphavirus subgenomic promoter.

21. The nucleic acid construct of any one of Claims 1 to 20, wherein at least one nonstructural protein (nsP), or a portion thereof, of the modified viral genome or srRNA is heterologous relative to the remainder of the modified viral genome or srRNA.

22. The nucleic acid construct of any one of Claims 1 to 21, further comprising a nucleic acid sequence encoding a heterologous nsP or a portion thereof.

23. The nucleic acid construct of any one of Claims 1 to 22, further comprising one or more untranslated regions (UTRs).

24. The nucleic acid construct of Claim 23, wherein at least one of the UTRs is a heterologous UTR.

25. The nucleic acid construct of any one of Claims 1 to 24, wherein the modified alphavirus genome or srRNA is of an alphavirus belonging to the Venezuelan equine encephalitis virus / Eastem Equine Encephalitis virus (VEEV / EEEV) group, or the Semliki Forest virus (SFV) group, or the Smdbis virus (SINV) group.

26. The nucleic acid construct of any one of Claims 1 to 25, wherein the modified alphavirus genome or srRNA is of an alphavirus belonging to the BFV complex, EEEV complex, MIDV complex, NDUV, complex, SFV complex, VEEV complex, WEEV complex.

27. The nucleic acid construct of Claim 26, wherein the alphavirus is Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O’Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu virus (NDUV), Madariaga virus (MADV), or Buggy Creek virus.

28. The nucleic acid construct of Claim 27, wherein the alphavirus is VEEV, EEEV, CHIKV, or SINV.

29. The nucleic acid construct of any one of Claims 1 to 28, wherein the coding sequence for the polypeptide construct comprises, in N-terminus to C-terminus direction:(a) (i) a coding sequence for IL-IRA, (ii) a connector sequence encoding an IRES, and (iii) a coding sequence for IL-18BP;(b) (i) a coding sequence for IL- IRA, (ii) a connector sequence encoding a P2A autoproteolytic peptide, and (iii) a coding sequence for IL-18BP;(c) (i) a coding sequence for IL-18BP, (ii) a connector sequence encoding an IRES, and (iii) a coding sequence for IL- IRA; or(d) (i) a coding sequence for IL-18BP, (ii) a connector sequence encoding a P2A autoproteolytic peptide, and (iii) a coding sequence for IL- IRA.

30. The nucleic acid construct of any one of Claims 1 to 29, wherein the polypeptide construct comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 7-10.

31. The nucleic acid construct of any one of Claims 1 to 30, wherein the coding sequence for the polypeptide construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 11-14.

32. The nucleic acid construct of Claim 31 , wherein the nucleic acid construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 15.

33. A recombinant cell comprising a nucleic acid construct according to any one of Claim 1 to 32.

34. The recombinant cell of Claim 33, wherein the recombinant cell is a eukaryotic cell.

35. The recombinant cell of Claim 34, wherein the eukaryotic cell is an animal cell.

36. The recombinant cell of Claim 35, wherein the animal cell is a vertebrate animal cell or an invertebrate animal cell.

37. The recombinant cell of Claim 35, wherein the animal cell is a mammalian cell.

38. The recombinant cell of Claim 35, wherein the animal cell is an insect cell.

39. The recombinant cell of Claim 38, wherein the insect cell is a mosquito cell.

40. The recombinant cell of any one of Claims 35 to 39, wherein the recombinant cell is an immune cell.

41. The recombinant cell of Claim 40, wherein the immune cell is a B cell, a monocyte, a natural killer (NK) cell, a natural killer T (NKT) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell (DC), a macrophage, a regulatory T cell, a helper T cell (TH), a cytotoxic T cell (TCTL), a memory T cell, a gamma delta (yd) T cell, a hematopoietic stem cell, or a hematopoietic stem cell progenitor.

42. The recombinant cell of Claim 41, wherein the immune cell is a B cell, a T cell, a macrophage, or a dendritic cell (DC).

43. A cell culture comprising at least one recombinant cell according to any one of Claims 33 to 42, and a cell culture medium.

44. A transgenic animal comprising a nucleic acid construct according to any one of Claims 1 to 32.

45. The transgenic animal of Claim 44, wherein the animal is a vertebrate animal or an invertebrate animal.

46. The transgenic animal of Claim 44, wherein the animal is a mammal.

47. The transgenic animal of Claim 46, wherein the mammal is a non-human mammal.

48. The transgenic animal of Claim 44, wherein the animal is an insect.

49. The transgenic animal of Claim 48, wherein the transgenic insect is a transgenic mosquito.

50. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and: a) a nucleic acid construct according to any one of Claims 1 to 32; and / or b) a recombinant cell according to any one of Claims 33 to 42.

51. The pharmaceutical composition of Claim 50, wherein the composition is formulated with a delivery vehicle into a delivery system, wherein the delivery system comprises a polymeric nanoparticle, a lipid-based nanoparticle (LNP), a liposome, a viral replicon particle (VRP), a physiologic buffer, a microsphere, an immune stimulating complex (ISCOM), a conjugate of bioactive ligand, or a combination of any thereof.

52. The pharmaceutical composition of Claim 51, wherein the polymeric nanoparticle comprises a cationic polymer, a non-cationic polymer, or a combination thereof.

53. The pharmaceutical composition of Claim 52, wherein the cationic polymer comprises a naturally-derived cationic polymer.

54. The pharmaceutical composition of Claim 53, wherein the naturally-derived cationic polymer comprises chitosan, gelatin, dextran, cellulose, cyclodextrin, or a combination thereof.

55. The pharmaceutical composition of Claim 52, wherein the cationic polymer comprises a synthetic cationic polymer.

56. The pharmaceutical composition of Claim 55, wherein the synthetic cationic polymer comprises a polyethyleneimine (PEI), poly-L-lysine (PLL), a poly(amino acid) (PAA), a poly(amidoamine) (PAMAM), a poly(cystamine bisacrylamide-co-4-amino-l-butanol) (pABOL), a poly(amino-co-ester) (PAE), poly(2-N,N-dimethylaminoethylmethacrylate, a poly(beta-amino ester) (PBAE), an imidazole-containing polymer, a tertiary-amine containingpolymer, poly(2-(dimethylamino)ethyl methacrylate), poly-N-(2-hydroxy- propyl)methacrylamide, a polyamidoamine dendrimer, a cationic glycopolymer, or derivatives thereof.

57. The pharmaceutical composition of Claim 52, wherein the non-cationic polymer is negatively-charged (i.e., anionic) or electronically neutral.

58. The pharmaceutical composition of Claim 57, wherein the non-cationic polymer comprises a polyethylene glycol (PEG), a polyester (e.g., polylactic acid (PLA), poly (lactic-co-glycolic acid) (PLGA), poly glycolic acid (PGA), polycaprolactone (PCL)), and polysarcosine (pSar), or derivatives thereof.

59. The pharmaceutical composition of any one of Claims 52 to 58, wherein the polymer is water-soluble and / or biodegradable.

60. The pharmaceutical composition of any one of Claims 52 to 58, wherein the polymeric nanoparticle comprises one or more of the following: poly-('Y-L-glutamylglutamine) (PGGA), poly-(Y-L-aspartylglutamine) (PGAA), poly-L-lactic acid (PLLA), poly-(lactic acid-co-glycolic acid) (PLGA), polyalkylcyanoacrylate (PACA), polyanhydrides, polyhydroxyacids, polypropylfumerate, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, [N-(2-hydroxypropyl)methacrylamide] (HPMA) copolymer, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polyurea, polyamine polyepsilon- caprolactone (PCL), and copolymers thereof.

61. The pharmaceutical composition of Claim 51, wherein the LNP delivery system comprises a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid.

62. The pharmaceutical composition of Claim 51, wherein the lipid is present in mass ratio of lipid to RNA from about 100:1 to about 4:1.

63. The pharmaceutical composition of Claim 51, wherein the lipid-based nanoparticles have an average diameter of about 25 nm to about 1000 nm.

64. The pharmaceutical composition of any one of Claims 50 to 63, wherein the composition is formulated as a biotherapeutic.

65. A method for modulating a pharmacodynamic effect in a subject, the method comprising administering to the subject a composition comprising: a) a nucleic acid construct according to any one of Claims 1 to 32; b) a recombinant cell according to any one of Claims 33 to 42; and / or c) a pharmaceutical composition according to any one of Claims 50 to 64.

66. The method of Claim 65, wherein the pharmacodynamic effect comprises eliciting an immune response in the subject.

67. The method of Claim 65, wherein the pharmacodynamic effect comprises one or more of the following: immunogenicity effect, a biomarker response, a therapeutic effect, a prophylactic effect, a desired effect, an undesired effect, an adverse effect, and effect in a disease model.

68. A method for improving / prolonging kinetics of expression of IL-IRA and / or IL-18BP, or for enhancing the endogenous expression of IL-IRA and / or IL-18BP in a subject, the method comprising administering to the subject a composition comprising: a) a nucleic acid construct according to any one of Claims 1 to 32; b) a recombinant cell according to any one of Claims 33 to 42; and / or c) a pharmaceutical composition according to any one of Claims 50 to 64.

69. A method for preventing and / or treating a health condition in a subject in need thereof, the method comprising administering to the subject a composition comprising: a) a nucleic acid construct according to any one of Claims 1 to 32; b) a recombinant cell according to any one of Claims 33 to 42; and / or c) a pharmaceutical composition according to any one of Claims 50 to 64.

70. The method of Claim 69, wherein the health condition is an autoimmune disease, an inflammatory disease, or a cardiovascular disease.

71. The method of any one of Claims 68 to 70, wherein the subject has or is suspected of having a health condition associated with an autoimmune disease, an inflammatory disease, or a cardiovascular disease.

72. The method of any one of Claims 68 to 71, wherein the administered composition elicits an immune response in the subject.

73. The method of any one of Claims 68 to 72, wherein the administered composition modulates production of one or more pro-inflammatory molecules in the subject.

74. The method of Claim 73, wherein the one or more pro-inflammatory molecules comprises interleukin-1 alpha (LFNa), inteleukin-1 beta ( FNP), interleukin- 18 (IL-18), interleukin-6 (IL- 6), interferon gamma (IFNy), cytokines, TNF-a, GM-CSF, and MIPla, granzyme B, granzyme A, perforin, or a combination of any thereof.

75. The method of any one of Claims 68 to 74, wherein the composition is administered to the subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapies.

76. The method of Claim 75, wherein the at least one additional therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery.

77. A kit for modulating a pharmacodynamic effect, eliciting an immune response, and / or for the prevention and / or treatment of a health condition, the kit comprising one or more of the following: a) a nucleic acid construct according to any one of Claims 1 to 32; b) a recombinant cell according to any one of Claims 33 to 42; and / or c) a pharmaceutical composition according to any one of Claims 50 to 64.

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